TITLE OF THE INVENTION
GLYCOPEGYLATION METHODS AND PROTEINS/PEPTIDES PRODUCED BY
THE METHODS
BACKGROUND OF THE INVENTION
Most naturally occurring peptides contain carbohydrate moieties attached to the
peptide via specific linkages to a select number of amino acids along the length of the
primary peptide chain. Thus, many naturally occurring peptides are termed
"glycopeptides." The variability of the glycosylation pattern on any given peptide has
enormous implication;; for the function of that peptide. For example, the structure of
the N-linked glycans on a peptide can impact various characteristics of the peptide,
including the protease susceptibility, intracellular trafficking, secretion, tissue targeting,
biological half-life and antigenicity of the peptide in a cell or organism. The alteration
of one or more of these characteristics greatly affects the efficacy of a peptide in its
natural setting, and also affects the efficacy of the peptide as a therapeutic agent in
situations where the peptide has been generated for that purpose.
The carbohydrate structure attached to the peptide chain is known as a "giycan"
molecule. The specific glycan structure present on a peptide affects the solubility and
aggregation characteristics of the peptide, the folding of the primary peptide chain and
therefore its functional or enzymatic activity, the resistance of the peptide to proteolytic
attack and the control of proteolysis leading to the conversion of inactive forms of the
peptide to active forms. Importantly, terminal sialic acid residues present on the glycan
molecule affect the length of the half life of the peptide in the mammalian circulatory
system. Peptides whose glycans do not contain terminal sialic acid residues are rapidly
removed from the circulation by the liver, an event which negates any potential
therapeutic benefit of the peptide.
The glycan structures found in naturally occurring glycopeptides are typically
divided into two classes, N-Iinked and O-Iinked glycans.
Peptides expressed in eukaryotic cells are typically N-glycosylated on
asparagine residues at sites in the peptide primary stioicture containing the sequence
asparagin6-X-serincol) ("PEG") is an exemplary polymer that has been
conjugated to peptides. The use of PEG to derivatize peptide therapeutics has been
demonstrated to reduce the immunogenicity of the peptides and prolong tlie clearance
time from the circulation. For example, U.S. Pat. No. 4,179,337 (Davis et al.) concerns
non-immunogenic peptides, such as enzymes and peptide hormones coupled to
polyethylene glycol (PEG) or polypropylene glycol. Between 10 and 100 moles of
polymer are used per moie peptide and at least 15% of the physiological activity is
maintained.
WO 93/15189 (Veronese et al.) concerns a method to maintain the activity of
polyethylene glj'col-modified proteolytic enzymes by Imking the proteolytic enzyme to
a macromoleculiirized inhibitor. The conjugates are intended for medical applications.
The principal mode of attachment of PEG, and its derivatives, to peptides is a
non-specific bonding through a peptide amino acid residue. For example, U.S. Patent
No. 4,088,538 discloses an enzymatically active polymer-enzyme conjugate of an
enzyme covalently linked to PEG. Similarly, U.S. Patent No. 4,496,689 discloses a
covalently attached complex of a-1 protease inhibitor with a polymer such as PEG or
methoxypoly(ethylene glycol) ("mPEG"). Abuchowski e/* a/. (J. Biol. Chem. 252: 3578
(1977) discloses the covalent attachment of mPEG to an amine group of bovine sen:Lm
albumin. U.S. Patent No. 4,414,147 discloses a method of rendering interferon less
hydrophobic by conjugating it to an anhydride of a dicarboxylic acid, such as
poly(ethyiene succinic anhydride). PCT WO 87/00056 discloses conjugation of PEG
and poly(oxyethylated) polyols to such proteins as interferon-p, interleuldn-2 and
immunotoxins. EP 154,316 discloses and clauns chemically modified iymphokines,
such as IL-2 containing PEG bonded directly to at least one primary amino group of the
lymphokine. U.S. Patent No. 4,055,635 discloses pharmaceutical compositions of a
water-soluble complex of a proteolytic enzyme linked covalently to a polymeric
substance such as a polysaccharide.
Another mode of attaching PEG to peptides is through the non-specific
oxidation of glycosyl residues on a peptide. The oxidized sugar is utilized as a locus
for attaching a PEG moiety to the peptide. For example, M'TimkuIu (WO 94/05332)
discloses the use of a hydrazine- or amino-PEG to add PEG to a glycoprotein. The
glycosyl moieties aj-e randomly oxidized to the corresponding aldehydes, which arc
subsequently coupled to the amino-PEG. See also. Bona et al. (WO 96/40731), where a
PEG is added to an immunoglobulin molecule by enzymatically oxidizing a glycan on
the immunoglobulin and then contacting the glycan with an amino-PEG molecule.
In each of the methods described above, poIy(ethylene glycol) is added in a
random, non-specific manner to reactive residues on a peptide backbone. For the
production of therap'eutic peptides, it is clearly desirable to utilize a derivatization
strategy that results in the formation of a specifically labeled, readily characterizable,
essentially homogeneous product.
Two princip£il classes of enzymes are used in the synthesis of carbohydrates,
glycosyltransferases (e.g., sialyltransferases, oligosaccharyltransferases, N-
acetylglucosaminyltransferases), and glycosidases. The glycosidases are fiirther
classified as exoglycosidases (e.g., P-raaixnosidase, |3-glucosidase), and
endoglycosidases (e.g., Endo-A, Endo-M). Each of these classes of enzymes has been
successfully used synthetically to prepare carbohydrates. For a genersil review, see.
Grout et al., Ciirr. Opin. Chem. Biol. 2: 98-111 (1998).
Glycosyltransferases modify the oligosaccharide structures on peptides.
Glycosyltransferases ijre effective for producing specific products with good
stereochemical and regiochemical control. Glycosyltransferases have been used to
prepare oligosaccharides and to modify terminal N- and 0-linked carbohydrate
structures, particularly on peptides produced in mammalian cells. For example, the
terminal oligosaccharides of glycopeptides have been completely sialylated and/or
fucosylated to provide more consistent sugar structures, which improves glycopeptide
pharmacodynamics and a variety of other biological properties. For example, (3-1,4-
galactosyltransferase is used to synthesize lactosamine, an illustration of the utility' of
glycosyltransferases in the synthesis of carbohydrates (see, e.g., Wong et al., J. Org.
Chem. 47: 5416-5418 (1982)). Moreover, numerous synthetic procedures have made
use of a-sialyltransferases to transfer sialic acid from cytidiiie-5'-monophospho-N-
acet^'Ineuraminic acid to the 3-OH or 6-OH of galactose {see, e.g., Kevin et al, Chem.
Eur. J. 2: 1359-1362 (1996)). Fucosyltransferases are used in synthetic pathways to
transfer a fiicose unit from guanosine-5'-diphosphofticose to a specific hydroxyl of a
saccharide acceptor. For example, Ichikawa prepared sialyl Lewis-X by a method that
involves the fucosylation of sialylated lactosamine with a cloned fucosyltransferase
(Ichikawa et al.,J. Am. Chem. Soc. 114: 9283-9298 (1992)). For a discussion of recent
advances in glycoconjugate synthesis for therapeutic use see, Koeller et al.. Nature
Biotechnology 18: 835-841 (2000). See also, U.S. Patent No. 5,876,980; 6,030,815;
5,728,554; 5,922,577; and WO/9831826.
Glycosidases can also be used to prepare saccharides. Glycosidases normally
catalyze the hydrolysis of a glycosidic bond. However, under appropriate conditions,
they can be used to form this linkage. Most glycosidases used for carbohydrate
synthesis are exoglycosidases; the glycosyl transfer occurs at the non-reducing termmus
of the substrate. The glycosidase binds a glycosyl donor in a glycosyl-enzyme
izitermediate that is either intercepted by water to yield the hydrolysis product, or by an
acceptor, to generate a new glycoside or oligosaccharide. An exemplary pathway using
an exoglycosidase is the synthesis of the core trisaccharide of all N-linked
glycopeptides, including the P-mannoside linkage, which is formed by the action of (5-
mannosidase (Singh et al, Chem. Commun. 993-994 (1996)).
In another exemplary application of the use of a glycosidase to form a
glycosidic linkage, a mutant glycosidase has been prepared in which the nonnal
nucleophilic amino acid within the active site is changed to a non-nucleophilic amino
acid. The mutant enzyme does not hydrolyze glycosidic linkages, but can still form
them. Such a mutant glycosidase is used to prepare oligosaccharides using an a-
glycosyl fluoride donor and a glycoside acceptor molecule (Wittiers et al, U.S. Patent
No. 5,716,812).
Although the;ir use is less common than that of the exoglycosidases,
endoglycosidases are also utilized to prepare carbohydrates. Methods based on the use
of endoglycosidases have the advantage that an oligosaccharide, rather than a
monosaccharide, is Iransferred. Oligosaccharide fragments have been added to
substrates using enc/o-p-N-acetylglucosamines such as endo-F, endo-M (Wang et al.,
TeU-ahedron Lett. 37: 1975-1978); andHaneda er a/., Carbohydr. Res. 292: 61-70
(1996)).
In addition to their use in preparing carbohydrates, the enzymes discussed above
are applied to the synthesis of glycopeptides as well. The synthesis of a homogenous
glycoform of ribonuclease B has been published (Witte K. et al., J. Am. Chem. Soc.
119: 2114-2118 (1997)). The high mannose core of ribonuclease B was cleaved by
treating the glycopeptide with endoglycosidase H. The cleavage occurred specifically
between the two core GlcNAc residues. The tetrasaccharide sialyl Lewis X was then
enzymatically rebuilt on the remaining GlcNAc anchor site on the now homogenous
protein by the sequential use of p-l,4-galactosyltransferase, a-2,3-sialyltransferase and
a-l,3-fucosyltraiisferase V. However, while each enzymatically catalyzed step
proceeded in excelkint yield, such procedures have not been adapted for the generation
of glycopeptides on an industrial scale.
Methods corabining both chemical and enzymatic synthetic elements are also
known in the art. For example, Yamamoto and coworkers (Carbohydr. Res. 305: 415-
422 (1998)) reported the chemoenzymatic synthesis of the glycopeptide, glycosylated
Peptide T, using an endoglycosidase. The N-acetylglucosaminyl peptide was
s>Tithesized by purely chemical means. The peptide was subsequently enzymatically
elaborated with the oligosaccharide of human transferrin peptide. The saccharide
portion was added to the peptide by treating it with an endo-P-N-
acetylglucosaniinidase. Thie resulting glycosylated peptide was highly stable and
resistant to proteolysis when compared to the peptide T and N-acetylglucosammyl
peptide T.
The use of gl3'^cosyltransferases to modify peptide structure with reporter groups
has been explored. For example, Brossmer et al. (U.S. Patent No. 5,405,753) discloses
the formation of a fluorescent-labeled cytidine monophosphate ("CMP") derivative of
sialic acid and the use; of the fluorescent glycoside in an assay for sialyl transferase
activity and for the fluorescent-labeling of cell surfaces, glycoproteins and peptides.
Gross et al. {Analyt. Biochem. 186: 127 (1990)) describe a similar assay. Bean et al.
(U.S. Patent No. 5,432,059) discloses an assay for glycosylation deficiency disorders
utilizing reglycosylation of a deficiently glycosylated protein. The deficient protein is
reglycosylated with a fluorescent-labeled CMP glycoside. Each of the fluorescent
sialic acid derivatives is substituted with the fluorescent moiety at either the 9-position
or at the amine that is normally acetylated in sialic acid. The methods using the
fluorescent sialic acid derivatives are assays for the presence of glycosyltransferases or
for non-glycosylated or improperly glycosylated glycoproteins. The assays are
conducted on small amounts of enzyme or glycoprotein in a sample of biological
origin. The enzymatic derivatization of a glycosylated or non-glycosylated peptide on
a preparative or industrial scale using a modified sialic acid has not been disclosed or
suggested in the prior iirt.
Considerable effort has also been directed towards the modification of cell
surfaces by altering glycosyl residues presented by those surfaces. For example,
Fukuda and coworkers have developed a method for attaching glycosides of defined
structure onto cell surfaces. The method exploits the relaxed substrate specificity of a
fucosyltransferase that can transfer fucose and fucose analogs bearing diverse glycosyl
substrates (Tsuboi et al., J. Biol Chem. 271: :r.'2i3 (1996)).
Enzymatic methods have also been used to activate glycosyl residues on a
glycopeptide towards subsequent chemical elaboration. The glycosyl residues are
typically activated using galactose oxidase, which converts a terminal galactose residue
to the corresponding aldehyde. The aldehyde is subsequently coupled to an amine-
containing modifying group. For example, Casares et al. {Nature Biotech. 19: 142
(2001)) have attached doxorubicin to the oxidized galactose residues of a recombinant
MHCII-peptide chimera.
Glycosyl residues have also been modified to contain ketone groups. For
example, Mahal and co-workers (Science 276; 1125 (1997)) have prepared N-
levulinoyl mannosaniine ("ManLev"), which has a ketone functionality at the position
normally occupied b;y' the acetyl group in the natural substrate. Cells were treated with
the ManLev, thereby incorporating a ketone group onto the cell surface. See, also
Saxon et al, Science 287: 2007 (2000); Hang et al, J. Am. Chem. Soc. 123: 1242
(2001); Yareraa et al, J. Biol. Chem. 273: 31168 (1998); and Charter et al,
Glycobiology 10: 1049(2000).
The methods of modifying cell surfaces have not been applied in the absence of
a cell to modify a glycosylated or non-glycosylated peptide. Further, the methods of
cell surface modificai:ion are not utilized for the'enzymatic incorporation preformed
modified glycosyl donor moiety into a peptide. Moreover, none of the cell surface
modification methodij are practical for producing glycosyl-modified peptides on an
industrial scale.
Despite the efforts directed toward the enzymatic elaboration of saccharide
structures, there remains still a need for an industrially practical method for the
modification of glycosylated and non-glycosylated peptides with modifying groups
such as water-soluble polymers, therapeutic moieties, biomolecules and the like. Of
particular interest are methods in which the modified peptide has improved properties,
which enhance its use as a therapeutic or diagnostic agent. The present invention
fulfills these and other needs.
SUMMARY OF THE INVENTION
The invention includes a multitude of methods of remodeling a peptide
to have a specific glycan structure attached thereto. Although specific glycan structures
are described herein, the invention should not be construed to be limited to any one
particular structure. In addition, although specific peptides are described herein, the
invention should not be limited by the nature of the peptide described, but rather should
encompass any and all suitable peptides and variations thereof.
The description which follows discloses the preferred embodiments of
the invention and provides a written description of the claims appended hereto. The
invention encompasses any and all variations of these embodiments that are or become
apparent following a reading of the present specification,
The invention includes a cell-fi-ee, in vitro method of remodeling a
peptide comprising poly(ethylene glycol), the peptide having the formula:
wherein
AA is a terminal or internal amino acid residue of the peptide;
X'-X^ is a saccharide covalently linked to the AA, wherein
X' is a first glycosyl residue; and
9 1 • 1
X is a second glycosyl residue covalently linked to X , wherem X and
X^ are selected from monosaccharyi and oligosaccharyl residues;
the method comprisLag:
(a) removing X"^ or a saccharyl subunit thereof from the peptide,
thereby forming a truncated glycan.
In one aspect, the invention fiirther comprises formation of a truncated
glycan by removing a Sia residue.
hi one embodiment of the invention, a peptide has the formula:
wherein
X^X^.X^X^X^andX" , are independently selected monosacchary]
or oligosaccharyl residues; and
a, b, c, d, e, and x are independently selected from the integers 0, 1 and
2.
In one aspect of the invention, an oligosaccharyl residue is a member
selected from GlcNAc-Gal-Sia and GlcNAc-Gal. In another aspect, at least one
oligosaccharide member is selected from a, b, c, d, e and x is 1 or 2. In yet another
aspect, the removing of step (a) produces a truncated glycan in which at least one of a,
b, c, e and x are 0.
The invention includes a method of remodeling a peptide wherein X^, X^
and X are members independently selected from (mannose)z and (mannose)z-(X^)
wherein
X is cL glycosyl moiety selected from mono- and oligo-saccharides; and
z is an integer between 1 and 20, wherein
when z is 3 or greater, each (mannose)z is independently selected from
linear and branched structures.
In one aspect, a is selected from the group consisting of GlcNAc and
xylose. In another aspect, X , X and X are (mannose)u, wherein u is selected from the
iiitegers between 1 and 20, and when u is 3 or greater, each (mannose)u is
independently selecte^d from linear and branched structures.
The tn\'ention also includes a method of remodeling a peptide, wherein
the peptide has the foiTnula:
wherein
r, s, and t are integers independently selected from 0 and 1.
Ln an embodiment of the invention, a peptide has the formula:
wherein
X^ and X^° are independently selected monosaccharyl or oligosaccharyl
residues and m, n and f are integers independently selected from 0 and 1.
In one cispect, a peptide has the formula:
wherein
X'^ is a member selected from:
wherein
s and i aie integers independently selected from 0 and 1.
In anothe;r aspect, a peptide has the formula:
wherein
are independently selected glycosyl residues; and
g, h, i, j, k, and p are independently selected from the integers 0 and 1.
In yet another aspect of the invention, at least one of g, h, i, j, k and p is
I. In another aspect, X'"* and X'^ are members independently selected from GIcNAc
and Sia and i and k aie independently selected from the integers 0 and 1. In still
another aspect, at least one of i and k is 1, and if k is 1, g, h, and j are 0.
The invention also includes a method of remodeling a peptide, wherein
the method comprises contacting the truncated glycan with at least one
glycosyltransferase ajid at least one glycosyl donor under conditions suitable to transfer
the at least one glycosyl donor to the truncated glycan, thereby remodeling the peptide
comprising poly(ethylene glycol).
In one aspect, a glycosyl donor comprises a modifying group covalently
linked thereto.
The invention also includes a method of remodeling a peptide, the
method comprising removing X', thereby exposing AA. In one aspect, a method
includes contacting AA with at least one glycosyltransferase and at least one glycosyl
donor under conditions suitable to transfer said at least one glycosyl donor to AA,
thereby remodeling said peptide comprising poly(ethylene glycol).
In one aspect, at least one glycosyl donor comprises a modifying group
covalently linked thereto. In another aspect, a modifying group is poly(ethylene
glycol). In one embodiment, a poly(ethylene glycol) has a molecular weight
distribution that is essentially homodisperse.
The invention includes a method of remodeling a peptide, wherein, prior
to contacting the truncated glycan with at least one glycosyltransferase and at least one
glycosyl donor under conditions suitable to transfer the at least one glycosyl donor to
the truncated glycan, thereby remodeling the peptide comprising poly(ethylene glycol),
a group added to the saccharide during post-translational modification is removed.
In one aspect, a removed group is a member selected from phosphate,
sulfate, carboxylate ajid esters thereof
The im^ention includes a method of remodeling a peptide wherein a
peptide has the formula;
wherein
Z is a member selected from O, S, NH and a cross-linker.
The invention also includes a method of remodeling a peptide, wherein
the peptide has the formula;
wherein
are independently selected glycosyl moieties; and
r and x are integers independently selected from 0 and 1.
In one aspect of the invention, X" and X''^ are (mannose)q, wherein q is
selected from the integers between 1 and 20, and when q is three or greater, (mannose),q
is selected from line;ir and branched structures.
[ 0 The invention includes a pharmaceutical composition comprising a
pharmaceutically acceptable diluent and a remodeled peptide according to a cell-free, m
vitro method of remodeling a peptide comprising poly(ethylene glycol), the peptide
having the formula:
15 wherein
AA IS a terminal or internal amino acid residue of the peptide;
X'-?C^ is a saccharide covalently linked to the AA, wherein
X' is a first glycosyl residue; and
X^ is a second glycosyl residue covalently linked to X', wherein X' and
20 X are selected from monosaccharyl Eind oligosacchar^d residues;
the method c;omprismg:
(a) removing X or a saccharyl subumt thereof jfrom the peptide,
thereby formmg a truncated glycan.
The invention also includes a cell-free, in vitro method of remodeling a
peptide comprising poly(ethylene glycol), the peptide having the formula:
wherein
AA is a terminal or internal amino acid residue of the peptide;
X' is a glycosyl residue covalently linked to the AA, selected from
monosaccharyl and oligosaccharyl residues; and
u is an integer selected from 0 and 1,
the method comprising:
contacting the peptide with at least one glycosyltransferase and at least
one glycosyl donor under conditions suitable to transfer at least one glycosyl donor to
the truncated glycan, thereby remodeling the peptide.
In one aspect, at least one glycosyl donor comprises a modifying group
covalently linked tJiereto. In another aspect, the modifying group is poly(ethylene
glycol). In yet another aspect, the poly(ethylene glycol) has a molecular weight
distribution that is essentially homodisperse.
The invention also includes a pharmaceutical composition comprising a
pharmaceutically acceptable diluent and a remodeled peptide according to a cell-free, in
vitro method of remodeling a peptide comprising poly(ethylene glycol), the peptide
having the formula:
wherein
AA is a terminal or internal amino acid residue of the peptide;
X' is a glycosyl residue covalently linked to the AA, selected from
monosaccharyl and oligosaccharyl residues; and
u is cin integer selected from 0 and 1,
the method c:omprising:
contacting the peptide with at least one glycosyltransferase and at least
one glycosyl donor under conditions suitable to transfer at least one glycosyl donor to
the truncated glycan, thereby remodeling the peptide.
BRIEF DESCRIPTION OF THE DRAWINGS
For the purpose of illustrating the invention, there are depicted in the drawings certain
embodiments of the invention. However, the invention is not limited to the precise
arrangements and instrum<5ntaUties of the embodiments depicted in the drawings.
Figure 1 is a scheme depicting a trimannosyl core glycan (left side) and the enzymatic
process for the generation of a glycan havuig a bisecting GlcNAc (right side).
Figure 2 is a scheme depicting an elemental trimannosyl core structure and complex
chains in various degrees of completion. The in vitro enzymatic generation of an elemental
trimannosyl core structure ftom a complex carbohydrate glycan structure which does not
contain a bisecting GlcNAc residue is shown, as is the generation of a glycan structure
therefrom which contains a bisecting GlcNAc. Symbols: squares: GlcNAc; light circles:
Man; dark circles: Gal; triangles: NeuAc.
Figure 3 is a scheme for the enzymatic generation of a sialylated glycan structure
(right side) beginning v^tli a glycan having a trimarmosyl core and a bisecting GlcNAc (left
side).
Figure 4 is a scheme of a typical high marmose containing glycan structure (left side)
and the enzymatic process for reduction of this structure to an elemental trimarmosyl core
structure. In this scheme, X is raannose as a monosaccharide, an oligosaccharide or a
polysaccharide.
Figure 5 is a diagram of a fucose and xylose containing N-linked glycan structure
typically produced in plant cells.
Figure 6 is a diagram of a fticose containing N-linked glycan structure typically
produced in insect cells. Note that the glycan may have no core fiicose, it amy have a single
core iiucose with either linkage, or it may have a single core fticose having a preponderance
of one linkage.
Figure 7 is a scheme depicting a variety of pathways for the trimming of a high
mannose structure and the synthesis of complex sugar chains therefrom. Symbols: squares:
GlcNAc; circles: Man; diamonds: fticose; pentagon: xylose.
Figure 8 is a scheme depicting in vitro strategies for the synthesis of complex
structures ft-om an elemental trimarmosyl core structure. Symbols: Squares: GlcNAc; light
circles; Man; dark circles: Gal; dark triangles: NeuAc; GnT: N-acetyl
glucosaminyltransferase; GalT: galactosyltransferase; ST: sialyltransferase.
Figure 9 is a scheme depicting two in vitro strategies for the synthesis of
monoantennaiy glycans, and the optional glycoPEGylation of the same. Dark squares:
GlcNAc; dark circles: Man; light circles: Gal; dark triangles: sialic acid.
Figure 10 is a scheme depicting two in vilro strategies for the synthesis of
monoantennary glycans., and the optional glycoPEGylation of the same. Dark squares:
GlcNAc; dark circles: Man; light circles: Gal; dark triangles: sialic acid.
Figure 11 is a scheme depicting various complex structures, which may be
synthesized from an eksmental trimannosyl core structure. Symbols: Squares: GlcNAc; light
circles: Man; dark circles: Gal; triangles: NeiAc; diamonds: flicose; FT and FucT:
flicosyltransferase; GalT: galactosyltransferase; ST: sialyltransferase; Le: Lewis antigen;
SLe; sialylated Lewis Etntigen.
Figure 12 is an exemplary scheme for preparing 0-linked glycopeptides originating
with serine or threonine. Optionally, a water soluble polymer (WSP) such as poly(ethylene
glycol) is added to the final glycan structure.
Figure 13 is a series of diagrams depicting the four types of 0-giycan structures,
termed cores 1 througli 4. The core structure is outlined in dotted lines.
Figure 14, comprising Figure 14A and Figure 14B, is a series of schemes showing an
exemplary embodiment of the invention in which carbohydrate residues comprising complex
carbohydrate structures and/or high marmose high mannose structures are trimmed back to
the first generation biantennary structure. Optionally, fucose is added only after reaction with
GnT I. A modified sugar bearing a water-soluble polymer (WSP) is then conjugated to one
or more of the sugar residues exposed by the trimming back process.
Figure 15 is a scheme similar to that shown in Figure 4, in which a high mannose or
complex structure is "trimmed back" to the mannose beta-linked core and a modified sugar
bearing a water soluble polymer is then conjugated to one or more of the sugar residues
exposed by the trimming back process. Sugars are added sequentially using
glycosyltransferases.
Figure 16 is a scheme similar to that shown in Figure 4, in which a high mannose or
complex structure is trimmed back to the GlcNAc to which the first mannose is attached, and
a modified sugar bearing; a water soluble polymer is then conjugated to one or more of the
sugar residues exposed tiy the trimming back process. Sugars are added sequentially using
glycosyltransferases.
Figure 17 is a scheme similar to that shown in Figure 4, in which a high mannose or
cpomplex structure is trimmed back to the first GlcNAc attached to the Asn of the peptide,
following which a water soluble polymer is conjugated to one or more sugar residues which
have subsequently been added on. Sugars are added sequentially using glycosyltransferases.
Figure 18, comprising Figure 18 A and 18B, is a scheme in which an N-linked
carbohydrate is optionaliiy trimmed back from a high mannose or cpmplex structure, and
subsequently derivatized: with a modified sugar moiety (Gal or GlcNAc) bearing a water-
soluble polymer.
Figure 19, comprising Figure 19A and 19B, is a scheme in which an N-linked
carbohydrate is trimmed back from a high mannose or complex structure and subsequently
derivatized with a sialic acid moiety bearing a water-soluble polymer. Sugars are added
sequentially using glycosyltransferases.
Figure 20 is a scheme in which an N-linked carbohydrate is optionally trimmed back
from a high mannose oor complex structure and subsequently derivatized with one or more
sialic acid moieties, and terminated with a sialic acid derivatized with a water-soluble
polymer. Sugars are added sequentially using glycosyltransferases.
Figure 21 is a scheme in which an 0-linked saccharide is "trimmed back" and
subsequently conjugated, to a modified sugar bearing a water-soluble polymer. In the
exemplary scheme, the c;arbohydrate moiety is "trimmed back" to the first generation of the
biantennary structure.
Figure 22 is an exemplary scheme for trimming back the carbohydrate moiety of an
0-linked glycopeptide to produce a marmose available for conjugation with a modified sugar
having a water-soluble fiolymer attached thereto.
Figure 23, comprising Figure 23A to Figure 23C, is a series of exemplary schemes.
Figure 23 A is a scheme that illustrates addition of a PEGylated sugar, followed by the
addition of a non-modified sugar. Figure 23B is a scheme that illustrates the addition of more
that one kind of modified ougar onto one glycan. Figure 23C is a scheme that illustrates tiie
addition of different modified sugars onto O-linked glycans and N-linked glycans.
Figure 24 is a diagram of various methods of improving the therapeutic fimction of a
peptide by glycan remodeling, including conjugation.
Figure 25 is a sei: of schemes for glycan remodeling of a therapeutic peptide to treat
Gaucher Disease.
Figure 26 is a sclaeme for glycan remodeling to generate glycans having a terminal
mannose-6-phosphate moiety.
Figure 27 is a diagram illustrating the array of glycan structures found on CHO-
produced glucocerebrosidase (Cereayme'^'**) after sialylation.
Figure 28, comprising Figure 28A to Figure 28Z and Figure 28AA to Figure 28CC, is
a list of peptides useful in the methods of the invention.
Figure 29, comprising Figures 29A to 29G, provides exemplary schemes for
remodeling glycan structures on granulocyte colony stimulating factor (G-CSF). Figure 29A
is a diagram depicting tjie G-CSF peptide indicating the amino acid residue to which a glycan
is bonded, and an exemplary glycan formula linked thereto. Figure 29B to 29G are diagrcims
of contemplated remodeling steps of the glycan of the peptide in Figure 29 A based on the
type of cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 30, comprising Figures 30A to 30EE sets forth exemplary schemes for
remodeling glycan struc:tures on interferon-alpha. Figure 30A is a diagram depicting the
interferon-alpha isoform 14c peptide indicating the amino acid residue to which a glycan is
bonded, and an exempliiry glycan formula linked thereto. Figure 30B to 30D are diagram.s of
contemplated remodeling steps of the glycan of the peptide in Figure 30A based on the type
of cell the peptide is expressed in and the desired remodeled glycan structure. Figure 30E is
a diagram depicting the interferon-alpha isoform 14c peptide indicating the amino acid
residue to which a glyc' and are built out
to the Gal and then glycoPEGylated with SA-PEG 1 kDa. Mono-20k refers to EPO
constructs where the glycans are mono-antennary and are built out to the Gal and then
glycoPEGylated with SA-PEG 20 kDa.
Figure 139, comprising Figures 1j9A and 139B, depicts the analysis of glycans
enzymatically released from EPO expressed in insect cells (Protein Sciences, Lot # 060302).
Figure 139 A depicts the HPLC analysis of the released glycans. Figure 139B depicts the
MALDI analysis of the released glycans. Diamonds represent fucose, and squares represent
GlcNAc, circles represent mannose.
Figure 140 depicts the MALDI analysis of glycans released from EPO after the GnT-
I/GalT-1 reaction. The structures of the glycans have been determined by comparison of title
peak spectrum with that of standard glycans. The glycan structures are depicted beside the
peaks. Diamonds represent fucose, and squares represent GlcNAc, circles represent
maimose, stars represent galactose.
Figure 141 depicts the SDS-PAGE analysis of EPO after the GnT-I/GalT-1 reaction,
Superdex 75 purification, ST3Gal3 reaction with SA-PEG (10 kDa) and SA-PEG (20 kDa).
Figure 142 depicts the results of the TF-1 cell in vitro bioassay of PEGylated mono-
antennary EPO.
Figure 143, comprising Figures 143A and 143B, depicts the analysis of glycan
released from EPO after the GnT-I/GnT-II reaction. Figure 143 A depicts the HPLC analysis
of the released glycans, where peak 3 represents the bi-antermary GlcNAc glycan. Figure
143B depicts the MALDI analysis of the released glycans. The structures of the glycans have
been determined by comparison of the peak spectrum witli that of standard glycans. The
glycan structures are depicted beside the peaks. Diamonds represent fticose, and squares
represent GlcNAc, circles represent maimose.
Figure 144, comprising Figures 144A and 144B, depict the HPLC analysis of glycans
released from EPO after the GalT-1 reaction. Figure 144A depicts the glycans released after
the small scale GalT-1 reaction. Figure 144B depicts the glycans released after the large
scale GalT-1 reaction. In both figures, Peak 1 is the bi-antennary glycan with terminal
galactose moieties and Peak 2 is the bi-antennary glycan ^^dthout terminal galactose moieties.
Figure 145 depicts the Superdex 75 chromatography separation of EPO species after
the GalT-1 reaction. Peak 2 contains EPO with bi-antennary glycans with terminal galactose
moieties.
Figure 146 depicts the SDS-PAGE analysis of each of the products of tihe
glycoremodeling process to make bi-antermary glycans with terminal galactose moieties.
Figure 147 depicts the SDS-PAGE analysis of EPO after ST3Gal3 sialylation or
PEGylation with SA-PEG (1 kDa) or SA-PEG (10 kDa).
Figure 148 depicts the HPLC analysis of glycans released from EPO after the GnT-
I/GnT-II reaction. The structures of the glycans have been determined by comparison of the
peak retention with that of standard glycans. The glycan structures are depicted beside the
peaks. Diamonds represent fucose, and squares represent GlcNAc, circles represent
marmose.
Figure 149 depicts the HPLC analysis of glycans released from EPO after the GnT-V
reaction. The structures of the glycans have been determined by comparison of the peak
retention with that of standard glycans. The glycan structures are depicted beside the peaiks.
Diamonds represent fucose, and squares represent GlcNAc, circles represent marmose.
Figure 150 depicts the HPLC analysis of glycans released from EPO after the GalT-1
reaction. The structures of the glycans have been determined by comparison of the peak
retention with that of standard glycans. The glycan structures are depicted beside the peiiks.
Diamonds represent facose, and squares represent GlcNAc, circles represent mamiose, open
circles represent galactose and triangles represent sialic acid.
Figure 151 depicts the HPLC analysis of glycans released from EPO after the
ST3Gal3 reaction. The structures of the glycans have been determined by comparison of the
peak retention with that of standard glycans. The glycan structures are depicted beside the
peaks. Diamonds represent fucose, and squares represent GlcNAc, circles represent mannose,
open circles represent galactose and triangles represent sialic acid.
Figure 152 dejiicts the HPLC analysis of glycans released fi-om EPO after the
ST6Gall reaction. The structures of the glycans have been determined by comparison of the
peak retention with that of standard glycans. The glycan structures are depicted beside the
peaks.
Figure 153 depicts the results of the TF-1 cells in vitro bioassay of EPO with bi-
aiitennary and triantennary glycans. "Di-SA" refers to EPO with bi-antennary glycans that
terminate in sialic acid. "Di-SA lOK PEG" refers to EPO with bi-antennary glycans that
terminate in siaUc acid derivatized with PEG (10 kDa). "Di-SA IK PEG" refers to EPO with
bi-antennary glycans that terminate in sialic acid derivatized with PEG (1 kDa). '"Tri-S.^ ST6
+ ST3" refers to EPO with tri-antennary glycans terminating in 2,6-SA capped with 2,3-SA.
"Tri-SA ST3" refers to EPO with tri-antennary glycans terminating in 2,3-SA
Figure 154 is an image of an lEF gel depicting the pi of the products of the
desialylation procedure. Lanes 1 and 5 are lEF standards. Lane 2 is Factor IX protein. Lane
3 is rFactor EX protein. Lane 4 is tlie desialylation reaction of rFactor IX protein at 20 hr.
Figure 155 is an image of an SDS-PAGE gel depicting the molecular weight of Factor
IX conjugated with eitlier SA-PEG (1 kDa) or SA-PEG (10 kDa) after reaction with CMP-
SA-PEG. Lanes 1 and 6 are SeeBlue +2 molecular weight standards. Lane 2 is rF-DC. Lane
3 is desialylated rF-IX. Lane 4 is rFactor IX conjugated to SA-PEG (1 kDa). Lane 5 is
rFactor IK conjugated to SA-PEG (10 kDa).
Figure 156 is an image of an SDS-PAGE gel depicting the reaction products of direct-
sialylationof Factor-DC and sialic acid capping of Factor-IX-SA-PEG. Lane 1 is protein
standards, lane 2 is blank; lane 3 is rFactor-IX; lane 4 is SA capped rFactor-K-SA-PEG (10
kDa); lane 5 is rFactor-IX-SA-PEG (10 kDa); lane 6 is ST3Gall; lane 7 is ST3Gal3; lanes 8,
9, 10 are rFactor-IX-SA-PEG(lO kDa) with no prior sialidase treatment.
Figure 157 is an image of an isoelectric focusing gel (pH 3-7) of asialo-Factor Vila.
Lane 1 is rFactor Vila; lanes 2-5 are asialo-Factor Vila.
Figure 158 is a graph of a MALDI spectra of Factor Vila.
Figure 159 is a graph of a MALDI spectra of Factor VIIa-PEG (1 kDa).
Figure 160 is a graph depicting a MALDI spectra of Factor Vlla-PEG (10 kDa).
Figure 161 is an image of an SDS-PAGE gel of PEGylated Factor Vila. Lane 1 is
asialo-Factor Vila. Lane 2 is the product of the reaction of asialo-Factor Vila and CMP-SA-
PEG(1 kDa) with ST3Gal3 after 48 hr. Lane 3 is the product of the reaction of asialo-Factor
Vila and CMP-SA-PEG (1 kDa) with ST3Gal3 after 48 hr. Lane 4 is the product of the
reaction of asialo-Facior Vila and CMP-SA-PEG (10 kDa) with ST3Gal3 at 96 hr.
Figure 162 is cin image of an isoelectric focusing (lEF) gel depicting the products of
the desialylation reaction of human pituitary FSH. Lanes 1 and 4 are isoelectric focusing
(lEF) standards. Lane 2 is native FSH. Lane 3 is desialylated FSH.
Figure 163 is an image of an SDS-PAGE gel of the products of the reactions to make
PEG-sialylation of rFSH. Lanes 1 and 8 are SeeBlue+2 molecular weight standards. Lane 2
is 15 |ig of native FSII. Lane 3 is 15 ^ig of asialo-FSH (AS-FSH). Lane 4 is 15 jig of the
products of the reaction of AS-FSH with CMP-SA. Lane 5 is !_¦ yig of the products of the
reaction of AS-FSH with CMP-SA-PEG (1 kDa). Lane 6 is 15 ^g of the products of the
reaction of AS-FSH with CMP-SA-PEG (5 kDa). Lane 7 is 15 ^g of the products of the
reaction of AS-FSH with CMP-SA-PEG (10 kDa).
Figure 164 is an image of an isoelectric focusing gel of the products of the reactions
to make PEG-sialylation of FSH. Lanes 1 and 8 are lEF standards. Lane 2 is 15 [ig of native
FSH. Lane 3 is 15 ng of asialo-FSH (AS-FSH). Lane 4 is 15 ng of the products of the
reaction of AS-FSH with CMP-SA. Lane 5 is 15 ng of the products of the reaction of AS-
FSH with CMP-SA-PEG (1 kDa). Lane 6 is 15 jig of the products of the reaction of AS-FSH
with CMP-SA-PEG (5 k]3a). Lane 7 is 15 |4.g of the products of the reaction of AS-FSH with
CMP-SA-PEG (10 kDa).
Figure 165 is an image of an SDS-PAGE gel of native non-recomblnant FSH
produced in human pituitary cells. Lanes 1, 2 and 5 are SeeBlue''"'^+2 molecular weight
standards. Lanes 3 and 4 are native FSH at 5 ^xg and 25 |ig, respectively.
Figure 166 is an image of an isoelectric focusing gel (pH 3-7) depicting the products
of the asialylation reaction of rFSH. Lanes 1 and 4 are lEF standards. Lane 2 is native rFSH.
Lane 3 is asialo-rFSH.
Figure 167 is an image of an SDS-PAGE gel depicting the results of the PEG-
sialylation of asialo-rFSH. Lane 1 is native rFSH. Lane 2 is asialo-FSH. Lane 3 is the
products of the reaction of asialo-FSH and CMP-SA. Lanes 4-7 are the products of the
reaction between asialoFSH and 0.5 mM CMP-SA-PEG (10 kDa) at 2 hr, 5 hr, 24 hr, and 48
hr, respectively. Lane 8 is the products of the reaction between asialo-FSH and 1.0 mM
CMP-SA-PEG (10 kDa) at 48 hr. Lane 9 is the products of the reaction between asialo-FSH
and l.O mM CMP-SA-PEG (1 kDa) at 48 hr.
Figure 168 is an image of an isoelectric focusing gel showing the products of PEG-
sialylation of asialo-rFSH with a CMP-SA-PEG (1 kDa). Lane 1 is native rFSH. Lane 2 is
asialo-rFSH. Lane 3 is the products of the reaction of asialo-rFSH and CMP-SA at 24 hr.
Lanes 4-7 are the products of the reaction of asialo-rFSH and 0.5 mM CMP-SA-PEG (1 kE>a)
at 2 hr, 5 hr, 24 hr, and 48 hr, respectively. Lane 8 is blank. Lanes 9 and 10 are the products
of the reaction at 48 hr of asialo-rFSH and CMP-SA-PEG (10 kDa) at 0.5 mM and 1.0 mM,
respectively.
Figure 169 is graph of the pharmacoki.ietics of rFSH and rFSH-SA-PEG (1 kDa and
10 kDa). This graph illustrates the relationship between the time a rFSH compound is m the
blood stream of the rat, and the mean concentration of the rFSH compoxmd in the blood for
glycoPEGylated rFSH £is compared to non-PEGylated rPSH.
Figure 170 is a graph of the results of the FSH bioassay using Sertoli cells. This
graph illustrates the relationship between the FSH concentration in the Sertoli cell incubation
medium and the amouni: of 17-p estradiol released from the Sertoli cells.
Figure 171 is a graph depicting the results of the Steelman-Pohley bioassay of
glycoPEGylated and non-glycoPEGylated FSH. Rats were subcutaneously injected with
human chorionic gonadotropin and varying amounts of FSH for three days, and the average
ovarian weight of the trtjatment group determined on day 4. rFSH-SA-PEG refers to
recombinant FSH that has been glycoPEGylated with PEG (1 kDa). rFSH refers to non-
glycoPEGylated FSH. Each treatment group contains 10 rats.
Figure 172, comprising Figures 172A and 172B, depicts the chromatogram of INF-p
elution from a Superdex-75 coluirm. Figure 172A depicts the entire chromatogram. Figure
172B depicts the boxed area of Figure 172A containing peaks 4 and 5 in greater detail.
Figiire 173, comprising Figures 173A and 173B, depict MALDI analysis of glycans
enzymatically released from INF-p. Figure 173 A depicts the MALDI analysis glycans
released from native INF-p. Figure 173B depicts the MALDI analysis of glycans released
from desialylated INF-p. The structures of the glycans have been determined by comparison
of the peak spectrum with that of standard glycans. The glycan structures are depicted beside
the peaks. Squares repre;sent GlcNAc, triangles represent fucose, circles represent mannose,
diamonds represent galactose and stars represent sialic acid.
Figure 174 depicts the lectin blot analysis of the sialylation of the desialylated INF-p.
The blot on the right side is detected with Maackia amurensis agglutinin (MAA) labeled with
digoxogenin (DIG) (Roche Applied Science, Indianapolis, IL) to detect a2,3-sialylation. The
blot on the left is detected with Erthrina cristagalli lectin (EICL) labeled with biotin (Vector
Laboratories, Burlingame, CA) to detect exposed galactose residues.
Figure 175 depicts the SDS-PAGE analysis of the products of the PEG (10 kDa)
PEGylation reaction of ENF-p. "-PEG" refers to INF-P before the PEGylation reaction.
"+PEG" refers to INF-P after the PEGylation reaction.
Figure 176 depicts the SDS-PAGE analysis of the products of the PEG (20 kDa)
PEGylation reaction of n'can structure.
"N-linked" oligosaccharides are those oligosaccharides that are linked to a peptide
backbone through asparagine, by way of an asparagine-N-acetylglucosaniine linkage. N-
linked oligosaccharides are also called "N-glyeans." All N-lmked oligosaccharides have a
common pentasaccharide core of Man3GlcNAc2. They differ in the presence of, and in the
number of branches (also called antennae) of peripheral sugfirs such as N-acetylglucosamine,
galactose, N-acetylgalactosamine, fucose and sialic acid. Optionally, this structure may also
contain a core fucose molecule and/or a xylose molecule.
An "elemental trimannosyl core structure" refers to a glycan moiety comprising solely
a trimannosyl core structure, with no additional sugars attached thereto. When the term
"elemental" is not included in the description of the "trimannosyl core structure," then the
glycan comprises the trimannosyl core structure with additional sugars attached thereto.
Optionally, this structure may also contain a core fucose molecule and/or a xylose molecule.
The term "elemental trimannosyl core glycopeptide" is used herein to refer to a
glycopeptide having glycan structures comprised primarily of an elemental trimannosyl core
structure. Optionally, this structure may also contain a core fucose molecule and/or a xylose
molecule.
"O-linked" oligosaccharides are those oligosaccharides that are linked to a peptide
backbone through threonine, serine, hydroxyproline, tyrosine, or other hydroxy-containing
amino acids.
All oligosaccharides described herein are described with the name or abbreviation for
the non-reducing saccharide (i.e.. Gal), followed by the configuration of the glycosidic bond
(a or P), the ring bond (1 or 2), the ring position of the reducing saccharide involved m the
bond (2, 3, 4, 6 or 8), and then the name or abbreviation of the reducing saccharide (i.e.,
GlcNAc). Each saccharide is preferably a pyranose. For a review of standard glycobiology
nomenclature see. Essentials of Glycobiology Varki et al. eds., 1999, CSHL Press.
The term "sialic add" refers to any member of a family of nine-carbon carboxylated
sugars. The most common member of the sialic acid family is N-acet>'l-neuraminic acid (2-
keto-5-acetamido-3,5-dideoxy-D-glycero-D-gaJactononulopyranos-l-onic acid (often
abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is N-glycolyl-
neurcunini; acid (NeuSGc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated.
A third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano ei al.
(1986) j; Biol. Chem.261: 11550-11557; Kanamori era/., J: Biol. C/zem. 265: 21811-21819
(1990)). Also included are 9-substituted sialic acids such as a 9-0-Ci-C6 acyl-Neu5Ac like
9-0-lactyl-Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deox:y-
Neu5Ac. For review of the sialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);
Sialic Acids: Chemistiy, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New
York (1992)). The synthesis and use of sialic acid compounds in a sialylation procedure is
disclosed in international application WO 92/16640, published October 1, 1992.
A peptide having "desired glycosylation", as used herein, is a peptide that comprises
one or more oligosaccharide molecules which are required for efficient biological activit;^^ of
the peptide.
A "disease" is a state of health of an animal wherein the animal cannot maintain
homeostasis, and wherein if the disease is not ameliorated then the animal's health continues
to deteriorate.
The "area under the curve" or "AUG", as used herein in the context of administering a
peptide dmg to a patient, is defined as total area under the curve that describes the
concentration of drug in systemic circulation in the patient as a function of time from zero to
infinity.
The term "half-life" or "t V2", as used herein in the context of administering a peptide
drug to a patient, is defined as the time required for plasma concentration of a drug in a
patient to be reduced by one half. There may be more than one half-life associated with ithe
peptide drug depending on multiple clearance mechanisms, redistribution, and other
mechanisms well knov/n in the art. Usually, alpha and beta half-lives are defined such that
the alpha phase is associated with redistribution, and the beta phase is associated with
clearance. However, with protein drugs that are, for the most part, confined to the
bloodstream, there can be at least two clearance half-lives, For some glycosylated peptides,
rapid beta phase clearance may be mediated via receptors on macrophages, or endothelial
cells that recognize terminal galactose, N-acetylgalactosarnine, N-acetylglucosaniine,
mannose, or fucose. Slower beta phase clearance may occur via renal glomerular filtration
for molecules with an effective radius < 2 nm (approximately 68 kD) and/or specific or non-
specific uptake and metabolism in tissues. GlycoPEGylafion may cap terminal .Mgajs (e.g.
galactose or N-acetylgalactosamine) and thereby block rapid alpha phase clearance via
receptors that recognize tliese sugars. It may also confer a larger effective radius and thereby
decrease the volume of distribution and tissue uptake, thereby prolonging the late beta phase.
Thus, the precise impact of glycoPEGylation on alpha phase and beta phase half-lives will
var)' depending upon the: size, state of glycosylation, and other parameters, as is well kno\\Ti
in the art. Further explanation of "half-life" is found in Phfirmaceutical Biotechnology
(1997, DFA Crommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp 101 -
120).
The term "residence time", as used herein in the context of administering a peptide
drug to a patient, is defined as the average time that drug stays in the body of the patient after
dosing.
An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has
been separated fi-om sequences which flank it in a naturally occurring state, e.g., a DNA
fragment which has been removed from the sequences which are normally adjacent to the
fragment, e.g., the seque;nces adjacent to the fragment in a genome in which it naturally
occurs. The term also applies to nucleic acids which have been substantially purified from
other components which naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, which natxirally accompany it in the cell. The term therefore includes, for example,
a recombinant DNA which is incorporated into a vector, into an autonomously replicating
plasmid or virus, or intci tlie genomic DNA of a prokaryote or eukaryote, or which exists as a
separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or
restriction enzyme digestion) independent of other sequences. It also includes a recombinant
DNA which is part of a hybrid nucleic acid encoding additional peptide sequence.
A "polynucleotide" means a single strand or parallel and anti-parallel strands of a
nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-sfranded
nucleic acid.
The term "nucleic acid" typically refers to large polynucleotides. The term
"oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50
nucleotides.
Conventional notation is used herein to describe polynucleotide sequences: the left-
hand end of a single-stranded polynucleotide sequence is the 5 end; the left-hand direction of
a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of
5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription
direction. The DNA strand having the same sequence as an mRNA is referred to as the
"coding strand"; sequences on the DNA strand which are located 5' to a reference point on the
DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a
reference point on the DNA are referred to as "downstream sequences."
"Encoding" refers to the inherent property of specific sequences of nucleotides in a
polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of
other polymers and macromolecules in biological processes having either a defmed sequence
of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the
biological properties resulting therefrom. Thus, a nucleic acid sequence encodes a protein if
transcription and translation of mRNA corresponding to that nucleic acid produces the
protein in a cell or other biological system. Both the coding strand, the nucleotide sequence
of which is identical to the mRNA sequence and is usually provided in sequence listings, and
the non-coding strand, use;d as the template for transcription of a gene or cDNA, can be
referred to as encoding the; protein or other product of that nucleic acid or cDNA.
Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence"
includes all nucleotide sequences that are degenerate versions of each other and that encode
the same amino acid sequeince. Nucleotide sequences that encode proteins and RNA may
include introns.
"Homologous" as used herein, refers to the subunit sequence similarity between two
polynieric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or
two RNA molecules, or between two peptide molecules. When a subunit position in both of
the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of
two DNA molecules is occupied by adenine, then they are homologous at that position. The
homology between two sequences is a direct ftinction of the number of matching or
homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of
the positions Ln two compound sequences are homologous then the two sequences are 50%
homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, tlie two
sequences share 90%) homology. By way of example, the DNA sequences 3 ATTGCC5' and
3'TATGGC share 50% homology.
As used herein, "homology" is used synonymously witli "identity."
The determination of percent identity between two nucleotide or amino acid
sequences can be accomplished using a mathematical algorithm. For example, a
mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and
Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and
Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated
mto the NBLAST and :XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403-
410), and can be accessed, for example at the National Center for Biotechnology Infonnation
(NCBI) world wide web site having the universal resource locator
"http://www.ncbi.nlm.iiih.gov/BLAST/". BLAST nucleotide searches can be performed with
the NBLAST program (designated "blastn" at the NCBI web site), using the following
parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward
= 1; expectation value 10.0; and word size = 11 to obtain nucleotide sequences homologous
to a nucleic acid described herein. BLAST protein searches can be performed with the
XBLAST program (designated "blastn" at the NCBI web site) or the NCBI "blastp" program,
using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to
obtain amino acid sequences homologous to a protein molecule described herein. To obtain
gapped aligrmients for comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-
Blast can be used to perform an iterated search which detects distant relationships between
molecules (Id.) and relationships between molecules which share a common pattern. Wlien
utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.riih.gov.
The percent id(;ntity between two sequences can be determined using techniques
similar to those described above, with or without allowing gaps. In calculating percent
identity, typically exact matches are counted.
A "heterologous nucleic acid expression unit" encoding a peptide is defined as a
nucleic acid having a coding sequence for a peptide of interest operably linked to one or
more expression control sequences such as promoters and/or repressor sequences wherein at
least one of the sequences is heterologous, i. e., not normally found in the host cell.
By describing two polynucleotides as "operably linked" is meant that a single-
stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged
within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is
able to exert a physiological effect by which it is characterized upon the other. By way of
example, a promoter operably linked to the coding region of a nucleic acid is able to promote
transcription of the coding region.
As used herein, the term "promoter/regulatory sequence" means a nucleic acid
sequence which is required for expression of a gene product operably linked to the
promoter/regulator sequence, hi some instances, this sequence may be the core promoter
sequence and in other instances, this sequence may also include an enhancer sequence and
other regulatory elements which are required for expression of the gene product. The
promoter/regulatory sequence may, for example, be one which expresses the gene product in
a tissue specific manner.
A "constitutive promoter is a promoter which drives expression of a gene to which it
is operably linked, in a constant manner in a cell. By way of example, promoters which drive
expression of cellular housekeeping genes are considered to be constitutive promoters.
An "inducible" jjromoter is a nucleotide sequence which, when operably linked wiiJi a
polynucleotide which encodes or specifies a gene product, causes the gene product to be
produced in a Uving cell substantially only when an inducer which corresponds to the
promoter is present in the cell.
A "tissue-specific" promoter is a nucleotide sequence which, when operably linked
with a polynucleotide which encodes or specifies a gene product, causes the gene product to
be produced in a living cell substantially only if the cell is a cell of the tissue type
corresponding to the promoter.
A "vector" is a composition of matter which comprises an isolated nucleic acid and
which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous
vectors are known in tiae art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
Thus, the tenn "vector" includes an autonomously replicating plasmid or a virus. The leim
should also b-; construed to include non-plasmid and non-viral compounds which facilu.':tc
transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes,
and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the like.
"Expression vector" refers to a vector comprising a recombinant polynucleotide
comprising expression control sequences operatively linked to a nucleotide sequence to be
expressed. An expression vector comprises sufficient cis-acting elements for expression;
other elements for expression can be supplied by the host cell or in an in vitro expression
system. Expression vectors include all those known in the jirt, such as cosniids, plasmids
(e.g., naked or contained in liposomes) and viruses that incorporate the recombinant
polynucleotide.
A "genetically engineered" or "recombinant" cell is a cell having one or more
modifications to the genetic material of the cell. Such modifications are seen to include, but
are not limited to, insertions of genetic material, deletions of genetic material and insertion of
genetic material that is eixtrachromasomal whether such material is stably maintained or not.
A "peptide" is an oligopeptide, polypeptide, peptide, protein or glycoprotein. The use
of the term "peptide" herein includes a peptide having a sugar molecule attached thereto
when a sugar molecule is attached thereto.
As used herein, "native form" means the form of the peptide when produced by the:
cells and/or organisms in which it is found in nature. When the peptide is produced by a
plurality of cells and/or organisms, the peptide may have a variety of native forms.
"Peptide" refers to a polymer in which the monomers are amino acids and are joined
together through amide bonds, alternatively referred to as a peptide. Additionally, uimatui'al
amino acids, for example, (3-alanine, phenylglycine and homoarginine are also included.
Amino acids that are not nucleic acid-encoded may also be used in the present invention.
Furthermore, amino aci^ds that have been modified to include reactive groups, glycosylation
sites, polymers, therapeutic moieties, biomolecules and the like may also be used in the
invention. All of the amino acids used in the present invention may be either the D - or L -
isomer thereof. The L -isomer is generally preferred, hi addition, other peptidomimetics are
also useful in the present invention. As used herein, "peptide" refers to both glycosylated and
unglycosylated peptides. Also included are peptides that ai-e incompletely glycosylated by a
system that expresses tfie peptide. For a general review, see, Spatola, ,\. F., in Chemis ir^'
AND Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstem, eds.. Marcel
Dekker, New York, p. 267 (1983).
The term "peptide conjugate," refers to species of the invention in which a peptide is
conjugated with a modified sugar as set forth herein.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well
as amino acid analogs and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-
carboxyglutamate, and 0-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is
linked to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified
R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical
structure as a naturally oc(;urring amino acid. Amino acid mimetics refers to chemical
compounds that have a structure that is different from the general chemical structure of an
amino acid, but that function in a manner similar to a naturally occurring amino acid.
As used herein, amino acids are represented by the fiill name thereof, by tlie three
letter code corresponding thereto, or by the one-letter code corresponding thereto, as
indicated in the following Table 1 :
Table 1. Amino acids, and the three letter and one letter codes.
The present invention also provides for analogs of proteins or peptides which
)rise a protein as identified above. Analogs may differ from naturally occurring proteins
ptides by conservative amino acid sequence differences or by modifications which do
ffect sequence, or by both. For example, conservative amino acid changes may be
;, which altliough they alter the primary sequence of the protein or peptide, do not
lally alter its function. Conservative amino acid substitutions typically include
itutions within the following groups:
glycine, alanine;
valine, isoleucine, leucine;
asp;irtic acid, glutamic acid;
aspiiragine, glutamine;
serine, threonine;
lysine, arginine;
phenylalanine, tyrw^Lne.
Modifications (whi(jh do not normally alter primary sequence) include in vivo, or in
vitro, chemical derivatization of peptides, e.g., acetylation, or carboxylation. Also included
are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns
of a peptide during its syntliesisand processing or in further processing steps; e.g., by
exposing the peptide to en2ymes which affect glycosylation, e.g., mammalian glycosylating
or deglycosylating enzymciS. Also embraced are sequences wliich have phosphorylated
amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
It will be appreciated, of course, that the peptides may incorporate amino acid
residues which are modified without affecting activity. For example, the termini may be
derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or
stabilize the N- and C-termini from "undesirable degradation", a term meant to encompass
any type of enzymatic, chemical or biochemical breakdown of the compound at its termini
which is likely to affect the function of the compound, i.e. sequential degradation of the
compound at a terminal end thereof.
Blocking groups include protecting groups conventionally used in the art of peptide
chemistry which will not adversely affect the in vivo activities of the peptide. For example,
suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-
terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or
unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted
forms thereof, such as the acetamidomethyl (Acm), Fmoc or Boc groups. Desamino analogs
of amino acids are also useful N-terminal blocking groups, and can either be coupled to the
N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal
blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not,
include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower
alkyl groups such as methyl, ethyl and propyl, and amide-forrning amino groups such as
primary amines (-NH?), and mono- and di-alkylamino groups such as methylamino,
ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-
terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also
useful C-terminal blocking groups and can be either coupled to the peptide's C-terminal
residue or used in place oi it. Further, it will be appreciated that the free amino and carboxyl
groups at the termini can. be removed altogether from the peptide to yield desamino and
descarboxylated forms tliereof without affect on peptide activity.
Other modifications can also be incorporated without adversely affecting the activity
and these include, but are not limited to, substitution of one or more of the amino acids in tlie
natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may
include one or more D-amino acid resides, or may comprise amino acids which are all in the
D-form. Retro-inverso forms of peptides in accordance with the present invention are also
contemplated, for example, inverted peptides in which all amino acids are substituted with D-
amino acid forms.
Acid addition salts of the present invention are also contemplated as functional
equivalents. Thus, a peptide in accordance with the present invention treated with an
inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or
an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic,
succinic, maleic, fumaric, tataric, citric, benzoic, cinnamic, mandelic, methanesulfonic,
ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the
peptide is suitable for use in the invention.
Also included a]:e peptides which have been modified using ordinary molecular
biological techniques so as to improve their resistance to proteolytic degradation or to
optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs
of such peptides Include those containing residues other th;m naturally occurring L-amino
acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of
the invention are not limited to products of any of the specific exemplary processes listed
herein.
As used herein, the term "MALDI" is an abbreviation for Matrix Assisted Laser
Desorption Ionization. During ionization, SA-PEG (sialic acid-poly(ethylene glycol)) can be
partially eliminated from the N-glycan structure of the glycoprotein.
As used herein, the term "glycosyltransferase," refers to any enzyme/protein that has
the ability to transfer a donor sugar to an acceptor moiety.
As used herein, the term "modified sugar," refers to a naturally- or non-nalurally-
occuiTing carbohydrate that is enzymatically added onto iiii amino acid or a glycosyl residue
of a peptide in a process of the invention. The modified sugar is selected from a number of
enzyme substrates including, but not limited to sugar nucleotides (mono-, di-, and tri-
phosphates), activated sugjirs {e.g., glycosyl halides, glycosyl mesylates) and sugars that are
neither activated nor nucleotides.
The "modified sugar" is covalently fiinctionEilized with a "modifying group." Useful
modifying groups include, but are not limited to, water-soluble polymers, therapeutic
moieties, diagnostic moielies, biomolecules and the like. The locus of functionalization v^th
the modifying group is selected such that it does not prevent the "modified sugar" from being
added enzymatically to a peptide.
The term "water-soluble" refers to moieties that have some detectable degree of
solubility in water. Methods to detect and/or quantify water solubility are well known in the
art. Exemplary water-soluble polymers include peptides, saccharides, poly(ethers),
poly(amines), poly(carbC'Xylic acids) and the like. Peptides can have mixed sequences or be
composed of a single amino acid, e.g. poly(lysine). Similarly, saccharides can be of mixed
sequence or composed of a single saccharide subunit, e.g., dextran, amylose, chitosan, and
poly(sialic acid). An exemplary poly(ether) is poly(ethyIene glycol). Poly(ethylene imine) is
an exemplary polyamine, and poly(aspartic) acid is a representative poiy(carboxylic acid).
"Poly(alkylene oxide)" refers to a genus of compounds having a polyether backbone.
Poly(alkylene oxide) species of use in the present invention include, for example, straight-
and branched-chain species. Moreover , exemplary poly(alkylene oxide) species can
tenninate in one or more reactive, activatable, or inert groups. For example, poly(ethylene
glycol) is a poly(alkylene oxide) consisting of repeating ethylene oxide subunits, which may
or may not include additional reactive, activatable or inert moieties at eitlier terminus. Useful
poly(alkylene oxide) species include those in which one terminus is "capped" by an inert
group, e.g., monomethoxy-poly(alkylene oxide). When the molecule is a branched species, i
may include multiple reactive, activatable or inert groups at the termini of the alkylene oxide
chains and the reactive groups may be either the same or different. Derivatives of straight-
chain poly(alkylene oxide) species that are heterobifiinctional are also known in the ait.
The term, "glycosyl linking group," as used herein refers to a glycosyl residue tc
wliich an agent (e.g., water-soluble polymer, therapeutic moiety, biomolecule) is covalently
attached. In the methods of the invention, the "glycosyl linking group" becomes covalently
attached to a glycosylated or unglycosylated peptide, thereby linking the agent to an amino
acid and/or glycosyl residue on the peptide. A "glycosyl linking group" is generally derived
from a "modified sugar" by the enzymatic attachment of the "modified sugar" to an amino
acid and/or glycosyl residue of the peptide. More specifically, a "glycosyl linking group," as
used herein, refers to a moiety that covalently joins a "modifying group," as discussed herein,
and an amino acid residue of a peptide. The glycosyl linking group-modifying group adduct
has a structure that is a. substrate for an enzyme. The enzymes for which the glycosyl linJIdng
group-modifying group adduct are substrates are generally those capable of transferring a
saccharyl moiety onto an amino acid residue of a peptide, e.g, a glycosyltransferase, amidase,
glycosidase, trans-sialidase, etc. The "glycosyl linking group" is interposed between, and
covalently joins a "modifying group" and an amino acid residue of a peptide.
An "intact glyc;osyl linking group" refers to a linking group that is derived from a
glycosyl moiety in which the individual saccharide monomer that links the conjugate is not
degraded, e.g., oxidized, e.g., by sodium metaperiodate. "Intact glycosyl linking groups" of
the invention may be derived from a naturally occurring oligosaccharide by addition of
glycosyl unit(s) or removal of one or more glycosyl unit from a parent saccharide structuure.
An exemplary "intact glycosyl linking group" includes at least one intact, e.g., non-degrcided,
saccharyl moiety that is covalently attached to an amino acid residue on a peptide. The
remainder of the "linking group" can have substantially any structure. For example, the
modifying group is optionally linked directly to the intact saccharyl moiety. Alternatively,
the modifying group is linked to the intact saccharyl moiety via a linker arm. The linker arm
can have substantially any structure determined to be useful in the selected embodiment. In
an exemplary embodiment, the linker arm is one or more intact saccharyl moieties, i.e. "the
intact glycosyl linking group" resembles an oligosaccharide. Another exemplary intact
glycosyl linking group is one in which a saccharyl moiety attached, directly or indirectly, to
the intact saccharyl moiety is degraded and derivatized (e.g., periodate oxidation followed by
reductive amination). Still a further linker arm includes the modifying group attached to the
intact saccharyl moiety, directly or indirectly, via a cross-linker, such as those described
herein or analogues thereof.
"Degradation," as used herein refers to the removal of one or more carbon atoms from
a saccharyl moiety.
The terms "targeting moiety" and "targeting agent", as used herein, refer to species
that will selectively localize in a particular tissue or region of the body. The localization is
mediated by specific recognition of molecular determinants, molecular size of the targeting
agent or conjugate, ionic interactions, hydrophobic interactions! and the like. Other
mechanisms of targeting an agent to a particular tissue or region are known to those of skill in
the art.
As used herein, "therapeutic moiety" means any agent useful for therapy including,
but not limited to, antibiotics, anti-inflammatory agents, anti-tumor drugs, cytotoxins, and
radioactive agents. "Therajieutic moiety" includes prodrugs of bioactive agents, constructs in
which more than one therapeutic moiety is linked to a carrier, e.g., multivalent agents.
Therapeutic moiety also inc;ludes peptides, and constructs that include peptides. Exemplary
peptides include those disclosed in Figure 28 and Tables 6 and 7, herein. "Tlierapeutic
moiety" thus means any ag(;nt useful for therapy including, but not limited to, antibiotics,
anti-inflammatory agents, anti-tumor drugs, cytotoxins, and radioactive agents. "Therapeutic
moiety" includes prodrugs (jf bioactive agents, constructs in which more than one therapeutic
moiety is linked to a carrier, e.g., multivalent agents.
As used herein, "anti-tumor drug" means any agent useful to combat cancer including,
but not limited to, cytotoxins and agents such as antimetabolites, alkylating agents,
anthracyclines, antibiotics, imtimitotic agents, procarbazine, hydroxyurea, asparaginase,
corticosteroids, interferons land radioactive agents. Also encompassed within the scope of the
term "anti-tumor drug," are conjugates of peptides with anti-tumor activity, e.g. TNF-a.
Conjugates include, but are not limited to those formed between a therapeutic protein and a
glycoprotein of the invention. A representative conjugate is that formed between PSGL-1
and TNF-a.
As used herein, "a c;/totoxin or cytotoxic agent" means any agent that is detrimental to
cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin, etoposide, tenoposide, vmcristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dih>dioxy anthracinedione, mitoxantrone, mithrcimycin, actinomycin D, 1-
dehydrdtestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and
puromycin and analogs or homologs thereof. Other toxins include, for example, ricin, CC-
1065 and analogues, the duocarmycins. Still other toxins include diphtheria toxm, and snake
venom (e.g., cobra \'enom).
As used herein, "a radioactive agent" includes any radioisotope that is effective in
diagnosing or destroying a tumor. Examples include, but are not limited to, indium- 111,
cobaIt-60 and technetium. Additionally, naturally occurring radioactive elements such as
uranium, radium, and thorium, which typically represent mixtures of radioisotopes, are
suitable examples of a radioactive agent. The metal ions are typically chelated with an
organic chelating moiety.
Many useful chelating groups, crown ethers, cryptands and the like are known in the
art and can be incorporated into the compounds of the invention (e.g. EDTA, DTP A, DOTA,
NTA, HDTA, etc. and their phosphonate analogs such as DTPP, EDTP, HDTP, NTP, etc).
See, for example, Pitt et ai, "The Design of Chelating Agents for the Treatment of Iron
Overload," In, Inorganic Chemistry IN BfOLOGY AND MEDICINE; Martell, Ed.; Americcin
Chemical Society, Washington, D.C., 1980, pp. 279-312; Lindoy, The Chemistry of
Macrocyclic Ligand Complexes; Cambridge University Press, Cambridge, 1989; Dugas,
BIOORGANIC Chemistry; Springer-Verlag, New York, 1989, and references contained
therein.
Additionally, a manifold of routes allowing the attachment of chelating agents, crown
ethers and cyclodextrins to other molecules is available to tliose of skill in the art. See, for
example, Meares et al., "Properties of In Vivo Chelate-Tagged Proteins and Polypeptides."
In, Modification of Proteins: Food, Nutritional, and Pharmacological Aspects;"
Feeney, et ai, Eds., American Chemical Society, Washington, D.C., 1982, pp. 370-387;
Kasina el ai, Bioconjugaie Chem., 9: 108-117 (1998); Song et al, Bioconjugate Chem., 8:
249-255 (1997).
As used herein, "pharmaceutically acceptable carrier" includes any material, which
when combined with the conjugate retains the activity of the conjugate activity and is non-
reactive witli the subject's immune system. Examples include, but are not limited to, any of
the standard phamiaceutical carriers such as a phosphate buffered saline solution, water,
emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers
may also include sterile solutions, tablets including coated tablets and capsules. Typically
such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin,
stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums,
glycols, or other known excipients. Such carriers may also include flavor and color additives
or other ingredients. Compositions comprising such carriers are formulated by well known
conventional methods.
As used herein, "administering" means oral administration, administration as a
suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional,
intranasal or subcutaneous administration, intrathecal administration, or tlie implantation of a
slow-release device e.g., a mini-osmotic pump, to the subject.
The term "isolated " refers to a material that is substantially or essentially Joree firom
components, which are used to produce the material. For peptide conjugates of the invention,
the term "isolated" refers to material that is substantially or essentially free from components,
which normally accompany the material in the mixture used to prepare the peptide conjugate.
"Isolated" and "pure" are used interchangeably. Typically, isolated peptide conjugates of the
invention have a level of purity preferably expressed as a range. The lower end of the range
of purity for the peptide c;onjugates is about 60%, about 70% or about 80%) and the upper end
of the range of purity is a.bout 70%, about 80%, about 90%) or more than about 90%.
When the peptide conjugates are more than about 90% pure, their purities are also
preferably expressed as ei range. The lower end of the range of purity is about 90%, about
92%, about 94%, about 96% or about 98%. The upper end of the range of purity is about
92%, about 94%, about 96%), about 98% or about 100% purity.
Purity is determiiaed by any art-recognized method of analysis {e.g., band intensity on
a silver stained gel, polyacrylamide gel electrophoresis, HPLC, or a similar means).
"Commercial scale" as used herein means about one or more gram of final product
produced in the method,
"Essentially each member of the population," as used herein, describes a
characteristic of a population of peptide conjugates of the invention in which a selected
percentage of the modified sugars added to a peptide are added to multiple, identical acceptor
sites on liie peptide. "Essentially each member of the population" speaks to the
"homogeneity" of the sites on the peptide conjugated to a modified sugar and refers to
conjugates of the invention, which are at least about 80%, preferably at least about 90% and
more preferably at least about 95% homogenous.
"Homogeneity," refers to the structural consistency across a population of acceptor
moieties to which the raodified sugars are conjugated. Thias, in a peptide conjugate of the
iavention in which each modified sugar moiety is conjugated to an acceptor site having the
same structure as the acceptor site to which every other modified sugar is conjugated, the
peptide conjugate is said to be about 100% homogeneous. Homogeneity is typically
expressed as a range. The lower end of the range of homogeneity for tlie peptide conjugeites
is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%,
about 80%, about 90%, or more than about 90%.
When the peptide conjugates are more than or equal to about 90% homogeneous, their
homogeneity is also preferably expressed as a range. The lower end of the range of
homogeneity is about 90%o, about 92%, about 94%), about 96% or about 98%. The upper end
of the range of purity is about 92%, about 94%, about 96%, about 98% or about 100%)
homogeneity. The purity of the peptide conjugates is typically determined by one or more
methods known to those of skill in the art, e.g., liquid chromatography-mass spectrometry
(LC-MS), matrix assisted laser desorption time of flight mass spectrometry (MALDl-TOF),
capillary electrophoresis, and the like.
"Substantially uniform glycoform" or a "substantially uniform glycosylation pati;em,"
when referring to a glycopeptide species, refers to the percentage of acceptor moieties tliat
are glycosylated by tlie glycosyltransferase of interest {e.g., fucosyltransferase). For
example, in the case of a al,2 fucosyltransferase, a substantially uniform fucosylation pattern
exists if substantially all (as defmed below) of the Gaip 1,4-GlcNAc-R and sialylated
analogues thereof are fucosylated in a peptide conjugate of the invention. It will be
understood by one of skill in the art, that the starting material may contain glycosylated
acceptor moieties (e.g., fucosylated Gaipi,4-GlcNAc-R moieties). Thus, the calculated
percent glycosylation will include acceptor moieties that are glycosylated by the methods of
the invention, as well as those acceptor moieties already glycosylated in the starting material.
The term "substantially" in the above definitions of "substantially unifoiTn" generally
means at least about 40%), at least about 70%), at least about 80%), or more preferably at least
about 90%, and still more preferably at least about 95% of the acceptor moieties for a
particular glycosyltransferase are glycosylated.
Description of the Invention
I. Method to Remodel Glvcan Chains
The present invention includes methods and compositions for the in vitro addition
and/or deletion of sugars to or from a glycopeptide molecule in such a manner as to provide a
peptide molecule having a specific customized or desired glycosylation pattern, preferably
including the addition of a modified sugar thereto. A key feature of the invention therefore is
to take a peptide produced by any cell t}Ape and generate a core glycan structure on the
peptide, foUowmg which th(i glycan structure is then remodeled in vitro to generate a peptide
having a glycosylation pattern suitable for therapeutic use in a mammal.
The importance of tlie glycosylation pattern of a peptide is well known in the art as
are the limitations of present in vivo methods for the production of properly glycosylated
peptides, particularly when these peptides are produced using recombinant DNA
methodology. Moreover, until the present invention, it has not been possible to generate
glycopeptides having a desired glycan structure thereon, wherein the peptide can be produced
at industrial scale.
In the present invention, a peptide produced by a cell is enzymatically treated in vitro
by the systematic addition of the appropriate enzymes and substrates therefor, such that sugar-
moieties that should not be present on the peptide are removed, and sugar moieties, optionally
including modified sugars, that should be added to the peptide are added in a manner to
provide a glycopeptide having "desired glycosylation", as defined elsewhere herein.
A. Method to remodel N-linked glycans
In one aspect, the pi-esent invention takes advzintage of the fact that most peptides of
commercial or pharmaceutical interest comprise a common five sugar structure referred to
herein as the trimannosyl core, which is N-linked to asparagine at the sequence Asn-X-
Ser/Thr on a peptide chain. The elemental trimannosyl core consists essentially of two N-
acetylglucosamine (GlcNAc) residues and three mannose (Man) residues attached to a
peptide, i.e., it comprises tiiese five sugar residues and no additional sugars, except that it
may optionally include a fucose residue. The first GlcNAc is attached to the amide group of
the asparagine and the second GlcNAc is attached to the first via a pi,4 linkage. A maimose
residue is attached to the second GlcNAc via a pi,4 linkage and two mannose residues are
attached to this mannose via an al,3 and an al,6 linkage respectively. A schematic depiction
of a trimannosyl core structure is shown in Figure 1, left side. While it is the case that glycan
structures on most peptides comprise other sugars in addition to the trimannosyl core, the
trimannosyl core structure represents an essential feature of N-linked glycans on mammalian
peptides.
The present invention includes the generation of a peptide having a trimannosyl core
structure as a fundamental element of the structure of the glycan molecules contained
thereon. Given the variety of cellular systems used to produce peptides, whether the systems
are themselves naturally occurring or whether they involve recombinant DNA methodology,
the present invention provides methods whereby a glycan molecule on a peptide produced in
any cell type can be reduced to an elemental trimannosyl core structure. Once the elemental
trimannosyl core structure has been generated then it is possible using the methods described
herein, to generate in vitro, a desired glycan structure on the peptide which confers on the
peptide one or more properties that enhances the therapeutic effectiveness of the peptide.
It should be cl a fluorescent marker in an assay. Polymers that are not naturally
occurring sugars may be used. In addition, the use of an otherwise naturally occurring sugar
that is modified by covalent attachment of another entity (e.g., poly(ethylene glycol),
poly(propylene glycol), poly(aspartate), biomolecule, therapeutic moiety, diagnostic moiety,
etc.) is also contemplated. In another exemplary embodiment, a therapeutic sugar moiety is
conjugated to a linker cirm and the sugar-linker arm is subsequently conjugated to a peptide
via a method of the invention.
Methods and chemistry for activation of water-soluble polymers and saccharides as
well as methods for conjugating saccharides and polymers to various species are described in
the literature. Commonly used methods for activation of polymers include activation of
fimctional groups with cyanogen bromide, periodate, glutciraldehyde, biepoxides,
epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc. {see, R.
F. Taylor, (1991), Protein Immobilisation. FLf>OAMENTALS ani:) Applications, Marcel
Dekker, N.Y.; S. S. Wong, (1992), Chemistry OF Protein Conjugation and
Crosslinking, CRC Press, Boca Raton; G. T. Hermanson et al, (1993), Immobilized
Affinity Ligand Techniques, Academic Press, N.Y.; Dimn, R.L., et al, Eds. Pol^'meric
Drugs And Drug Delivery Systems, ACS Symposium Series Vol. 469, American
Chemical Society, Washington, D.C. 1991).
Routes for preparing reactive PEG molecules and forming conjugates using the
reactive molecules are known in the art. For example, U.S. Patent No. 5,672,662 discloses a
water soluble and isolatable conjugate of an active ester of a polymer acid selected from
linear or branched poly(alkylene oxides), poly(oxyethylated polyols), poly(olefinic alcohols),
and poly(acrylomorpholtne), wherein the polymer has about 44 or more recurring units.
U.S. Patent No. 6,376,604 sets forth a method for preparing a water-soluble 1-
benzotriazolylcarbonate ester of a water-soluble and non-peptidic polymer by reacting a
terminal hydroxyl of the polymer with di(l-benzotriazoyl)carbonate in an organic solvent.
The active ester is used, to form conjugates with a biologically active agent such as a protein
or peptide.
WO 99/45964 describes a conjugate comprising a biologically active agent and an
activated water soluble polymer comprising a polymer backbone having at least one terminus
linked to the polymer backbone through a stable linkage, wherein at least one terminus
comprises a branching moiety having proximal reactive groups linked to the branching
moiety, in which the biologically active agent is Imked to at least one of the proximal reactive
groups. Other branched poly(ethylene glycols) are described in WO 96/21469, U.S. Patent
No. 5,932,462 describes a conjugate formed with a branched PEG molecule that includes a
branched terminus that includes reactive functional groups. The free reactive groups are
available to react with a biologically active species, such as a protein or peptide, forming
conjugates between the poly(ethylene glycol) and the biologically active species. U.S. Patent
No. 5,446,090 describes a bifiinctional PEG linker and its use in forming conjugates having a
peptide at each of the PEG linker termini.
Conjugates that include degradable PEG linkages :ire described in WO 99/34833; and
WO 99/14259, as well as in U.S. Patent No. 6,348,558. Such degradable Hnkages are
applicable in the present invention.
Although both reactive PEG derivatives and conjugates formed using the derivatives
are known in the art, until the present invention, it was not recognized that a conjugate could
be formed between PE(j (or other polymer) and another species, such as a peptide or
glycopeptide, through an intact glycosyl linking group.
Many water-soluble polymers are known to those of skill in the art and are useful in
practicing the present invention. The term water-soluble polymer encompasses species such
as saccharides (e.g., dextran, amylose, hyalouronic acid, poly(sialic acid), heparans, heparins,
etc.); poly (amino acids), e.g., poly(glutamic acid); nucleic acids; synthetic polymers (e.g.,
poly(acrylic acid), poly(ethers), e.g., poIy(ethylene glycol); peptides, proteins, and the like.
The present invention may be practiced with any water-soluble polymer with the sole
limitation that the polyraer must include a point at which the remainder of the conjugate cim
be attached.
Methods for activation of polymers can also be found in WO 94/17039, U.S. Pat. No.
5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No'. 5,219,564, U.S. Pat. No. 5,122,614,
WO 90/13540, U.S. Pat. No. 5,281,698, and more WO 93/15189, and for conjugation
between activated poljnmers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),
hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No. 4,412,989),
ribonuclease and superoxide dismutase (Veronese at a!., App. Biochem. Biotech. 11: 141-45
(1985)).
Preferred water-soluble polymers are those in which a substantial proportion of the
polymer molecules in a sample of the polymer are of approximately the same molecular
weight; such polymers are "homodisperse."
The present invention is further illustrated by reference to a poly(ethyIene glycol)
conjugate. Several reviews and monographs on the fiinctionalization and conjugation of PEG
are available. See, for example, Harris, Macronol. Chem. Phys. C25: 325-373 (1985);
Scouten, Methods in Emymology 135: 30-65 (1987); Wong et al. Enzyme Microb. Technoi
14: 866-874 (1992); Delgado et al. Critical Reviews in Therapeutic Drug Carrier Systems 9:
249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al.,
Pharmazie, 57:5-29 (2002).
Poly(ethylene glj'col) molecules suitable for use in the invention include, but are not
limited to, those described by the following Formula 3:
R= H, alkyl, benzyl, aryl, acetal, OHC-, H2N-CH2CH2-, HS-CH2CH2-,
-sugar-nucleotide, protein, methyl, etbyl;
X, Y, W, U (independently selected) = O, S, NH, N-R';
R', R'" (independently selected) = alkyl, benzyl, aryl, alkyl aryl, pyridyl, substituted aryl,
arylalkyl, acylaryl;
n = 1 to 2000;
ra, q, p (independently selected) = 0 to 20
o = 0 to 20;
Z = HO, NH2, halogen, S-R'", activated esters,
I
-sugar-nucleotide, protein, imidazole, HOBT, tetrazole, halide; and
V = HO, NH2, halogen, S-R'", activated esters, activated amides, -sugar-nucleotide, protein.
In preferred embodiments, the poly(ethylene glycol) molecule is selected from tiie
following:
The poly(ethylene glycol) useful in forming the conjugate of the invention is eitlier linear or
branched. Branched poly(ethylene glycol) molecules suitable for use in the invention
include, but are not limited to, those described by the following Formula:
Formula 4:
R', R", R'" (mdependently selected) = H, alkyl, benzyl, aryl, acetal, OHC-, H2N-CH2CH2-,
HS-CH^CH2-, -(CH2)qCY-Z, -sugar-nucleotide, protein, methyl, ethyl, heteroaryl,
acylalkyl, acylaryl, acylalkylaryl;
X,Y, W, A, B (independently selected) = O, S, NH, N-R', (CH2)i;
n, p (independently selected) = 1 to 2000;
m, q, 0 (independently selected) = 0 to 20;
Z = HO, NH2, halogen, S-R'", activated esters,
-sugar-nucleotide, protein;
V = HO, NH2, halogen, Si-R'", activated esters, activated amides,
-sugar-nucleotide, protein.
The in vivo half-life, area under the curve, and/or residence time of therapeutic
peptides can also be enhanced with water-soluble polymers such as polyethylene glycol
(PEG) and polypropylene glycol (PPG). For example, chemical modification of proteins with
PEG (PEGyiation) increases their molecular size and decreases their surface- and functional
group-accessibility, each of which are dependent on the size of the PEG attached to the
protein. This results in an improvement of plasma half-lives and in proteolytic-stabiiity, and
a decrease in immunogenicity and hepatic uptake (Chaffee et al. J. Clin. Invest. 89: 1643-
1651 (1992); Pyatake/o/. Res. Commun. Chem. Pathol Pharmacol. 29: 113-127 (1980)).
PEGyiation of interleukin-2 has been reported to increase its antitumor potency in vivo (KatJ-e
etal. Proc. Natl. Acad. Sci. USA. 84: 1487-1491 (1987)) and PEGyiation of a F(ab')2 derived
jfrom the monoclonal antibody A7 has improved its tumor localization (Kitamura et al.
Biochem. Biophys. Res. Commun. 28: 1387-1394 (1990)).
In one preferred embodiment, the in vivo half-life of a peptide derivatized with a
water-soluble polymer by a method of the invention is increased relevant to the in vivo half-
life of the non-derivatized peptide. In another preferred embodiment, the area under the
curve of a peptide derivatized with a water-soluble polymer using a method of the invention
is increased relevant to the area under the curve of the non-derivatized peptide. In another
preferred embodiment, the residence time of a peptide derivatized with a water-soluble
polymer using a method of the invention is increased relevant to the residence time of the
non-derivatized peptide. Techniques to determine the in vivo half-life, the area under the
curv^e and the residence time are well known in the art. Descriptions of such techniques can
be found in J.G. Wagner, 1993, Pharmacokinetics for the Pharmaceutical Scientist,
Technomic Publishing Company, Inc. Lancaster PA.
The increase in peptide in vivo half-life is best expressed as a range of percent
increase in this quantty. The lower end of the range of percent increase is about 40%, about
60%, about 80%, about 100%, about 150% or about 200%. The upper end of the range is
about 60%, about 80%, about 100%, about 150%), or more than about 250%).
In an exemplary embodiment, the present invention provides a PEGylated follicle
stimulating hormone (Examples 23 and 24). In a further exemplary embodiment, the
invention provides a PEGylated transferrin (Example 42).
Other exemplar}' water-soluble polymers of use in the invention include, but are not
limited to linear or branched poIy(alkyIene oxides), poly(oxyethylated polyols), poIy(olefinic
alcohols), and poly(acrylomorpholine), dextran, starch, poIy(amino acids), etc.
b) Water-insoluble polymers
The conjugates of the invention may also include one or more water-insoluble
polymers. This embodiment of the invention is illustrated by the use of the conjugate as a
vehicle with which to deliver a therapeutic peptide in a contiroJled manner. Polymeric drug
delivery systems are known in the art. See, for example, Dunn et al., Eds. Polymeric
Drugs And Drug Delivery Systems, ACS Symposium Series Vol. 469, American
Chemical Society, Washington, D.C. 1991. Those of skill in the art will appreciate that
substantially any known drug delivery system is applicable to the conjugates of the present
invention.
Representative water-insoluble polymers include, but are not limited to,
polyphosphazines, poly(vinyi alcohols), polyamides, polycarbonates, polyaikylenes,
polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates,
polyvinyl ethers, polyvinyl; esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides,
polysiioxanes, polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),
poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate),
poly(isodecyl methacrylate), poIy(lauryI methacrylate), poIy(phenyl methacrylate),
poIy(methyi acrylate), poly(isopropyl acrylate), poiy(isobutyl acrylate), poIy(octadecyl
acrylate) polyethylene, polj'propylene, poly(ethylene glycol), poIy(ethylene oxide), poly
(ethylene terephtlialate), po[y(vinyI acetate), polyvinyl chloride, polystyrene, poly\'Lnyl
pyrrolidone, pluronics and pol)rvinylphenol and copolymers thereof.
Synthetically modified natural polymers of use in conjugates of the invention include,
but are not limited to, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, and nitrocelluloses. Particularly preferred members of tlie broad classes of
synthetically modified natural polymers include, but are not limited to, methyl cellulose,
ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl
methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose
acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt,
and pol>Tners of acrylic and methacrylic esters and alginic acid.
These and the; other polymers discussed herein can be readily obtained from
commercial sources such as Sigma Chemical Co. (St. Louis, MO.), Polysciences (Wanenton,
PA.), Aldrich (Milwaukee, WI.), Fluka (Ronkonkbma, NY), and BioRad (Richmond, C'A), or
else synthesized from monomers obtained from these suppliers using standard techniques.
Representative biodegradable polymers of use in the conjugates of the invention
ijiclude, but are not limited to, polylactides, polyglycolides and copolymers thereof,
poly(ethylene terephfhialate), poly(butyric acid), poIy(valeric acid), poly(lactide-co-
caprolactone), poly(Ia.ctide-co-gIycolide), polyanhydrides, polyorthoesters, blends and
copolymers thereof. <3f particular use are compositions that form gels, such as those
including collagen, pluronics and the like.
The polymers of use in the invention include "hybrid' polymers that include water-
insoluble materials having within at least a portion of theiir structure, a bioresorbable
molecule. An example of such a polymer is one that includes a water-insoluble copolymer,
which has a bioresorbable region, a hydrophilic region and a plurality of crosslinkable
functional groups per ]Dolymer chain.
For purposes of the present invention, "water-insoluble materials" includes materials
that are substantially insoluble in water or water-containing environments. Thus, although
certain regions or segments of the copolymer may be hydi'ophilic or even water-soluble, the
polymer molecule, as a whole, does not to any substantial measure dissolve in water.
For purposes of the present invention, the term "bioresorbable molecule" includes a
region that is capable of being metabolized or broken down and resorbed and/or eliminated
through normal excretory routes by the body. Such metabolites or break doA\ii products cire
preferably substantially non-toxic to the body.
The bioresorbable region may be either hydrophobic or hydrophilic, so long as the
copolymer composition as a whole is not rendered water-soluble. Thus, the bioresorbable
region is selected based on the preference that the polymer, as a whole, remains water-
insoluble. Accordingly, the relative properties, i.e., the kinds of functional groups contained
by, and the relative proportions of the bioresorbable region, and the hydrophilic region are
selected to ensure that useful bioresorbable compositions remain water-insoluble.
Exemplary resorbable polymers include, for example, synthetically produced
resorbable block copolymers of poly(a-hydroxy-carboxylic acid)/poly(oxyalkyIene, (see,
Cohn et al., U.S. Patent No. 4,826,945). These copolymers ai:e not crosslinked and cire water-
soluble so that the body can excrete the degraded block copolymer compositions. See,
Younes et al.,JBiomed. Mater. Res. 21: 1301-1316 (1987); £ind Cohn et al.,J Biomed.
Mater. Res. 22: 993-1009 (1988).
Presently preferred bioresorbable polymers include one or more components selected
from poly(esters), poly(hydroxy acids), poly(Iactones), poly(amides), poly(ester-amides),
poly (amino acids), poly(anhydrides), poly(orthoesters), poly(carbonates),
poly(phosphazines), poly(phosphoesters), poly(thioesters), polysaccharides and mixtures
thereof. More preferably still, the biosresorbable polymer includes a poly(hydroxy) acid
component. Of the poly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproic acid,
polybutyric acid, polyvaleric acid and copolymers and mixtares thereof are preferred.
In addition to forming fragments that are absorbed in vivo ("bioresorbed"), preferred
pol>'meric coatings for use in the methods of the invention can also form an excretable and/or
metabolizable fragment.
Higher order copolymers can also be used in the present invention. For example,
Casey et al, U.S. Patent No. 4,438,253, which issued on March 20, 1984, discloses tri-block
copolymers produced from the transesterification of poly(glycoIic acid) and an hydroxyl-
ended poly(alkylene glycol). Such compositions are disclosed for use as resorbable
monofilament sutures. The flexibility of such compositions is controlled by the incorporation
of an aromatic orthocaibonate, suchi as tetra-p-tolyl orthocarbonate into the copolymer
structure.
Other coatings based on lactic and/or glycolic acids can also be utilized. For exajnple,
Spinu, U.S. Patent No. 5,202,413, which issued on April 13, 1993, discloses biodegradable
multi-block copolymers having sequentially ordered blocks of polylactide and/or
polyglycolide produced by ring-opening polymerization of lactide and/or glycolide onto
either an oligomeric diol or a diamine residue followed by chain extension with a di-
ftinctional compound, such as, a diisocyanate, diacylchloride or dichlorosilane.
Bioresorbable regions of coatings useful in the present invention can be designed to
be hydrolytically and/or enzymatically cleavable. For purposes of the present invention,
" hydrolytically cleavable" refers to die susceptibility of the copoHmer, especially the
bioresorbable region, to hydrolysis in water or a water-containing environment. Similarly,
"enzymatically cleavable" as used herein refers to the susceptibility of the copolymer,
especially the bioresorbable region, to cleavage by endogenous or exogenous enzymes.
When placed within the body, the hydrophilic region can be processed into excretable
and/or metabolizable fragments. Thus, the hydrophilic region can include, for example,
polyethers, polyalkylene oxides, polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol),
poly(alk;yl oxazolines), polysaccharides, carbohydrates, peptides, proteins and copolymers
and mixtures thereof Furthermore, the hydrophilic region can also be, for example, a
poly(alkylene) oxide. Such poly(aUcylene) oxides can include, for example, poly(ethylene)
oxide, poly(propyIene) cxide and mixtures and copolymers thereof
Polymers that are components of hydrogels are also useful in the present invention.
Hydrogels are polymeric materials that are capable of absorbing relatively large quantities of
water. Examples of hydrogel forming compounds include, but are not limited to, polyacrylic
acids, sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine, gelatin,
carrageenan and other f)olysaccharides, hydroxyethylenemethacrylic acid (HEMA), as well as
derivatives thereof, and the like. Hydrogels can be produced that are stable, biodegradable
and bioresorbable. Moreover, hydrogel compositions can include subunits that exhibit one oi
more of these properties.
Bio-compatible hydrogel compositions whose integrity can be controlled through
crosslinking are known and are presently preferred for use in the methods of the invention.
For example, Hubbell et al., U.S. Patent Nos. 5,410,016, which issued on April 25, 1995 and
5,529,914, which issued on June 25, 1996, disclose water-soluble systems, which are
crosslinked block copolymers having a water-soluble central block segment sandwiched
between two hydrolytically labile extensions. Such copolymers are fiirther end-capped with
photopolymerizable acrylate functionalities. Wlien crosslinked, these systems become
hydrogels. The water soluble central block of such copolymers can include poly(ethylene
glycol); whereas, the hydrolytically labile extensions can be a poly(a-hydroxy acid), such as
polyglycolic acid or polylactic acid. See, Sawhney et al., Macromolecules 26: 581-587
(1993).
In another preferred embodiment, the gel is a thermoreversible gel. Thermore\'ersible
gels including components, such as pluronics, collagen, gelatin, hyalouronic acid.
Biomolecules useful in practicing the present inivention can be derived from any
source. The biomolecules can be isolated from natural sources or they can be produced by
synthetic methods. Peptides can be natural peptides or mutated peptides. Mutations :ages include, but are not limited
to, protein-sugar-linker-sugar-protein, protein-sugar-linker-protein and multivalent forms
thereof, and protein-suga]--linker-drug where the drug includes small molecules, peptides,
lipids, among others.
Site-specific and target-oriented delivery of therapeutic agents is desirable for the
purpose of treating a wide variety of human diseases, such as different types of malignancies
and certain neurological disorders. Such procedures are accompanied by fewer side effects
and a higher efficiacy of drug. Various principles have been relied on in designing these
delivery systems. For a review, see GzmQM, Advanced Drug Delivery Reviews 53:171-216
(2001).
One important consideration in designing a drug delivery system to target tissues
specifically. The discovery of tumor surface antigens has made it possible to develop
therapeutic approaches where tumor cells displaying definable surface antigens are
specifically targeted and Idlled. There are three main classes of therapeutic monoclonal
antibodies (antibody) that have demonstrated effectiveness in human clinical trials in treating
malignancies: (1) unconjugated MAb, which either directly induces gro\\'th inhibition and/or
apoptosis, or indirectly activates host defense mechanisms to mediate antitumor cytotoxicity;
(2) drug-conjugated MAb, which preferentially delivers a potent cytotoxic toxin to the tumor
cells and therefore minimizes the systemic cytotoxicity commonly associated with
conventional chemotherapy; and (3) radioisotope-conjugated MAb, which delivers a
sterilizing dose of radiation to the tumor. See review by Reff et al., Cancer Control 9:152-
166(2002).
In order to arm M\bs with the power to kill malignaiit cells, the MA.bs can be
connected to a toxm, which may be obtained from a plant, bacterial, or fungal source, to form
chimeric proteins called immunotoxins. Frequenny used plant toxins are divided into two
classes: (I) holotoxins (or class 11 ribosome inactivating proteins), such as ricin, abrin,
mistletoe lectin, and mo-53124692; US-06333410B1
"JP-1104183
'^ EP-00689845
'^EP-00689845
'^EP-00136791; EP-00087957
''us 50001112; US 5143906.
"WO-00136048
Conventional imnaunotoxins contain an MAb chemically conjugated to a toxin that is
mutated or chemically modified to minimized binding to nonnal cells. Examples include
anti-B4-blocked ricin, targeting CDS; and RFB4-deglycosylated ricin A chain, targeting
CD22. Recombinant imraunotoxins developed more recently are chimeric proteins
consisting of the variable region of an antibody directed against a tumor antigen fused to a
protein toxin using recombinant DNA technology. The toxin is also frequently genetically
modified to remove normal tissue binding sites but retain its cytotoxicity. A large number
internalized in early endosomes (Xing et al., 1998, Biochem. J. 336:667; Li et al., 2002,
Trends in Pharmcol. Sci. 23:206; Suhaila et al., 1998, J. Biol. Chem. 273:14355). Other
contemplated targeting agents that are related to transferrin include, but are not limited to.
lactotransferrin (lactoferrin), melanofransferrin (p97), ceruloplasmin, and divalent catiTnbols R', R", R^, R** and R^ represent H, polymers, a water-soluble poiymci.
therapeutic moiety, biomolecule or other moiety. The symbol R6 represents H, OH, or a
polymer. Alternatively, tliese symbols represent a linker that is linked to a polymer, water-
soluble polymer, therapeutic moiety, biomolecule or other moiety.
In another exemplary embodiment, a mannosamine is simultaneously acylated and
activated for a nucleophilic substitution by the use of chloroacetic anhydride as set forth in
Scheme 5. In each of the schemes presented in this section, i^ or Na"^ can be interchangeable
wherein the salt can be sodium, or can be any other suitable salt.
The resulting chloro-derivatized glycan is contacted with pyruvate in the presence of an
aldolase, forming a chloro-derivatized sialic acid. The corresponding nucleotide sugar is
prepared by contacted the sialic acid derivative with an appropriate nucleotide triphosphates
and a synthetase. The cliloro group on the sialic acid moiety is then displaced witli a
nucleophilic PEG derivative, such as thio-PEG.
In a further exemplary embodiment, as shown is Scheme 6, a mannosamine is
acylated with a bis-HOBT dicarboxylate, producing the coiTCsponding amido-alkyl-
carboxylic acid, which is subsequently converted to a sialic acid derivative. The sialic acid
derivative is converted to a nucleotide sugar, and the carboxylic acid is activated and reactCi'
with a nucleophilic PEG derivative, such as amino-PEG.
Scheme 6
In another exemplary embodiment, set forth in Scheme 7, amine- and carboxyl-
protected neuraminic acid is activated by converting the primary hydroxyl group to the
corresponding p-toluenesulfonate ester, and the methyl ester is cleaved. The activated
neuraminic acid is converted to the corresponding nucleotide sugar, and the activating group
is displaced by a nucleophilic PEG species, such as thio-PEG.
In yet a further exemplary embodiment, as set forth in Scheme 8, the primary
hydroxyl moiety of an Jimine- and carboxyl-protected neuraminic acid derivative is alkylated
using an electrophihc PEG, such as chloro-PEG. The metliyl ester is subsequently cleaved
and the PEG-sugar is converted to a nucleotide sugar.
Glycans other than sialic acid can be derivatized with PEG using the methods set forth
herein. The derivatized glycans, themselves, are also within the scope of the invention.
Thus, Scheme 9 provides an exemplary synthetic route to a PEGylat'-d galactose nucleotide
sugar. The primary hydjroxyl group of galactose is activated as the corresponding
toluenesulfonate ester, which is subsequently converted to a nucleotide sugar.
Scheme 10 sets forth an exemplary route for preparing a galactose-PEG derivative
thai is based upon a galactose-6-amine moiety. Thus, galactosamine is converted to a
nucleotide sugar, and the amine moiety of galactosamine is fiinctionalized with an active
PEG derivative.
Scheme 11 provides another exemplary route to galactose derivatives. The starting
point for Scheme 11 is galactose-2-amine, which is converted to a nucleotide sugar. The
amine moiety of the nucleotide sugar is the locus for attaching a PEG derivative, such as
Methoxy-PEG (mPEG) carboxylic acid.
Exemplary moieties attached to the conjugates disclosed herein include, but are not
limited to, PEG derivatives {e.g., acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-
PEG, aryl-PEG, alkyl-PEG), PPG derivatives (e.g., acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-
PPG carbamoyl-PPG, ar/l-PPG), polyapartic acid, polyglutjmiate, polylysine, therapeutic
moieties, diagnostic moieties, mannose-6-phosphate, heparin, heparan, SLe^ mannose,
mannose-6-phosphate. Sialyl Lewis X, FGF, VFGF, proteins (e.g., transferrin), chondroitin,
keratan, dermatan, dextrctn, modified dextran, amylose, bisphosphate, poly-SA, hyaluronic
acid, keritan, albumin, integrins, antermary oligosaccharides, peptides and the like. Methods
of conjugating the various modifying groups to a saccharide moiety are readily accessible to
those of skill in the art (POLY (ETHYLENE GLYCOL CHEMISTRY : BlOTECHNICAL AND
Biomedical Applications, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; Poly
(Ethylene Gl^'col) Chiemical and Biological Applications, J. Milton Han-is, Ed., ACS
Symposium Series No. 680, American Chemical Society, 1997; Hermanson, BlOCONJUGATE
Techniques, Academic Press, San Diego, 1996; and Dunn et ai, Eds. Polymeric Drugs
And Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical
Society, Washington, D.C. 1991).
Purification of sugars, nucleotide sugars and derivatives
The nucleotide sugars and derivatives produced by the above processes can be used
without purification. However, it is usually preferred to recover the product. Standard, well-
known techniques for reciovery of glycosylated saccharides such as thin or thick layer
chromatography, column, chromatography, ion exchange chromatography, or membrane
filtration can be used. It is preferred to use membrane filtration, more preferably utilizing a
reverse osmotic membrane, or one or more column chromatographic techniques for the
recovery as is discussed hereinafter and in the literature cited herein. For instance, membrcjie
filtration wherein the membranes have molecular weight cutoff of about 3000 to about 10,000
can be used to remove proteins for reagents having a molecular weight of less than 10,000
Da.. Membrane filtration or reverse osmosis can then be used to remove salts and/or purify
the product saccharides {i:ee, e.g., WO 98/15581). Nanofilter membranes are a class of
reverse osmosis membranes that pass monovalent salts but retain polj^alent salts and
uncharged solutes larger than about 100 to about 2,000 Daltons, depending upon the
membrane used. Thus, in a typical application, saccharides prepared by the methods of the
present invention will be retained in the membrane and contjuninating salts will pass through.
G. Cross-linking Groups
Preparation of the modified sugar for use in the methods of the present invention
includes attachment of a modifying group to a sugar residue iand forming a stable adduct,
which is a substrate for a glycosyltransferase. Thus, it is often preferred to use a cross-
linking agent to conjugate the modifying group and the sugar. Exemplary bifunctional
compounds which can be used for attaching modifying groups to carbohydrate moieties
include, but are not limited to, bifunctional poly(ethylene glycols), polyamides, polyethers,
polyesters and the like. General approaches for linking carbohydrates to other molecules are
known in the literature. See, for example, Lee et ai. Biochemistry 28: 1856 (1989); Bhatia
etai, Anal. Biochem. 178: 408 (1989); Janda et ai, J. Am. Chem. Soc. 112: 8886 (1990) and
Bednarski et ai, WO 92/18135. In the discussion that follows, the reactive groups are treated
as benign on the sns-^x moiety of the nascent modified sugar. The focus of the discussion is
for clarity of illustration. Those of skill in the art will apprcxiiate that the discussion is
relevant to reactive groups on the modifying group as well.
An exemplary strategy involves incorporation of a protected sulfhydryl onto the sugar
usmg the heterobifiinctional crossluiker SPDP (n-succinimidyl-3-(2-pyridyldithio)propionate
and then deprotecting tlie sulfliydryl for formation of a disulfide bond with another sulfh)'dryl
on the modifying group.
If SPDP detrimentally affects the ability of the modified sugar to act as a
glycosyltransferase substrate, one of an array of other crosslinkers such as 2-iminothioIaj,ie or
N-succinimidyl S-acetylthioacetate (SATA) is used to form a disulfide bond. 2-
iminothiolane reacts with primary amines, instantly incorporating an unprotected sulfhydryl
onto the amine-contammg molecule, SATA also reacts with primary amines, but
incorporates a protected sulfhydryl, which is later deacetylated usmg hydroxylamine to
produce a fi-ee sulfhydryl. In each case, the incorporated sulfhydryl is free to react with, other
sulftiydryls or protected sulfhydryl, like SPDP, forming the required disulfide bond.
The above-described strategy is exemplary, and not limiting, of linkers of use in the
mvention. Other crosslinkers are available that can be used in different strategies for
crosslinking the modifj'ing group to the peptide. For example, TPCH(S-(2-thiopyridyl)-L-
cysteine hydrazide and TPMPH ((S-(2-thiopyridyl) mercapto-propionohydrazide) react with
carbohydrate moieties that have been previously oxidized by mild periodate treatment, thus
forming a hydrazone bond between the hydrazide portion of the crosslinker and the periodatr
generated aldehydes. TPCH and TPMPH introduce a 2-pyridylthione protected sulfhydryl
group onto the sugar, which can be deprotected with DTT and then subsequently used for
conjugation, such as forming disulfide bonds between components.
If disulfide bonding is found unsuitable for producing stable modified sugars, other
crosslinkers may be used that incorporate more stable bonds between components. The
heterobifunctional crosslinkers GMBS (N-gama-malimidobutyryloxy)succinimide) and
SMCC (succinimidyl 4-(N-maleimido-methyl)cyclohexane) react with primary amines, thus
introducing a maleimide group onto the component. The maleimide group can subsequently
react with sulthydryls on the other component, which can be introduced by previously
mentioned crosslhikers, thus forming a stable thioether bond between Uie components. If
stiiic hindrance between components interferes with either component's acti.ity or the ability
of the modified sugaj: to act as a glycosyltransferase substrate, crosslinkers can be used which
introduce long space]- arms between components and include derivatives of some of the
previously mentioned crosslinkers {i.e., SPDP). Thus, there is an abundance of suitable
crosslinkers, which aire useful; each of which is selected depending on the effects it has on
optimal peptide conjugate and modified sugar production.
A variety of reagents are used to modify the components of the modified sugar ^vith
intramolecular chemical crosslinks (for reviews of crosslinking reagents and crosslinking
procedures see: Wold, F., Meth. Enzymol. 25: 623-651, 1972; Weetall, H. H., and Cooney, D.
A., In: Enzymes as Drugs. (Holcenberg, and Roberts, eds.) pp. 395-442, Wiley, New York,
1981; Ji, T. H., Meth. Enzymol. 91: 580-609, 1983; Mattson et al, Mol. Biol. Rep. \1:\61-
183, 1993, all of which are incorporated herein by reference). Preferred crosslinking reagents
ai-e derived fi-om various zero-length, homo-bifimctional, and hetero-bifunctional crosslinking
reagents. Zero-length crosslinking reagents include direct conjugation of two intrinsic
chemical groups with no introduction of extrinsic material. Agents tliat catalyze formation of
a disulfide bond belong to this category. Another example is reagents tliat induce
condensation of a carboxyl and a primary amino group to form an amide bond such as
carbodiimides, ethylchloroformate. Woodward's reagent K (2-ethyI-5-phenyIisoxazolium-3'
sulfonate), and carbonyldiimidazole. In addition to these chemical reagents, the enzyme
transglutaminase (glut£unyl-peptide y-glutamyltransferase; EC 2.3.2.13) may be used as zqvi.)
length crosslinking reagent. This enzyme catalyzes acyl transfer reactions at carboxamide
groups of protein-linked glutaminyl residues, usually with a primary amino group as
substrate. Preferred homo- and hetero-bifunctional reagents contain two identical or two
dissimilar sites, respectively, which may be reactive for amino, sulfhydryl, guanidino, indoi
or nonspecific groups.
2. Preferred Specific Sites in Crosslinking Reagents
a. Amino-Reactive Groups
In one preferred embodiment, the sites on the cross-linker are amino-reactive grc il -
Useful non-limiting examples of amino-reactive groups include N-hydroxysuccinimide
(NHS) esters, imidoesters, isocyanates, acylhalides, arylazides, p-nitrophenyl esters,
aldehydes, and sulfonyl chlorides.
NHS esters react; preferentially with the primary (including aromatic) amino groups of
a modified sugar component. The imidazole groups of histidines are known to compete with
primary amines for reaction, but the reaction products are unstable and readily hydrolyzed.
The reaction involves the nucleophilic attack of an amuie on the acid carboxyl of an NHS
ester to form an amide, releasing the N-hydroxysuccinimide. Thus, the positive charge of the
original amino group is lost.
Imidoesters are the most specific acylating reagents for reaction with the amine
groups of the modified sugar components. At a pH between 7 and 10, imidoesters react only
with primary amines. Primary amines attack imidates nucleophilically to produce an
intermediate that breaks down to amidine at high pH or to a new imidate at low pH. The new
imidate can react with another primary amine, thus crosslinking two amino groups, a case of
a putatively monofiinctional imidate reacting bifiinctionally. The principal product of
reaction with primary amines is an amidine that is a stronger base than the original amine.
The positive charge of th(J original amino group is therefore retained.
Isocyanates (and isothiocyanates) react with the primary amines of the modified sugar
components to form stable bonds. Their reactions with sulfhiydryl, unidazole, and tyrosyl
groups give relatively unstable products.
Acylazides are also used as amino-specific reagents in which nucleophilic arrdnes of
the affinity component attack acidic carboxyl groups under slightly alkaline conditions, e.g.
pH8.5.
Arylhalides such as l,5-difluoro-2,4-dinitrobenzene react preferentially with the
amino groups and tyrosine phenolic groups of modified sugar components, but also with
sulfhydryl and imidazole groups.
p-Nitrophenyl esters of mono- and dicarboxylic acids are also useful amino-reactive
groups. Although the reagent specificity is not very high, a- and e-amino groups appear to
react most rapidly.
Aldehydes such as glutaraldehydc react with primary .amines of modified sugar.
Although unstable Schiff bases are formed upon reaction of the amino groups with tlie
aldehydes of the aldehydes, glutaraldehydc is capable of modifying the modified sugar with
stable crosslinks. At pH 6-8, the pH of typical crosslinking conditions, the cyclic pol>Tners
undergo a dehydration to form a-P unsaturatec. yidehyde polymers. Schiff bases, however
are stable, when conjugated to another double bond. The resonant interaction of both double
bonds prevents hydrolysis of the Schiff linkage. Furthermore, amines at high local
concentrations can attack the ethylenic double bond to form a stable Michael addition
product.
Aromatic sulfonyl chlorides react with a variety of sites of the modified sugar
components, but reaction with the amino groups is the most important, resulting in a stable
sulfonamide linkage.
b. Sulfhydryl-Reactive Groups
In another preferred embodiment, the sites are sulfhydryl-reactive groups. Useful,
non-limiting examples of sulfhydryl-reactive groups include maleimides, alkyl halides,
pyridyl disulfides, and thiophthalimides.
Maleimides react preferentially with the sulfhydryl group of the modified sugar
components to form stable thioether bonds. They also react at a much slower rate with
primary amino groups and the imidazole groups of histidines. However, at pH 7 the
maleimide group can be considered a sulfhydryl-specific group, since at this pH the reaction
rate of simple thiols is 1000-fold greater than that of the corresponding amine.
Alkyl halides react with sulfliydryl groups, sulfides, imidazoles, and amino groups
At neutral to slightly alkaline pH, however, alkyl halides react primarily with sulfhydryl
groups to form stable thioether bonds. At higher pH, reaction with amino groups is favored
Pyridyl disulfides react with free sulfhydryls via disulfide exchange to give mixed
disulfides. As a result, pyridyl disulfides are the most specific sulfhydryl-reactive groups
Thiophthalimides react with free sulfhydryl groups to form disulfides.
c. Carboxyl-Reactive Residue
In another embodiment, carbodiimides soluble in both water and organic solvent,..
used as carboxyl-reactive reagents. These compounds react with free carboxyl groups
forming a pseudourea that can then coupled to available amines yielding an amide linkage
Procedures to modify a carboxyl group with carbodiimide is well know in the art (see,
Yamada etal., Bioche?}tistry 20: 4S36AS42, 1981).
3. Preferred Nonspecific Sites in Crosslinking Reagents
In addition to the use of site-specific reactive moieties, the present invention
contemplates the use of non-specific reactive groups to link: the sugar to the modif>'ing group.
Exemplary non-specific cross-linkers include photoactivatable groups, completely
inert in the dark, which are converted to reactive species upon absorption of a photon of
appropriate energy. In one preferred embodiment, photoaclivatable groups are selected from
precursors of nitrenes generated upon heating or photolysis of azides. Electron-deficient
nitrenes are extremely reactive and can react with a variety of chemical bonds including N -H,
0-H, C-H, and C=C. Although three types of azides (aryl, alkyl, and acyl derivatives) may
be employed, arylazides are presently preferred. The reactivity of arylazides upon photolysis
is better with N-H and O-H than C-H bonds. Electron-deficient arylnitrenes rapidly ring-
expcmd to form dehydroazepines, which tend to react with nucleophiles, rather than form C-H
insertion products. The reactivity of arylazides can be increased by the presence of electron-
withdrawing substituents such as nitro or hydroxyl groups in the ring. Such substituents push
the absorption maximum of arylazides to longer wavelength. Unsubstituted arylazides havi;
an absorption maximum in the range of 260-280 nm, while hydroxy and nitroarylazides
absorb significant light beyond 305 nm. Therefore, hydroxy ,and nitroarylazides are most
preferable since they allow to employ less harmful photolysis conditions for the affinity
component than unsubstimted arylazides.
In another preferred embodiment, photoactivatable groups are selected from
fluormated arylazides. The photolysis products of fluorinated arylazides are arylnitrenes, all
of which undergo the characteristic reactions of this group, including C-H bond insertion,
with high efficiency (Keana era/., J. Org. Chem. 55: 3640-3647, 1990).
In another embodiment, photoactivatable groups are selected from benzophenone
residues. Benzophenone reagents generally give higher crosslinking yields than arylazide
reagents.
In another embodiment, photoactivatable groups are selected from diazo compounds,
which form an electron-deficient carbene upon photolysis. These carbenes imdergo a vaiiety
of reactions including insertion into C-H bonds, addition to double bonds (including aromatic
systems), hydrogen attraction and coordination to nucleophilic centers to gi\e carbon ions
In still another embodiment, photoactivatable groups are selected from
diazopyruvates. For example, the p-nitrophenyl ester of p-nitrophenyl diazopyruvate reacts
with aliphatic amines to give diazopyruvic acid amides that undergo ultraviolet photolysis to
form aldehydes. The photolyzed diazopyruvate-raodified affinity component will react like
formaldehyde or glutaraldehyde forming crosslinks.
4. Homobifunctional Reagents
a. Homobifunctional crosslinkers reactive with primary amines
Synthesis, properties, and applications of amine-reactive cross-linkers are
conmiercially described in die literature (for reviews of crosslinking procedures and reagents,
see above). Many reagents are available {e.g.. Pierce Chemical Company, Rockford, 111.;
Sigma Chemical Compjiny, St. Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).
Preferred, non-limiting examples of homobifunctional NHS esters include
disuccmimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)
suberate (BS), disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate (sulfo-DST), bis-
2-(succinimidooxyc£irbonyloxy)ethylsulfone(BSOCOES), bis-2-(sulfosuccinimidooxy-
carbonyloxy)ethylsulfone (sulfo-BSOCOES), ethylene glycolbis(succinimidylsuccinate)
(EGS), ethylene glycolbis(sulfosuccininiidylsuccinate) (sulfo-EGS), ditliiobis(succLQimidyl-
propionate (DSP), and dithiobis(sulfosuccinimidylpropionate (suIfo-DSP). Preferred, non-
limiting examples of homobifunctional imidoesters include dimethyl malonimidate (DMM),
dimethyl succinimidate (DMSC), dimethyl adipimidate (DMA), dimethyl pimelimidate
(DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-oxydipropionimidate (DODP),
dimethyl-3,3'-(methylenedioxy)dipropionimidate(DMDP), dimethyl-,3'-
(dunethylenedioxy)dipropionimidate(DDDP), dimethyl-3,3'-(tetramethylenedioxy)-
dipropionimidate (DTDP), and dimethyl-3,3'-dithiobispropionimidate (DTBP).
Preferred, non-lim iting examples of homobifunctional isothiocyanates include: p-
phen)'lenediisothiocyanate (DITC), and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene
(DIDS).
Preferred, non-limiting examples of homobifunctional isocyanates include xylene-
diisocyanate, toluene-2,4-diisocyanate, toluene-2-isocyanate-4-isothiocyanate, 3-
methoxydiphenylmet,haiie-4,4'-diisocyanate, 2,2'-dicarboxy-4,4'-azophenyIdiisocyanate, and
hexamethylenediisoc yanate.
Preferred, non-lixniting examples of homobifimctional arylhalides include 1,5-
difluoro-2,4-dimtrobenzene (DFDNB), and 4,4'-difluoro-3,3'-dinitrophenyl-sulfone.
Preferred, non-limiting examples of homobifimctional aliphatic aldehyde reagents
include glyoxal, malondialdehyde, and glutaraJdehyde.
Preferred, non-limiting examples of homobifimctional acylating reagents include
nitrophenyl esters of dicarboxylic acids.
Preferred, non-limiting examples of homobifimctional aromatic sulfonyl chlorides
include phenoI-2,4-disulfonyl chloride, and a-naphthoI-2,4-disulfonyI chloride.
Preferred, non-limiting examples of additional amino-reactive homobifunctional
reagents include erythritolbiscarbonate which reacts with amines to give biscarbamates.
b. Homobifimctional Crosslinkers Reactive with Free Sulfhydryl
Groups
Synthesis, properties, and applications of such reagents are described in tlie literature
(for reviews of crosslinking procedures and reagents, see above). Many of the reagents are
commercially available (e.g.. Pierce Chemical Company, Rockford, HI; Sigma Chemical
Company, St. Louis, Mo.; Molecular Probes, Inc., Eugene, OR).
Preferred, non-limiting examples of homobifimctional maleimides include
bismaleimidohexane (BMH), N,N'-(I,3-phenylene) bismaleimide, N,N'-(1,2-
phenylene)bismaleimide, azophenyldimaleimide, and bis(N-maleimidomethyl)ether.
Preferred, non-limiting examples of homobifimctional pyridyl disulfides include 1,4-
di-3'-(2'-pyridyIdithio)propionamidobutane(DPDPB).
Preferred, non-limiting examples of homobifimctional alkyl halides include 2,2'-
dicarboxy-4,4'-diiodoacet1:ic amount. The catalytic amount of a particular enzyme
varies according to the concentration of that enzyme's substrate as well as to reaction
conditions such as temperature, time and pH value. Means for determining the catalytic
amount for a given enzyme under preselected substrate concentrations and reaction
conditions are well known to those of skill in the art.
The temperature at which an above-described process is carried out can range from
just above freezing to the temperature at which the most sensitive enzyme denatures.
Preferred temperature ranges are about 0 °C to about 55 °C, and more preferably about 20 ° C
to about 37 °C. In another exemplary embodiment, one or more components of the present
method are conducted at an elevated temperature using a thermophilic enzyme.
The reaction mixture is maintained for a period of time sufficient for the acceptor to
be glycosylated, thereby forming the desired conjugate. Some of the conjugate can often be
detected after a few hours, with recoverable amounts usually being obtained within 24 hours
or less. Those of skill in the art understand that the rate of reaction is dependent on a number
of variable factors {e.g, en;iyme concentration, donor concentration, acceptor concentration,
temperature, solvent volume), which are optimized for a selected system.
The present invention also provides for the industrial-scale production of modified
peptides. As used herein, ;m industrial scale generally produces at least one gram of fuiished,
purified conjugate.
In the discussion that follows, the invention is exemplified by the conjugation of
modified sialic acid moieties to a glycosylated peptide. The exemplary modified sialic acid is
labeled with PEG. The focus of the following discussion on the use of PEG-modified sialic
acid and glycosylated peptides is for clarity of illustration and is not intended to imply that
the invention is limited to the conjugation of these two partners. One of skill understands that
the discussion is generally applicable to the additions of modified glycosyl moieties other
than sialic acid. Moreover, the discussion is equally applicable to the modification of a
glycosyl unit with agents other than PEG including other water-soluble polymers, therapeutic
moieties, and biomolecules.
An enzymatic approach can be used for the selective introduction of PEGylated or
PPGylated carbohydrates onto a peptide or glycopeptide. The method utilizes modified
sugars containing PEG, PPG, or a masked reactive fiinctional group, and is combined witli
the appropriate glycosyltrcinsferase or glycosynthase. By selecting the glycosyltransferase
that will mcike the desired carbohydrate linkage and utilizing the modified sugar as the donor
substrate, the PEG or PPG caii be introduced directly onto the peptide backbone, onto
existing sugar residues of a glycopeptide or onto sugar residues that have been added to a
peptide.
An acceptor for the sialyltransferase is present on the peptide to be modified by the
methods of the present invention either as a naturally occurring structure or one placed there
recombinantly, enzymaticaliy or chemically. Suitable acceptors, mclude, for example,
galactosyl acceptors such as Galpl,4GlcNAc, Galpl,4GalNAc, GaI(31,3GalNAc, lacto-N-
tetraose, Gaipi,3GlcNAc, Gaipi,3Ara, Galpl,6GlcNAc, Galpl,4Glc (lactose), and other
acceptors known to those of skill in the art {see, e.g., Paulson et al, J. Biol. Chem. 253: 5617-
5624(1978)).
In one embodiment, an acceptor for the sialyltransferase is present on the peptide to
be modified upon in vivo synthesis of the peptide. Such peptides can be sialylated using the
claimed methods without prior modification of the glycosylation pattern of the peptide.
Alternatively, the methods of the invention can be used to sialylate a peptide that does not
include a suitable acceptor; one first modifies the peptide to include an acceptor by methods
known to those of skill in the art. In an exemplary embodiment, a GalNAc residue is added
by the action of a GalNAc transferase.
In an exemplary embodiment, the galactosyl acceptor is assembled by attaching a
galactose residue to an appropriate acceptor linked to the peptide, e.g., a GlcNAc. The
method includes incubating the peptide to be modified with a reaction mixture that contains .¦
suitable amount of a galactosyltransferase {e.g., galpl,3 or gaipi,4), and a suitable galactosyl
donor {e.g., UDP-galactose). The reaction is allowed to proceed substantially to completion
or, alternatively, the reaction is terminated when a preselected amount of the galactose
residue is added. Other methods of assembling a selected saccharide acceptor will be
apparent to those of skill in the art.
In yet another embodiment, peptide-linked oligosaccharides are first "trimmed,"
either in whole or in part, to expose either an acceptor for the sialyltransferase or a moiety to
which one or more appropriate residues can be added to obtain a suitable acceptor. Enzymes
such ai; glycosyltransferases and endoglycosidases {see, for example U.S. Patent No.
5,716,812) are useful for the attaching and trimming reactions. A detailed discussion of
"trimming" and remodeling N-linked and O-linked glycans is provided elsewhere herein.
In the discussion that follows, the method of the invention is exemplified by the use of
modified sugars having a water-soluble polymer attached thereto. The focus of tlie
discussion is for clarity of illustration. Those of skill will appreciate that the discussion is
equally relevant to those embodiments in which the modified sugar bears a therapeutic
moiety, biomolecule or the like.
An exemplary embodiment of the invention in which a carbohydrate residue is
"trimmed" prior to the addition of the modified sugar is set forth in Figure 14, which sets
forth a scheme in which high mannose is trimmed back to the first generation biantennary
structure. A modified sugar bearing a water-soluble polymer is conjugated to one or more of
the sugar residues exposed by the "triirmaing back." In one example, a water-soluble polymer
is added via a GlcNAc moiety conjugated to the water-soluble polymer. The modified
GlcNAc is attached to one or both of the terminal mannose residues of the biantennary
structure. Alternatively, an unmodified GlcNAc can be added to one or both of the termini of
the branched species.
In anotlier exemplary embodiment, a water-soluble polymer is added to one or botli of
the terminal mannose residues of the biantennary structure via a modified sugar having a
galactose residue, which is conjugated to a GlcNAc residue added onto the terminal mannose
residues. Alternatively, an unmodified Gal can be added to one or both terminal GlcNAc
residues.
In yet a further example, a water-soluble polymer is added onto a Gal residue using a
modified sialic acid.
Another exemplairy embodiment is set forth in Figtire 15, which displays a scheme
similar to that shown in Figure 14, in which the high mannose structure is "trimmed back" Us
the mannose fi-om which the biantennary structtire branches. In one example, a water-soluble
polymer is added via a GlcNAc modified with the polymer. Alternatively, an urmiodified
GlcNAc is added to the mannose, followed by a Gal with an attached water-soluble polymci
In yet another embodiment, unmodified GlcNAc and Gal residues are sequentially added to
the mannose, followed by a sialic acid moiety modified with a water-soluble polymer.
Figure 16 sets forth a further exemplary embodiment using a scheme similar to that
shown in Figure 14, in which high mannose is "trimmed back" to the GlcNAc to which the
first mannose is attached. The GlcNAc is conjugated to a Gal residue bearing a water-solul'
polymer. Alternatively, an unmodified Gal is added to the GlcNAc, followed by the addition
of a sialic acid modified with a water-soluble sugar. In yet a further example, the terminal
GlcNAc is conjugated with Gal and the GlcNAc is subsequently flicosylated with a modified
fucose bearing a water-soluble polymer.
Figure 17 is a scheme similar to that shown in Figure 14, in which high mannose is
trimmed back to the first GlcNAc attached to the Asn of the peptide. In one example, the
GlcNAc of the GlcNAc-(Fuc)a residue is conjugated with a GlcNAc bearing a water soluble
polymer. In another example, the GlcNAc of the GlcNAc-(Fuc)a residue is modified with
Gal, wliich bears a water soluble polymer. In a still further embodiment, the GlcNAc is
modified with Gal, followed by conjugation to the Gal of a sialic acid modified with a water-
soluble polymer.
Other exemplary embodiments are set forth in Figures 18-22. An illustration of the
array of reaction types wii±i which the present invention may be practiced is provided in each
of the aforementioned figures.
The Examples set forth above provide an illustration of the power of the methods set
forth herein. Using the methods of the invention, it is possible to "trim back" and build up a
carbohydrate residue of substantially any desired structure. The modified sugar can be added
to the termini of the carbohydrate moiety as set forth above, or it can be intermediate between
the peptide core and the terminus of the carbohydrate.
In an exemplary embodiment, an existing sialic acid is removed from a glycopeptide
using a sialidase, thereby unmasking all or most of the underlying galactosyl residues.
Alternatively, a peptide or glycopeptide is labeled with galactose residues, or an
oligosaccharide residue that terminates in a galactose unit. Following the exposure of or
addition of the galactose residues, an appropriate sialyltransferase is used to add a modified
sialic acid. The approach is summarized in Scheme 12.
In yet a further approach, summarized in Scheme 13, a masked reactive functionaii¦>
is present on the siahc acid. The masked reactive group is preferably unaffected by the
conditions used to attach the modified sialic acid to the peptide. After the covalent
attachment of the modified sialic acid to the peptide, the mask is removed and the peptide is
conjugated with an agent such as PEG, PPG, a therapeutic moiety, biomolecule or other
agent. The agent is conjugated to the peptide in a specific manner by its reaction with th.
urunasked reactive group on the modified sugar residue.
Any modified sagar can be used with its appropriate glycosyltransferase, depending
on the tenninal sugars of the oligosaccharide side chains of the glycopeptide (Table 4), As
discussed above, the teiTninal sugar of the glycopeptide required for introduction of the
PEGylated or PPGylated structure can be introduced naturally during expression or it can be
produced post expression using the appropriate glycosidase(s), glycosyltransferase(s) or mix
of ,glycosidase(s) and g]ycosyltransferase(s).
In a further exemplary embodiment, UDP-galactose-PEG is reacted with bovine mil]
P1,4-gaiactosyItransferase, thereby transferring the modified galactose to the appropriate
terminal N-acetylgiucosamine structirre. The terminal GlcNAc residues on the giycopeptide
may be produced during expression, as may occur in such expression systems as mammalia,
insect, plant or fungus, but also can be produced by treating the giycopeptide with a sialidast
and/or glycosidase and'or glycosyltransferase. as required.
In another exemplary embodiment, a GlcNAc transferase, such as GnT-I-IV, is
utilized to transfer PEGylated-GicNc to a mannose residue on a glycopeptide. In a still
further exemplary embodiment, the N- and/or 0-linked glycan structures are enzymatically
removed from a glycopeptide to expose an amino acid or a terminal glycosyl residue that is
subsequently conjugated with the modified sugar. For example, an endoglycanase is used to
remove the N-linked structures of a glycopeptide to expose a terminal GlcNAc as a GlcNAc-
linked-Asn on the glycopeptide. UDP-Gal-PEG and the appropriate galactosyltransferase is
used to introduce the PECr- or PPG-galactose functionality onto the exposed GlcNAc.
In an alternative embodiment, the modified sugar is added directly to the peptide
backbone using a glycosyltransferase known to transfer sugar residues to the peptide
backbone. This exemplaiy embodiment is set forth in Scheme 14. Exemplary
glycosyltransferases useful in practicing the present invention include, but are not limited to,
GalNAc transferases (GalNAc Tl-14), GlcNAc transferases, fiicosyltransferases.
glucosyltransferases, xylosyltransferases, mannosyltransferases and the like. Use of this
approach allows the direct addition of modified sugars onto peptides that lack any
carbohydrates or, alternatively, onto existing glycopeptides. In both cases, the addition of M;
modified sugar occurs at specific positions on the peptide backbone as defined by the
substrate specificity of the glycosyltransferase and not in a random manner as occurs durinp
modification of a protein's peptide backbone using chemical methods. An array of agents
can be introduced into proteins or glycopeptides that lack the glycosyltransferase substrate
peptide sequence by engineering the appropriate amino acid sequence into the peptide cha;
Scheme 14
In each of the exemplary embodiments set forth above, one or more additional
chemical or enzymatic modification steps can be utilized following the conjugation of tlu
modified sugar to the peptide. In an exemplary embodiment, an enzyme (e.g.,
fticosyltransferase) is used to append a glycosyl unit (e.g., fiicose) onto the terminal modified
sugar attached to the peptide. In another example, an enzj^natic reaction is utilized to "cap'
sites to which the modified sugar failed to conjugate. Alternatively, a chemical reaction is
utilized to alter the structure of the conjugated modified sugar. For example, the conjugated
modified sugar is reacted with agents that stabilize or destabilize its linkage with the peptide
component to which the modified sugar is attached. In another example, a component of the
modified sugar is deprotected following its conjugation to the peptide. One of skill will
appreciate that there is an array of enzymatic and chemical procedures that are useful in the
methods of the invention at a stage after the modified sugar is conjugated to the peptide.
Further elaboration of the modified sugar-peptide conjugate is within the scope of the
invention.
Peptide Tajrgeting With Mannose-6-Phosphate
In an exemplary embodimerit the peptide is derivatized with at least one mannose-6-
phosphate moiety. The m.annose-6-phosphate moiety targets the peptide to a lysosome of .n
ceil, and is useful, for example, to target therapeutic proteins to lysosomes for therapy of
lysosomal storage diseases;.
Lysosomal storage diseases are a group of over 40 disorders which are the result ol
defects in genes encoding enzymes that break down glycoUpid or polysaccharide waste
products within the lysosomes of cells. The enzjonatic products, e.g., sugars and lipids, aiv
then recycled into new products. Each of these disorders results from an inherited autoson .
or X-linked recessive trait which affects the levels of enzymes in the lysosome. Generall>
there is no biological or fimctional activity of the affected enzymes in the cells and tissues
affected individuals. Table 5 provides a list of representative storage diseases and the
enzymatic defect associated with the diseases. In such diseases the deficiency in enzyme
function creates a progressive systemic deposition of lipid or carbohydrate substrate in
lysosomes in cells in the body, eventually causing loss of organ fimction and death. The
genetic etiology, clinical m.anifestations, molecular biology and possibility of the lysoson
storage diseases are detailed in Scriver e{ ah, eds., The Metabolic and Molecul.a.r Ra.
OF Inherited Disease, T.sup.th Ed., Vol. II, McGraw Hill, (1995).
Table 5. Lysosomal storage diseases and associated enzjanatic defects
De Duve first suggested that replacement of the missing lysosomal enzyme with
exogenous biologically active enzyme might be a viable approach to treatment of lysosomal
storage diseases (De Duve, Fed. Proc. 23: 1045 (1964). Since that time, various studies ha-,
suggested that enzyme replacement therapy may be beneficial for treating various lysosoa
storage diseases. The best success has been shovra with individuals with type I Gaucher
disease, who have been treated with exogenous enzyme (P-glucocerebrosidase), prepared
from placenta (Ceredase"^'"*) or, more recently, recombinantly (Cerezyme'^*^. It has been
suggested that enzyme replacement may also be beneficial for treating Fabry's disease, as
well as other lysosomal storage diseases. See, for example, Dawson etai, Fed. Res. 7(8):
684-690 (1973) {in vitro) and Mapes et al, Science 169: 987 (1970) {in vivo). Clinical tri..
of enzyme replacement therapy have been reported for Fabry patients using inflisions of
normal plasma (Mapes et ai, Science 169: 987-989 (1970)), a-galactosidase A purified fn
placenta (Brady et al, N. Eng. J. Med. 279: 1163 (1973)); or a-galactosidase A purified Ir
spleen or plasma (Desnick et al, Proc. Natl Acad. Sci., USA 76: 5326-5330 (1979)) and h
demonstrated the 'biochemical effectiveness of direct enzyme replacement for Fabry disea
Tliese studies indicate the potential for eliminating, or significantly reducing, the pathological
glycolipid storage by repeated enzyme replacement. For example, in one study (Desnick et
al, supra), intravenous injection of purified enzyme resulted in a transient reduction in the
plasma levels of the stored lipid substrate, globotriasylceramide.
Accordingly, there exists a need in the art for methods for providing sufficient
quantities of biologically active lysosomal enzymes, such as human a-galactosidase A, to
deficient cells. Recently, recombinant approaches have attempted to address these needs, see,
e.g., U.S. Pat. No. 5,658,567; 5,580,757; Bishop et al.. Proc. Natl. Acad. ScL. USA. 83: 4859-
4863 (1986); Medine/a/., Proc. Natl Acad. Sci.. USA. 93: 7917-7922 (1996); Novo, F. J.,,
Gene Therapy. 4: 488-492 (1997); Ohshima et al, Proc. Natl Acad Sci., USA. 94: 2540-
2544 (1997); and Sugimoto et al, Human Gene Therapy 6: 905-915, (1995). Through the
mannose-6-phosphate mediated targeting of therapeutic peptides to lysosomes, the present
invention provides compositions and methods for delivering sufficient quantities of
biologically active lysosomal peptides to deficient cells.
Thus, in an exemplary embodiment, the present invention provides a peptide
according to Table 7 thai, is derivatized with marmose-6-phosphate (Figure 24 and Figure 1
The peptide may be recombinantly or chemically prepared. Moreover, the peptide can be thi.
fiill, natural sequence, or it may be modified by, for example, truncation, extension, or it mr -
include substitutions or deletions. Exemplary proteins that are remodeled using a method r
the present invention include glucocerebrosidase, P-glucosidase, a-galactosidase .A, acid-a-
glucosidase (acid maltase). Representative modified peptides that are in clinical use inclu.
but are not limited to, Ceredase'^'^, Cerezyme'^'^, and Fabryzyme™. A glycosyl group on
modified and clinically relevant peptides may also be altered utilizing a method of the
invention. The maimose-6-phosphate is attached to the peptide via a glycosyl linking grot;
In an exemplary embodiment, the glycosyl linking group is derived from sialic acid.
Exemplary sialic acid-denved glycosyl linking groups are set forth in Table 3, in which oj
or more of the "R" moieties is mannose-6-phosphate or a spacer group having one or more
mannose-6-phosphate moieties attached thereto. The modified sialic acid moiety is
preferably the terminal residue of an oligosaccharide linked to the surface of the peptide
(Figure 26)
In addition to the mannose-6-phosphate, the peptides of the invention may be further
derivatized with a moiety sucii as a water-soluble polymer, a therapeutic moiety, or an
additional targeting moiety. Methods for attaching these and other groups are set forth
herein. In an exemplary embodiment, the group other than mannose-6-phosphate is attached
to the peptide via a derivatized sialic acid derivative according to Table 3, in which one or
more of the "R" moieties is a group other than mannose-6-phosphate.
In an exemplary embodiment, a sialic acid moiety modified with a Cbz-protected
glycine-based linker arm is prepared. The coiresponding nucleotide sugar is prepared and the
Cbz group is removed by catalytic hydrogenation. The resulting nucleotide sugar has an
available, reactive amine tliat is contacted with an activated mannose-6-phosphate derivative,
providing a mannose-6-phosphate derivatized nucleotide sugar that is usefiil in practicing the
methods of the invention.
As shovvTi in the scheme below (scheme 15), an exemplary activated mannose-6-
phosphate derivative is formed by converting a 2-bromo-benzyl-protected phosphotriester
into the corresponding tiiflate, in situ, and reacting the triflate with a linker having a reactive
oxygen-containing moievy, forming an ether linkage between the sugar and the linker. The
benzyl protecting groups are removed by catalytic hydrogenation, and the methyl ester of the
linker is hydrolyzed, providing tlie corresponding carboxylic acid. The carboxylic acid is
activated by any method .known in the art. An exemplary activation procedure relies upon i ¦
conversion of the carboxylic acid to the N-hydroxysuccinimide ester.
[n another exemplary embodiment, as shown in tlie scheme below (scheme
16), a N-acetylated siahc acid is converted to an aniine by maiiipulation of the p} ruvyl
moiety. Thus, the primary hydroxyl is converted to a sulfonate ester and reacted with sodn.
azide. l"he azide is catalytically reduced to the corresponding amine. The sugar is
subsequently converted to its nucleotide analogue and coupled, through the amine group, to
the linker arm-denvatized mannose-6-phosphate prepared as discussed above.
Peptides useful to treat lysosomal storage disease can be derivatized with other
targetuig moieties including, but not limited to, transferrin (to deliver the peptide across the
blood-brain barrier, and to endosomes), carnitine (to deliver the peptide to muscle ceils), ant
phosphonates, e.g, bisphosphonate (to target the peptide to bone and other calciferous
tissues). The targeting moiety and therapeutic peptide are conjugated by any method
discussed herein or other\vise known in the art.
In an exemplary embodiment, the targeting agent and the therapeutic peptide are
coupled via a linker moiet}'. In this embodiment, at least one of the therapeutic peptide or tin?
targeting agent is coupled to the linker moiety via an intact glycosyl linking group according
to a method of the invention. In an exemplary embodiment, the linker moiet> includes a
poly(ether) such as poly(et]iylene glycol). In another exemplary embodiment, the linker
moiety includes at least one bond that is degraded in vivo, releasing the therapeutic peptide
from the targeting agent, following delivery of the conjugate to the targeted tissue or region
of the body.
In yet another exemplary embodiment, the in vivo distribution of the tlierapeutic
moiety is altered via altering a glycoform on the therapeutic moiety without conjugatmg the
therapeutic peptide to a targeting moiety. For example, the therapeutic peptide can be
shunted away from uptake by the reticuloendothelial system by capping a terminal galactose
moiety of a glycosyl group with sialic acid (or a derivative thereof) (Figures 24 and 27).
Sialylation to cover terminal Gal avoids uptake of the peptide by hepatic asialoglycoprotein
(ASGP) receptors, and may extend the half life of the peptide as compared with peptides
having only complex glycan chains, in tlie absence of sialylation.
II. Peptide/Glycopeptides of the Invention
In one embodiment, the present invention provides a composition comprising multij
copies of a single peptide having an elemental trimannosyl core as the primary glycan
structure attached there;to. hi preferred embodiments, the peptide may be a therapeutic
molecule. The natural form of the peptide may comprise complex N-linked giycans or may
be a high mannose glycan. The peptide may be a mammalian peptide, and is preferably a
human peptide. In some embodiments the peptide is selected from the group consisting oi
immunoglobulin, erythropoietin, tissue-type activator peptide, and others (See Figure 28).
Exemplary peptides whose giycans can be remodeled using the methods of the
invention are set forth in Figure 28.
Table 6. Preferred peptides for glycan remodeling
Factor VII clotting factor TNfF receptor-IgG Fc fusion (EnbreF^)
Factor IX clotting factor MAb-Her-2 (Herceptin'^'^)
Follicle Stimulating Hormone (FSH) MAb-F protein of Respiratory
Erythropoietin (EPO) Syncytial Virus (Synagis'^'^)
Granulocjie-macrophage colony MAb-CD20 (Rituxan"™)
stimulating factor (GM-CSF) MAb-TlMFa (RemicadeTM)
Interferon y MAb-Glycoprotein Ilb/IIIa (Reopro^'^)
tti protease inhibitor (0:1 antitr>7)sin)
Tissue-type plasminogen activator (TPA)
Glucocerebrosidase (CerezymeTM)_______________________________________________
A more detailed list of peptides useful in the invention and their source is provided in
Figure 28.
Other exemplary peptides that are modified by the methods of the invention include
members of the immunoglobulin family (e.g., antibodies, MHC molecules, T cell receptors,
and the like), intercellular receptors (e.g., integrins, receptors for hormones or growth factors
and the like) lectins, and c>tokines (e.g., interleukins). Additional examples include
tissue-type plasminogen activator (TPA), renin, clotting factors such as Factor VIII and
Factor IX, bombesin, thrombin, hematopoietic growth factor, colony stimulating factors, pe
peptides or they can be mutated peptides, produced by methods known in the art, such as site-
directed mutagenesis. Glycosylation of peptides is typically either Nf-linked or 0-linked. exemplary N-linkage is the attachment of the modified sugar to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-
threonine, where X is any amino acid except proline, are the recognition sequences tor
enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a peptide creates a potential glycosylation
site. As described elsewhere herein, 0-linJced glycosylation refers to the attachment of one
sugar {e.g., N-acetylgalactosamine, galactose, mannose, GIcNAc, glucose, flicose or xylose)
to a hydroxy side chain of a hydroxyamino acid, preferably serine or threonine, although 5-
hydroxyproline or 5-hydroxylysine may also be used.
Several exemplary embodiments of the invention are discussed below. While several
of these embodiments use peptides having names having trademarks, and other specific
peptides as the exemplary peptide, these examples are not confined to any specific peptide.
The following exemplary embodiments are contemplated to include all peptide equivalents
and variants of any peptide. Such variants include, but are not limited to, addmg and deleting
N-linked and O-linked glycosylation sites, and ftjsion proteins witli added glycosylation sites.
One of skill in the art will appreciate that the following embodiments and the basic methods
disclosed therein can be applied to many peptides with equal success.
In one exemplary embodiment, the present invention provides methods for modifying
Granulocyte Colony Stimulating Factor (G-CSF). Figures 29A to 29G set forth some
examples of how this is accomplished using tlie methodology disclosed herein. In Figure
29B, a G-CSF peptide that is expressed in a mammalian cell system is trimmed back using a
sialidase. The residues thus exposed are modified by the addition of a sialic acid-
poly(ethyIene glycol) moiety (PEG moiety), using an appropriate donor therefor and
ST3Gall. Figure 29C sets fortli an exemplary scheme for modifying a G-CSF peptide that is
expressed in an insect cell. The peptide is modified by adding a galactose moiety using an
appropriate donor thereof cind a galactosyltransferase. The galactose residues are
functionalized with PEG vi a a sialic acid-PEG derivative, througli the action of ST3Cia[ 1. In
Figure 29D, bacterial!}' expressed G-CSF is contacted with an N-acetylgalactosamine donor
and N-acetylgalactoscunine transferase. The peptide is fimctionaJized with PEG, using a
PEGylated sialic acid donor and a sialyltransferasc. In Figure 29E, mammalian cell
expressed G-CSF is contacled with a sialic acid donor that is modified with levulinic acid,
adding a reactive ketone to the sialic acid donor. After addition \o a glycosyl residue on the
glycan on the peptide, the ketone is derivatized with a moiety such as a hydrazine- or amine-
PEG. In Figure 29F, bacterially expressed G-CSF is remodeled by contactmg the peptide
with an endo-GalNAc enzyme under conditions where it fimctions in a synthetic, rather than
ahydroiytic manner, thereby adding a PEG-Gal-GalNAc molecule from an activated
derivative thereof. Figuj-e 29G provides another route for remodeling bacterially expressed
G-CSF. The polypeptide is derivatized with a PEGylated N-acetylgalactosamine residue b>'
contacting the polypeptide with an N-acetylgalactosamine transferase and an appropriate
donor of PEGylated N-acetylgalactosamine.
In anotlier exemplary embodunent, the invention provides methods for modifying
Interferon a-14C (IFNal4C), as shown in Figures 30A to SON. The various forms of IFNa
are disclosed elsewhere herein. In Figure SOB, IFNaMC expressed in mammalian cells is
first treated with sialidase to trim back the sialic acid units thereon, and then the molecule is
PEGjIated using ST3Gal3 and a PEGylated sialic acid donor. In Figure 30C, N-
acetylglucosamine is frrst added to IFNal4C which has been expressed m insect or fungal
cells, where the reaction is conducted via the action of GnT-I and/or II using an N-
acetylglucosamine donor. The polypeptide is then PEGylated using a galactosyltransferase
and a donor of PEG-galactose. In Figure SOD, IFNal4C expressed in yeast is first treated
with Endo-H to trim back the glycosyl units thereon. The molecules is galactosylated using a
galactosyltransferase and a galactose donor, and it is then PEGylated using STSGalS and a
donor of PEG-sialic acid. In Figure 3OF, IFNal4C produced by mammalian cells is modified
to inched a PEG moiety using STSGalS and a donor of PEG-sialic acid. In Figure 30G,
IFNal4C expressed in insect of fungal cells first has N-acetyiglucosamine added using one or
more of GnT-I, II, IV, and V, and an N-acetylgiucosamine donor. The protein is
subsequently galactosylated using an appropriate donor and a galactosyltransferase. Then,
IFNaUC is PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 30H, yeast
produced IFNaHC is first treated with mannosidases to trim back the mamiosyl groups. N-
acetylgJucosamine is then added using a donor of N-acetylglucosamine and one or more of
GriT-I, II, IV, and V. IFNal 4C is fiirther galactosylated using an appropriate donor and a
galactoj-yltransfcrase. Then, the polypeptide is PEGylated using ST3GaI3 and a donor of
PEG-sia!ic acid. In Figure 301, NSO cell expressed IFNaI4C is modified by capping
appropriate tcrniinai residues with a sialic acid donor that is modified with levulinic acid,
thereby adding a reactive ketone to the sialic acid donor. After addition to a glycosyl residue
of the peptide, the ketone is derivalized with a moiety such as a hydrazine- or amine- PEG.
In Figure 30J, IFNaHC expressed by mammalian cells is PEGylated using a donor of PEG-
sialic acid and a 2,8-sialyltransferase. In Figure 30K, IFNaHC produced by mammalian
cells is first treated with siialidase to trim back the terminal sialic acid residues, and then the
molecule is PEGylated using trans-sialidase and PEGylated sialic acid-lactose complex. In
Figure SOL, IFNal4C expressed in a mammalian system is sialylated using a donor of sialic
acid and a 2,8-siaIyltransferase. In Figure 30M, EFNaHC expressed in insect or fungal cells
first has N-acetylglucosamine added using an appropriate donor and GnT-I and/or II. The
molecule is then contacted with a galactosyltransferase and a galactose donor that is
derivatized with a reactive sialic acid via a linker, so that the polypeptide is attached to the
reactive sialic acid via the linker and the galactose residue. The polypeptide is then contacted
with STBGalB and transfeain, and thus becomes cormected with transferrin via the sialic acid
residue. In Figure SON, IFNaHC expressed in either insect or fungal cells is first treated
with endoglycanase to trim back the glycosyl groups, and is then contacted with a
galactosyltransferase and a galactose donor that is derivatized witli a reactive sialic acid via a
linker, so that the polypeptide is attached to the reactive sialic acid via the linker and the
galacto.se residue. The molecule is then contacted with STSGalS and transferrin, and thus
becomes cormected with transferrin via the sialic acid residue.
In another exemplar}' embodiment, the invention provides methods for modifying
Interferon a-2a or 2b (IFNa), as shown in Figures 30O to SOEE. In Figure BOP, IFNa
produced in mammalian cells is first treated with sialidase to trim back the glycosyl units, and
is then PEGylated using STSGalS and a PEGylated sialic acid donor. In Figure 30Q, IFNa
expressed in insect cells is first galactosylated using an appropriate donor and a
galactosyltransferase, and is then PEGylated using STSGall and a PEGylated sialic acid
donor. Figure 30R offers another method for remodeling IFNa expressed in bacteria:
TEGylaicd N-acetylgalactosamine is added to the protein using an appropriate donor andN-
acetylgalactosamine transferase. In Figure SOS, IFNa expressed in mammalian cells is
modified by capping appropriate terminal residues with a sialic acid donor that is modified
Willi leviilinic acid, adding a reactive ketone to the sialic acid donor. After addition lo a
glycosyl residue of the peptide, the ketone is derivatized with a moiety such as a hydrazine-
or amine- PEG. In Figure 301, IFNa expressed in bacteria is PEGylated using a modified
enzyme Endo-N-acetylgaiactosamidase, which functions in a synthetic instead of a hydrolytic
maimer, and using a N-acetylgalactosamine donor derivatized with a PEG moiety. In Figure
30U, N-acetylgalactosajnine is first added IFNa using an appropriate donor and N-
acetylgalactosaraine transferase, and then is PEGylated usmg a sialyltransferase and a
PEGylated sialic acid donor. In Figure 30V, IFNa expressed in a mammalian system is first
treated with sialidase to trim back the sialic acid residues, and is then PEGylated using a
suitable donor and STBGall and/or ST3Gal3. In Figure 30W, IFNa expressed in mammalian
cells is first treated with sialidase to trim back the sialic acid residues. The polypeptide is
then contacted with ST3(jalI and two reactive sialic acid residues that are connect via a
linker, so that the polypeptide is attached to one reactive sialic acid via the linker and the
second sialic acid residue The polypeptide is subsequently contacted with ST3GaI3 and
transferrin, and thus becomes connected with transferrin via the sialic acid residue. In Figure
30Y, IFNa expressed in mammalian cells is first treated with sialidase to trim back the sialic
acid residues, and is then PEGylated using ST3Gall and a donor of PEG-sialic acid. In
Figure 30Z, IFNa produced by insect cells is PEGylated using a galactosyltransferase and a
donor of PEGylated galactose. In Figure 30AA, bacterially expressed IFNa fu^st has N-
acetylgalactosamine added using a suitable donor and N-acetylgalactosamine transferase.
The protein is then PEGylated using a sialyltransferase and a donor of PEG-sialic acid. In
Figure 30CC, IFNa expressed in bacteria is modified in another procedure: PEGylated N-
acetylgalactosamine is added to the protein by N-acetylgalactosamine transferase usmg a
donor of PEGylated N-acetylgalactosamine. In Figure 30DD, IFNa expressed in bacteria is
remodeled in yet another scheme. The polypeptide is first contacted with N-
acetyigalactosamine transferase and a donor of N-acetylgalactosamine that is derivatized with
a reactive sialic acid via a linker, so that IFNa is attached to the reactive sialic acid via the
linker and the N-acetylgalactosamine. IFNa is then contacted with ST3Gal3 and asialo-
transferrin so that it becomes connected with transferrin via the sialic acid residue. Then,
IFNa is capped with sialic acid residues using ST3Gal3 and a sialic acid donor. An
additional method for modifying bacterially expressed IFNa is disclosed in Figure 30EE,
where IFNa is fust exposed to NHS-CO-linker-SA-CMP and is then coimected to a reactive
sialic acid via the linker. It is subsequently conjugated with transferrin using ST3Gal3 and
trans ferrm.
The methods for remodeling INN omega are essentially identical to those presented
here for IFN alpha except that the attachment of the glycan to the IFN omega peptide occurs
at annno acid residue lOl in SEQ ID NO:75. The nucleotide and amino acid sequences for
IFN omega are presented herein as SEQ ID NOS:74 and 75. Methods of making and using
IFN omega are found in U.S. Patent No. 4,917,887 and 5,317,089, and in EP Patent No.
0170204-A.
In another exemplary embodiment, the invention provides methods for modifying
Interferon j3 (IFN-p), as shown in Figures 31A to 31S. In Figure 3IB, IFN-p expressed in a
mammalian system is first, treated with sialidase to trun back the terminal sialic acid residues.
The protein is then PEGylated using ST3Gal3 and a donor of PEGylated sialic acid. Figure
3IC is a scheme for modifying IFN-P produced by insect cells. First, N-acetylglucosamine is
added to IFN-p using an a]3propriate donor and GnT-I and/or -11. The protein is then
galactosylated using a galactose donor and a galactosyltransferase. Finally, IFN-p is
PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 3 ID, IFN-P expressed
in yeast is first treated with Endo-H to trim back its glycosyl chains, and is then
galactosylated using a galactose donor and a galactosyltransferase, and is then PEGylated
using ST3Gal3 and a donor of PEGylated sialic acid. In Figure 3 IE, IFN-P produced by
mammalian cells is modified by PEGylation using ST3Gal3 and a donor of sialic acid already
derivatized with a PEG moiety. In Figure 3 IF, IFN-p expressed in insect cells fu-st has N-
acetylglucosamine added by one or more of GnT-I, II, IV, and V using a N-
acetylgiucosamine donor, ajid then is galactosylated using a galactose donor and a
galactosyltransferase, and is tlien PEGylated using ST3Gal3 and a donor of PEG-sialic acid.
In Figuie 31G, IFN-p expressed in yeast is first treated with mannosidases to trim back the
mannosyl units, then has N-acetylglucosamine added using a N-acetylgiucosamine donor and
one or more of GnT-I, II, IV. and V. The protein is fiuther galactosylated using a galactose
donor and a galactosyltransfcrase, and then PEGylated using ST3Gal3 and a PEG-sialic acid
donor. In Figure 31H, mammalian cell expressed IFN-P is modified by capping appropriate
terminal residues with a sialic acid donor tliat is modified with levoilinic acid, adding a
reactive ketone to the sialic acid donor. After addition to a glycosyl residue of the peptide,
the ketone is derivatized with a moiety such as a hydrazine- or amine- PEG. In Figure 311,
IFN-P expressed in a mammalian system is PEGylated using a donor of PEG-sialic acid ajid u
2,8-sialyltransferase. In Figure 3IJ, IFN-p expressed by mammalian cells is first treated with
sialidase to trim back its termiiial sialic acid residues, and then PEGylated using trans-
sialidase and a donor of PEGylated sialic acid. In Figiure 3 IK, IFN-P expressed in
mammalian cells is first treated with sialidase to trim back terminal sialic acid residues, then
PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and then sialylated using ST3Gal3
and a sialic acid donor. In Figure 3IL, IFN-P expressed in mammalian cells is first treated
with sialidase and galactosidase to trim back the glycosyl chaias, then galactosylated using a
galactose donor and an a-galactosyltransferase, and then PEGylated using ST3Gal3 or a
sialyltransferase and a donor of PEG-sialic acid. In Figure 31M, IFN-P expressed in
mammalian cells is first treated with sialidase to trim back the glycosyl units. It is then
PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and is then sialylated using
ST3Gal3 and a sialic acid donor. In Figure 3 IN, IFN-P expressed in mammalian cells is
modified by capping apj^ropriate terminal residues with a sialic acid donor that is modified
with levulinic acid, adding a reactive ketone to the sialic acid donor. After addition to a
glycosyl residue of the peptide, the ketone is derivatized with a moiety such as a hydrazine-
or amine- PEG. In Figu)-e 310, EFN-P expressed in mammalian cells is sialylated using a
sialic acid donor and a 2,8-sialyltransferase. In Figure 31Q, IFN-P produced by insect cells
first has N-acetylglucosamine added using a donor of N-acetylglucosamine and one or more
of GnT-I, II, IV, and V, cind is further PEGylated using a donor of PEG-galactose and a
galactosyltransferase. In Figure 3IR, IFN-P expressed in yeast is first treated with
endoglycanase to trim back the glycosyl groups, then galactosylated using a galactose donor
and a galactosyltransferase, and then PEGylated using ST3Gal3 and a donor of PEG-sialic
acid. In Figure 31S, IFN-P expressed in a mammalian system is first contacted with ST3Gal3
and two reactive sialic acid residues connected via a linker, so that the polypeptide is attached
to one reactive sialic acid via the linker and the second sialic acid residue. The polypeptide is
then contacted with ST3Gral3 and desialylated transferrin, and thus becomes connected with
transferrin via the sialic acid residue. Then, IFN-P is further sialylated using a sialic acid
donor and ST3Gal3.
In anodier exempliiry embodiment, the invention provides methods for modifying
Factor VII or Vila, as shown in Figures 32 A to 32D. In Figure 32B, Factor VII or Vila
produced by a manimalian system is first treated with sialidase to trim back the terminal
sialic acid residues, and dien PEGylated using ST3Gal3 and a donor of PEGylated sialic acid.
Figure 32C, Factor VII or Vila expressed by mammalian cells is first treated with sialidase to
trim back the terminal sialic acid residues, and then PEGylated using ST3Gal3 and a donor of
PEGylated sialic acid. Further, the polypeptide is sialylated with ST3Gal3 and a sialic acid
donor. Figure 32D offers another modification scheme for Factor VII or Vila produced by
manmialian cells: the polypeptide is first treated with sialidase and galactosidase to trim back
its sialic acid and galactose residues, then galactosylated using a galactosyltransferase and a
galactose donor, and then PEGylated using ST3Gal3 and a donor of PEGylated sialic acid.
In another exemplary embodiment, the invention provides methods for modifying
Factor DC, some examples of which are included in Figvu^es 33 A to 33G. In Figure 33B,
Factor IX produced by mammalian cells is first treated with sialidase to trim back the
terminal sialic acid residues, and is then PEGylated with ST3Gal3 using a PEG-sialic acid
donor. In Figure 33C, Factor IX expressed by mammalian cells is first treated with sialidase
to trim back the terminal sialic acid residues, it is then PEGylated using ST3Gal3 and a PEG-
sialic acid donor, and fiirther sialylated using ST3Gall and a sialic acid donor. Another
scheme for remodeling mammalian cell produced Factor IX can be found in Figure 33D. The
polypeptide is first treated with sialidase to trim back tlie terminal sialic acid residues, then
galactosylated using a galactose donor and a galactosyltransferase, fiirther sialylated using a
sialic acid donor and ST3Gal3, and then PEGylated using a donor of PEGylated sialic acid
and ST3Gall. In Figure 33E, Factor IX that is expressed in a mammalian system is
PEGylated tlirough the process of sialylation catalyzed by ST3Gal3 using a donor of PEG-
sialic acid. In Figure 33F, Factor IX expressed in mammalian cells is modified by capping
appropriate terminal residues with a sialic acid donor tliat is modified with levulinic acid,
adding a reactive ketone ta the sialic acid donor. After addition to a glycosyl residue of the
peptide, tlic ketone is derivatized with a moiety such as a hydrazine- or amine- PEG. Figure
33G provides an additional method of modifying Factor IX. The polypeptide, produced by
mammalian cells, is PEGylated using a donor of PEG-sialic acid and a 2,8-sialyltransferase.
In another exemplar}' embodiment, the invention provides methods for modification
of Follicle Stimulating Homione (FSH). Figures 34A to 34J present some examples. In
Figure 34B, FSH is expreiised in a mammalian system ;md modified by treatment of sialidaso
to trim back terminal sialic acid residues, followed by PEGylation using ST3Gal3 and a
donor of PEG-sialic acipeptide is attached
to a reactive sialic acid via the linker and a second sialic acid residue. The polypeptide is
then contacted with ST3Gall and desialylated chorionic gonadotrophin (CG) produced in
CHO, and thus becomes connected with CG via the second sialic acid residue. Then, FSH is
sialylated using a sialic acid donor and ST3Gal3 and/or ST3Gall.
In another exemplary embodiment, the in\ention provides methods for modifying
ervthropoietin (EPO), Figures 35A to 35AA set forth some examples which are relevant to
the remodeling of both wild-type and mutant EPO peptides. In Figure 35B, EPO expressed
in various mammalian systems is remodeled by contacting the expressed protein with a
siaUdase to remove terminal sialic acid residues. The resulting peptide is contacted with a
sialyltransferase and a CMP-sialic acid that is derivatized with a PEG moiety. In Figure 35C,
EPO that is expressed in insect cells is remodeled with N-acetylglucosamine, using GnT-1
and/or GnT-II. Galactose is then added to the peptide, using galactosyltransferase. PEG
group is added to the remodeled peptide by contacting it with a sialyltransferase and a CMP-
sialic acid that is derivati2;ed with a PEG moiety. In Figure 35D, EPO that is expressed in a
mammalian cell system is remodeled by removing terminal sialic acid moieties via the action
of a sialidase. The terminal galactose residues of the N-linked glycosyl units are "capped"
with sialic acid, using ST3Gal3 and a sialic acid donor. The terminal galactose residues on
the 0-Iinked glycan are fimctionalized with a sialic acid bearing a PEG moiety, using an
appropriate sialic acid donor and ST3Gall, In Figure 35E, EPO that is expressed in a
mammalian cell system is remodeled by flmctionalizing the N-linked glycosyl residues with a
PEG-derivatized sialic acid moiety. The peptide is contacted with ST3Gal3 and an
appropriately modified sialic acid donor. In Figure 35F, EPO that is expressed in an insect
cell system, yeast or fungi, is remodeled by adding at least one N-acetylglucosamine residues
by contacting the peptide with a N-acetylglucosamine donor and one or more of GnT-I, GnT-
II, and GnT-V. The peptide is then PEGylated by contacting it with a PEGylated galactose
donor and a galactosyltransferase. In Figure 35G, EPO that is expressed in an insect cell
system, yeast or fungi, is remodeled by the addition of at least one N-acetylglucosamine
residues, using an appropriate N-acetylglucosamine donor and one or more of GnT-I, GnT-11,
and GnT-V. A galactosidase that is altered to operate in a synthetic, rather than a hydrolytic
manner is used to add an activated PEGylated galactose donor to the N-acetyiglucosamine
residues. In Figure 35H, EPO that is expressed in an insect cell system, yeast or fungi, is
remodeled by the addition of at least one terminal N-acetylglucosamine-PEG residue. The
peptide is contacted with GnT-I and an appropriate N-acetlyglucosamine donor that is
derivatized with a PEG moiety. In Figure 351, EPO that is expressed in an insect cell system.,
yeast or fungi, is remodeled by adding one or more terminal galactose-PEG residues. The
peptide is contacted witli CrnT-I and an appropriate N-acetylglucosamine donor that is
derivatized with a PEG moiety. The peptide is then contacted with galacto.syjtransferase and
an appropriate galactose donor that is modified with a PEG moiety. In Figure 3 5 J, EPO
expressed in an insect cell system, yeast or fungi, is remodeled by the addition of one more
terminal sialic acid-PEGr residues. The peptide is contacted with an appropriate N-
acetylglucosamine donor and GnT-I. The peptide is fiirther contacted with
galactosyltransferase and an appropriate galactose donor. The peptide is then contacted with
ST3Gal3 and an appropiiate sialic acid donor that is derivatized with a PEG moiety. In
Figure 35K, EPO expressed in an insect cell system, yeast or fimgi, is remodeled by the
addition of terminal sialic acid-PEG residues. The peptide is contacted with an appropriate
N-acetylglucosamine donor and one or more of GnT-I, GnT-II, and GnT-V. The peptide is
then contacted with galactosyltransferase and an appropriate galactose donor. The peptide is
further contacted with STSGaB and an appropriate sialic acid donor that is derivatized with a
PEG moiety. In Figure ;55L, EPO expressed in an insect cell system, yeast or fimgi, is
remodeled by the addition of one or more terminal a2,6-sialic acid-PEG residues. The
peptide is contacted witli an appropriate N-acetylglucosamine donor and one or more of GnT-
I, GnT-II, and GnT-V. irhe peptide is further contacted with galactosyltransferase and an
appropriate galactose donor. The peptide is then contacted with a2,6-sialyltransferase and cm
appropriately modified sialic acid donor. In Figure 35M, EPO expressed in a mammalian cell
system is remodeled by addition of one or more terminal sialic acid-PEG residues. The
peptide is contacted with a sialidase to remove terminal sialic acid residues. The peptide is
fiirther contacted with a sialyltransferase and an appropriate sialic acid donor. The peptide is
further contacted with a sialyltransferase and an appropriate sialic acid donor that is
derivatized with a PEG moiety, hi Figure 35N, EPO expressed in a mammalian cell system
is remodeled by the addilion of one or more terminal sialic acid-PEG residues. The peptide is
contacted with a sialyltransferase and an appropriate sialic acid donor that is derivatized with
a PEG moiety. In Figure 350, EPO expressed in a mammalian cell system is remodeled by
the addition of one or mere terminal a2,8-sialic acid-PEG residues to primarily O-linked
glyc;ms. The peptide is contacted with a2,8-sialyltransferase and an appropriate sialic acid
donor that is derivatized with a PEG moiety. In Figure 35P, EPO expressed in a mammalian
cell is remodeled by the f.ddition of one or more temiinal a2,8-sialic acid-PEG residues to O-
liiiked and N-linked glycans. The peptide is contacted with a2,8-sialyltransferase and an
appropriate sialic acid donor that is derivatized with a PEG moiety. In Figure 35Q, EPO
expressed in yeast or ftingi is remodeled by the addition of one or more terminal siaUc acid-
PEG residues. The peptide is contacted with mannosidases to remove terminal mannose
residues. Next, the peptide is contacted with GnT-I and an appropriate N-acetylglucosamine
donor. The peptide is further contacted with galactosyltransferase and an appropriate
galactose donor. The peptide is then contacted with a sialyltransferase and an appropriate
sialic acid donor that is derivatized with a PEG moiety. In Figure 35R, EPO expressed in
yeast or fungi is remodeled by the addition of at least one terminal N-acetylglucosamine-PEG
residues. The peptide is contacted with marmosidases to remove terminal maimose residue.
The peptide is then contacted with GnT-I and an appropriate N-acetylglucosamine donor that
is derivatized with a PEG moiety. In Figure 35S, EPO expressed in yeast or fungi is
remodeled by the additon of one mor more terminal sialic acid-PEG residues. The peptide is
contacted with mannosidase-I to remove a2 mannose residues. The peptide is further
contacted with GnT-I and an appropriate N-acetylglucosamine donor. The peptide is then
contacted with galactosyltransferase and an appropriate galacose donor. The peptide is tlien
contacted with a sialyltransferase and an appropriate sialic acid donor that is derivatized with
a PEG moiety. In Figure 35U, EPO expressed in yeast or fungi is remodeled by addition of
one or more galactose-PEG residues. The peptide is contacted with endo-H to trim back
glycosyl groups. The p€;ptide is then contacted with galactosyltransferase and an appropriate
galactose donor that is derivatized with a PEG moiety. In Figure 35V, EPO expressed in
yeast or fungi is remodeled by the addition of one or more terminal sialic acid-PEG residues.
Tlie peptide is contacted with endo-H to trim back glycosyl groups. The peptide is fiuther
contacted with galactosyltransferase and an appropriate galactose donor. The peptide is tlieti
contacted with a sialyltrctnsferase and an appropriate sialic acid donor that is derivatized udth
a PEG moiety. In Figure 35W, EPO expressed in an insect cell system is remodeled by tlie
addition of terminal galactose-PEG residues. The peptide is contacted with marmosidases to
remove terminal maimose residues. The peptide is then contacted with galactosyltrajisferasc
and cm appropriate galaciose donor that is derivatized with a PEG moeit}-. In Figure 35Y. a
mutant EPO called "novel erytliropoiesis-stimulating protein" or NESP, expressed in NSu
murine myeloma cells is remodeled by capping appropriate terminal residues with a sialic
acid donor that is modified witli Ie\ailimc acid, adding a reactive ketone t.i tlie sialic acid
donor. After addition to a glycosyl residue of the peptide, the ketone is derivatized with a
moiety such as a hydrazine- or amine-PEG. In Figure 35Z, mutant EPO, i.e. NESP,
expressed in a mammalian cell system is remodeled by addition of one or more terminal
siahc acid-PEG residues. PEG is added to the glycosyl residue on the giycan using a PEG-
modified siahc acid and an a 2,8-siaIyltransferase. In Figure 35AA, NESP expressed in a
mammalian cell system is remodeled by the addition of terminal sialic acid residues. The
sialic acid is added to the glycosyl residue using a sialic acid donor and an a2,8-
sialyltransferase.
In another exemphuy embodiment, the invention provides methods for modifying
granulocyte-macrophage colony-stimulating factor (GM-CSF), as shown in Figures 36A to
36K. In Figure 36B, GM-CSF expressed in mammalian cells is first treated with sialidase to
trim back the sialic acid residues, and then PEGylated using ST3Gal3 and a donor of PEG-
siaHc acid. In Figure 36C, GM-CSF expressed in mammalian ceils is first treated with
sialidase to trim back the sialic acid residues, then PEGylated using ST3GaI3 and a donor of
PEG-sialic acid, and then is further sialylated using a sialic acid donor and ST3Gall and/or
ST3Gal3. In Figure 36D, GM-CSF expressed in NSO cells is first treated with sialidase and
a-galactosidase to trim back the glycosyl groups, then sialylated using a sialic acid donor and
ST3Gal3, and is then PEG3dated using ST3GaIl and a donor of PEG-sialic acid. In Figrire
36E, GM-CSF expressed in mammalian cells is first treated with siahdase to trim back sialic
acid residues, then PEGylaied using ST3Gal3 and a donor of PEG-sialic acid, and then is
further sialylated using ST3Gal3 and a sialic acid donor. In Figure 36F, GM-CSF expressed
in manunalian cells is modified by capping appropriate terminal residues with a sialic acid
donor that is modified with levulinic acid, adding a reactive ketone to the sialic acid donor.
After addition to a glycosyl residue of the peptide, the ketone is derivatized with a moiety
such as a hydrazine- or amine- PEG. In Figure 36G, GM-CSF expressed in mammalian cells
is sialylated using a sialic acid donor and a 2,8-sialyltransferase. In Figure 361, GM-CSF
expressed in insect cells is modified by addition of N-acetylglucosamine using a suitable
donor and one or more of GnT-I, II, IV, and V, followed by addition of PEG)^Iated galactose
using a suitable donor and a galactos3'ltransferase. In Figuie 36J, yeast expressed GM-CSF is
first treated with endoglycar,ase and/or maiuiosidase to trim back the glycosyl luiits. and
subsequently PEGylated using a galactosyltransferase and a donor of PEG-galactosc. In
Figure 36K, GM-CSF expressed in mammalian cells is first treated with sialidase to trim
back sialic acid residues, and is subsequently sialylated using ST3Gal3 and a sialic acid
donor. The polypeptide is then contacted wdth STBGall and two reactive sialic acid residues
connected via a linker, so that the polypeptide is attached to one reactive sialic acid via ttie
linker and second sialic acid residue. The polypeptide is further contacted wdth STSGaB and
transferria, and thus becomes connected with transferrin.
In another exemplary embodiment, the invention provides methods for modification
of Interferon gamma (IFNy). Figures 37 A to 3 7N contain some examples. In Figure 3 7B,
IFNy expressed in a variety of mammalian cells is first treated with sialidase to trim back
terminal sialic acid residues, and is subsequently PEGylated using ST3Gal3 and a donor of
PEG-sialic acid, hi Figure 37C, IFNy expressed in a mammalian system is first treated with
sialidase to trim back terminal sialic acid residues. The polypeptide is then PEGylated using
ST3Gal3 and a donor of PEG-sialic acid, and is fiarther sialylated with ST3Gal3 and a donor
of sialic acid. In Figure 3 7D, mammalian cell expressed IFNy is first treated with sialidase
and a-galactosidase to trim back sialic acid and galactose residues. The polypeptide is then
galactosylated using a galactose donor and a galactosyltransferase. Then, IFNy is PEGylated
using a donor of PEG-sialic acid and ST3Gal3. In Figure 37E, IFNy that is expressed in a
mammalian system is first treated with sialidase to trim back terminal sialic acid residues.
The polypeptide is then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and is
further sialylated with ST3Gal3 and a sialic acid donor. Figure 37F describes another method
for modifying IFNy expressed in a mammalian system. The protein is modified by capping
appropriate tefrninal residues with a sialic acid donor that is modified with levulinic acid,
addmg a reactive ketone to the sialic acid donor. After addition to a giycosyl residue of the
peptide, the ketone is derivatized with a moiety such as a hydrazme- or amine- PEG. In
Figure 37G, IFNy expressed in mammalian cells is remodeled by addition of sialic acid using
a sialic acid donor and an a 2,8-sialyltransferase. In Figure 371, IFNy expressed in insect or
fungal cells is modified by addition of N-acetylglucosainine using an appropriate donor and
one or more of GnT-I, II, I\'', and V. The protein is fiirther modified by addition of PEG
moieties using a donor of PlSGylated galactose and a galactosyltransferase. Figure 37.1 offers
a method for modifying IFNy expressed in yeast. The polypeptide is first treated witli
endoglycanase to trim back the sacchiiride chains, and then galactosylated usuig a galactose
donor and a galactosyltransferase. Then, IFN7 is PEGylated using a donor of PEGylated
sialic acid and ST3Gal3. In Figure 37K, IFNy produced by mammalian cells is modified as
follows: the polypeptide is first contacted with ST3Gal3 and a donor of sialic acid that is
derivatized with a reactive galactose via a linker, so that the polypeptide is attached to the
reactive galactose via the linker and sialic acid residue. The polypeptide is then contacted
with a galactosyltransferaiie and transferrin pre-treated with endoglycanase, and thus becomes
coimected with transferrin via the galactose residue. In the scheme illustrated by Figvire 37L.
IFNy, which is expressed m a mammalian system, is modified via the action of ST3Gal3:
PEG}'lated sialic acid is transferred from a suitable donor to IFNy. Figure 37M is an example
of modifying IFNy expressed in insect or fungal cells, where PEGylation of the polypeptide
is achieved by transferring PEGylated N-acetylglucosamine from a donor to IFNy using GnT-
I and/or II. In Figure 37N, IFNy expressed in a mammalian system is remodeled with
addition of PEGylated sialic acid using a suitable donor and an a 2,8-siaIyltransferase.
In another exempliry embodiment, the invention provides methods for modifying ai
anti-trypsin (a I-protease inhibitor). Some such examples can be found in Figures 38 A to
38N. In Figure 3 SB, ai anti-trypsin expressed in a variety of mammalian cells is first treated
with sialidase to trim back sialic acid residues. PEGylated sialic acid residues are then added
using an appropriate donor, such as CMP-SA-PEG, and a sialyltransferase, such as ST3Gal3.
Figure 38C demonstrates another scheme of ai anti-trypsin modification, ai anti-trypsin
expressed in a mammalia]! system is first treated with sialidase to trim back sialic acid
residues. Sialic acid residues derivatized with PEG are tlien added using an appropriate
donor and a sialyltransferase, such as ST3Gal3. Subsequently, the molecule is further
modified by the addition of sialic acid residues using a sialic acid donor and ST3Gal3.
Optionall)', mammalian cell expressed ai anti-trypsin is first treated with sialidase and a-
galactosidase to trim back temiinal sialic acid and a-linkage galactose residues. The
polyjjeptide is tlien galactosylated using galactosyltransferase and a suitable galactose donor.
Further, sialic acid derivatized with PEG is added by the action of ST3Gal3 using a
PEGylated sialic acid donor. In Figure 38D, ai anti-trypsin expressed in a mammalian
system first has the temiiaal sialic acid residues trimmed back using sialidase. PEG is then
added to N-linked glycosyl residues via the action of ST3Gal3, which mediates tlie transfer ^ ~
PEGylated sialic acid from a donor, such as CMP-SA-PEG, to ai anti-trypsin. More sialic
acid residues are subsequently attached using a sialic acid donor and ST3Gal3. Figure 38E
illustrates another process through which ai anti-trypsin is remodeled, ai anti-trypsin
expressed in mammalian cells is modified by capping appropriate terminal residues with a
sialic acid donor that is modified with levulinic acid, adding a reactive ketone to the sialic
acid donor. After addition to a glycosyl residue of the peptide, the ketone is derivatized with
a moiety such as a hydrazine- or amine- PEG. In Figure 38F, yet another method of ai anti-
trypsin modification is disclosed, aj anti-trypsin obtained from a mammalian expression
system is remodeled with addition of sialic acid using a sialic acid donor and an a 2,8-
sialyltransferase. hi Figure 38H, ai anti-trypsin is expressed in insect or yeast cells, and
remodeled by the addition of terminal N-acetylglucosamine residues by way of contacting the
polypeptide with UDP-N-acetylglucosamine and one or more of GnT-1,11, FV, or V. Then,
the ]3olypeptide is modified with PEG moieties using a donor of PEGylated galactose and a
galactosyltransferase. ki Figure 381, ai anti-trypsin expressed in yeast cells is treated first
with endoglycanase to trim back glycosyl chains. It is then galactosylated with a
galactosyltransferase and a galactose donor. Then, the polypeptide is PEGylated using
ST3Gal3 and a donor of PEG-sialic acid, hi Figure 38J, ai anti-trypsin is expressed in a
mammalian system. The polypeptide is first contacted with ST3GaI3 and a donor of sialic
acid that is derivatized v/ith a reactive galactose via a linker, so that the polypeptide is
attached to the reactive galactose via the linker and sialic acid residue. The polypeptide is
then contacted witli a galactosyltransferase and transferrin pre-treated with endoglycanase,
and tlius becomes connected with transferrin via the galactose residue. In Figure 38L, ai
anti-trypsin expressed in yeast is first treated with endoglycanase to trim back its glycosyl
groups. The protein is then PEGylated using a galactosyltransferase and a donor of galactose
witli a PEG moiety. In Figure 38M, ai anti-trypsin expressed in plant cells is treated with
hexosaminidase, mannosidase, and xylosidase to trim back its glycosyl chains, and
subsequently modified witii N-acetylglucosamine derivatized with a PEG moiety, using N-
acetylglucosamine transferase and a suitable donor. In Figure 38N, ai anti-tr>'psin expressed
in nianimalian cells is modified by adding PEGylated sialic acid residues using ST3Gal3 and
a donor of sialic acid derivatized with PEG.
In another exemplary embodiment, the invention provides methods for modifying
gkxoccrebrosidase (P-glucosidase, Cerezyme'^'^' or Ceredase''"''*), as showTi in Figures 39A to
39K. In Figure 39B, Cerezyme"™ expressed in a mammalian system is first treated with
sialidase to trim back terminal sialic acid residues, and is then PEGylated using ST3GaI3 and
a donor of PEG-sialic acid. In Figure 39C, CerezymeT'^ expressed in mammalian cells is first
treated with sialidase to trim back the sialic acid residues, then has mannose-6-phosphate
group attached using ST3Gal3 and a reactive sialic acid derivatized with mannose-6-
phosphate, and then is siaiylated using ST3Gal3 and a sialic acid donor. Optionally, NSO
cell expressed Cerezyme"^" is first treated with sialidase and galactosidase to trim back the
glycosyl groups, and is then galactosylated using a galactose donor and an a~
galactosyltransferase. Then, mannose-6-phosphate moiety is added to the molecule using
ST3Gal3 and a reactive sialic acid derivatized with mannose-6-phosphate. In Figure 39D,
Cerezyme^M expressed in mammalian cells is first treated with sialidase to trim back the
sialic acid residues, it is then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and
is then siaiylated using ST3Gal3 and a sialic acid donor. In Figure 39E, Cerezyme''''^
expressed in mammalian cells is modified by capping appropriate terminal residues with a
sialic acid donor that is modified with levulinic acid, adding a reactive ketone to the sialic
acid donor. After addition to a glycosyl residue of the peptide, the ketone is derivatized with
a moiety such as one or mcire mannose-6-phosphate groups. In Figure 39F, Cerezyme'^^
expressed in mammalian cells is siaiylated using a sialic acid donor and a 2,8-
sialyltransferase. In Figure 39H, CerezymeT*^ expressed in insect cells first has N-
acetylglucosamine added using a suitable donor and one or more of GnT-I, II, IV, and V, and
then is PEGylated using a galactosyltransferase and a donor of PEG-galactose. In Figure 391,
Cerez)me'''^' expressed in yeast is first treated with endoglycanase to trim back the glycosyl
groups, then galactosylated using a galactose donor and a galactosyltransferase, and then
PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 39JK, CerezymeTM
expressed in mammalian ceils is first contacted with ST3Gal3 and two reacti\'e sialic acid
residues connected via a linker, so that the polypeptide is attached to one reactive sialic acid
\'ia the linker and the second sialic acid residue. The polypeptide is then contacted with
ST3GaJ3 and desialylated transferrin, and thus becomes connected with transferrin. Then,
the polypeptide is siaiylated using a sialic acid donor and ST3Gal3.
In another exemplar}- embodiment, the invention provides metliods for modifying
Tissue-Type Plasminogen Activator (TPA) and its mutant. Several specific modification
schemes are presented in Figures 40A to 40W. Figure 40B illustrates one modification
procedure: after TPA is expressed by mammalian cells, it is treated with one or more of
mannosidase(s) and sialidase to trim back mannosyl and/or sialic acid residues. Terminal N-
acetylglucosamine is then added by contacting the polypeptide with a suitable donor of N-
acetylglucosamine and one or more of GnT-I, II, FV, and V. TPA is further galactosylated
using a galactose donor and a galactosyltransferase. Then, PEG is attached to the molecule
by way of sialylation catalyzed by ST3Gal3 and using a donor of sialic acid derivatized with
a PEG moiety. In Figuie 40C, TPA is expressed in insect or fiingal cells. The modification
includes the steps of addition of N-acetylglucosamine using an appropriate donor of N-
acetylglucosamine and GnT-I and/or II; galactosylation using a galactose donor and a
galactosyltransferase; and attachment of PEG by way of sialylation using ST3Gal3 and a
donor of sialic acid derivatized with PEG. In Figure 40D, TPA is expressed in yeast and
subsequently treated with e;ndoglycanase to trim back the saccharide chains. The polypeptide
is further PEGylated via the action of a galactosyltransferase, which catalyzes the transfer of
a PEG-galactose from a doQor to TPA. In Figure 40E, TPA is expressed in insect or yeast
cells. The polypeptide is then treated with a- and P- mannosidases to trim back terminal
mannosyl residues. Further, PEG moieties are attached to the molecule via transfer of PEG-
galactose from a suitable donor to TPA, which is mediated by a galactosyltransferase. Figure
40F provides a different method for modification of TPA obtained from an insect or yeast
system: tlie polypeptide is remodeled by addition of N-acetylglucosamine using a donor of N-
acetylglucosamine and GnT-I and/or II, followed by PEGylation using a galactosyltransferase
and a donor of PEGylated galactose. Figure 40G offers another scheme for remodeling TPA
expressed in insect or yeast cells. Terminal N-acetylglucosamine is added using a donor of
N-acetylglucosamine and GnT-I and/or II. A galactosidase that is modified to operate in a
ssTidietic, rather than a hydrol>tic maimer, is utilized to add PEGylated galactose from a
proper donor to the N-acety [glucosamine residues. In Figure 401, TPA expressed in a
mammalian system is first treated with sialidase and galactosidase to trim back sialic acid and
galacto-se residues. The polypeptide is further modified by capping appropriate terminal
residues with a sialic acid donor that is modified with levoilinic acid, adding a reactive ketOHL-
to the sialic acid donor. After addition to a glycosyl residue of the peptide, the ketone is
derivatized with a moiety sucii as a hydrtizine- or amine- PEG. In Figure 40J, TPA, which is
expressed in a manimaliein system, is remodeled following this scheme: first, the polypeptide
is treated with a- and P- mannosidases to trim back the terminal mannosyl residues; sialic
acid residues are then attached to terminal galactosyl residues using a sialic acid donor and
ST3Gal3; ftirther, TPA is PEGylated via the transfer of PEGylated galactose from a donor to
a N-acetylglucosaminyl residue catalyzed by a galactosyltransferase. In Figure 40K, TPA is
expressed in a plant system. The modification procedure in this example is as follows: TPA
is first treated with hexosjiminidase, mannosidase, and xylosidase to trim back its glycosyl
groups; PEGylated N-acetylglucosamine is then added to TPA using a proper donor and N-
acetylglucosamine transferase. In Figure 40M, a TPA mutant (TNK TPA), expressed in
mammalian cells, is remodeled. Terminal sialic acid residues are first trimmed back using
sialidase; ST3Gal3 is then used to transfer PEGylated sialic acid from a donor to TNK TPA,
such that the polypeptide is PEGylated. In Figure 40N, TNK TPA expressed in a mammalian
system is first treated with sialidase to trim back terminal sialic acid residues. The protein is
then PEGylated using CMP-SA-PEG as a donor and ST3Gal3, and fiirther sialylated using a
sialic acid donor and ST3Gal3. In Figure 40O, NSO cell expressed TNK TPA is first treated
with sialidase and a-galactosidase to trim back terminal sialic acid and galactose residues.
TNK ITA is then galactosylated using a galactose donor and a galactosyltransferase. The
last step in this remodeling scheme is transfer of sialic acid derivatized witli PEG moiety
from a donor to IT^IK TPA using a sialyltransferase such as ST3Gal3. In Figure 40Q, TNK
TPA is expressed in a mammalian system and is first treated with sialidase to trim back
terminal sialic acid residues. The protein is then PEGylated using ST3Gal3 and a donor of
PEGylated sialic acid. Then, the protein is sialylated using a sialic acid donor and ST3Gal3.
In Figure 40R, TNK TPA e;'
addition of N-acetylglucosamuie using an appropriate donor and one or more of GnT-I, II,
IV, and V. The protein is fuither modified by addition of PEG moieties using a donor of
PEGylated galactose and a galactosyitransferase. In Figure 40V, TNK TPA is expressed in
yeast. The polypeptide is first treated with endoglycanase to trim back its glycosyl chains
and then PEGylated using a galactose donor derivatized with PEG and a
galactosyitransferase. In Figure 40W, TNK TPA is produced in a mammalian system. The
polypeptide is first contacted with ST3Gal3 and a donor of sialic acid that is derivatized with
a reactive galactose via a linker, so that the polypeptide is attached to the reactive galactose
via the linker and sialic acid residue. The polypeptide is then contacted with a
galactosyitransferase and anti-TNF IG chimera produced in CHO, and thus becomes
connected with the chimera via the galactose residue.
In another exemplary embodiment, the invention provides methods for modifying
Interleukin-2 (IL-2). Figvu'es 41A to 41G provide some examples. Figure 41B provides a
tvvo-step modification scheme: IL-2 produced by mammalian cells is first treated with
sialidase to trim back its terminal sialic acid residues, and is then PEGylated using ST3Gal3
and a donor of PEGylated sialic acid. In Figure 41C, insect cell expressed IL-2 is modified
first by galactosylation using a galactose donor and a galactosyitransferase. Subsequently,
IL-2 is PEGylated using ST3Gal3 and a donor of PEGylated siaUc acid. In Figure 41D, IL-2
expressed in bacteria is modified with N-acetylgalactosamine using a proper donor and N-
acetylgalactosamine transferase, followed by a step of PEGylation with a PEG-sialic acid
donor and a sialyltransferase. Figure 4 IE offers another scheme of modifying IL-2 produced
by a mammalian system. The polypeptide is modified by capping appropriate terminal
residues with a sialic acid donor tliat is modified with levulinic acid, adding a reactive ketone
to the sialic acid donor. After addition to a glycosyl residue of the peptide, the ketone is
derivatized witli a moiety such as a hydrazine- or amine- PEG. Figure 41F illustrates an
example of remodeling lL-2 expressed by E. coli. The polypeptide is PEGylated using a
reactive N-acetylgaiactos;imine complex derivatized with a PEG group and an enzyme that i;
modified so that it functions as a synthetic enzyme rather tlian a hydrolytic one. In Figure
41G, IL-2 expressed by bacteria is modified by addition of PEGylated N-acetj'lgalactosamine
using a proper donor and N-acetylgalactosamine transferase.
In another exempiary embodiment, the invention provides methods for modifying
Factor VIII, as shown in Figures 42A to 42N. In Figure 42B, Factor VIII expressed in
mmnmalian cells is first treated with sialidase to trim back the sialic acid residues, and is then
PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 42C, Factor VUI
expressed in mammalian cells is first treated with sialidase to trim back the sialic acid
residues, then PEGylated using ST3Gal3 and a proper donor, and is then further sialylated
using ST3Gall and a sialic acid donor.
In Figure 42E, mammalian cell produced Factor VUI is modified by the single step of
PEGylation, using.ST3Gal3 and a donor of PEGylated sialic acid. Figure 42F offers another
example of modification of Factor VIII that is expressed by mammalian cells. The protein is
PEGylated using ST3Gall and a donor of PEGylated sialic acid. In Figure 42G, mammalian
cell expressed Factor VIII is remodeled foUowmg another scheme: it is PEGylated usmg a
2,8-sialyltransferase and a donor of PEG-sialic acid. In Figure 42 I, Factor VIII produce by
mammalian cells is modified by capping appropriate terminal residues with a sialic acid
donor that is modified withi levulinic acid, addmg a reactive ketone to the sialic acid donor.
Afier addition to a glycosyi residue of the peptide, the ketone is derivatized with a moiety
such as a hydrazine- or amine- PEG. In Figure 42J, Factor VIII expressed by mammalian
cells is first treated with Endo-H to trim back glycosyi groups. It is then PEGylated using a
galactosyltransferase and a donor of PEG-galactose. In Figure 42K, Factor VIII expressed in
a mammalian system is firsi; sialylated using ST3Gal3 and a sialic acid donor, then treated
with Endo-H to trim back tlie glycosyi groups, and then PEGylated with a
galactosyltransferase and a donor of PEG-galactose. In Figure 42L, Factor VIII expressed in
a manmialian system is first treated witli mannosidases to trim back terminal mannosyl
residues, then has an N-acetylglucosamine group added using a suitable donor and GnT-I
and/or K, and then is PEGylated usmg a galactosyltransferase and a donor of PEG-galactose.
Ill Figure 42M, Factor VIII expressed in mammalian cells is first treated with mannosidases
to trim back mannosyl units, then has N-acetylglucosamine group added using N-
acetylglucosamine transferase and a suitable donor. It is further gaiactosylated using a
galactosyltransferase and a galactose donor, and then sialylated using ST3GaI3 and a sialic
acid donor. In Figure 42N, Factor VIII is produced by mammalian cells and modified as
follows; it is first treated with mannosidases to trim back the terminal mannosyl groups. A
PEGylated N-acetylglucosamine group is then added using GnT-I and a suitable donor of
PEGylated N-acetylglucosamine.
In another exemplary embodiment, the invention provides methods for modijfying
urokinase, as shown in Figures 43A to 43M. hi Figure 43B, uroldnase expressed in
mammalian cells is fust b-eated vvith sialidase to trim back sialic acid residues, and is then
PEGylated usmg ST3Ga]3 and a donor of PEGyiated sialic acid. In Figme 43C, urokinase
expressed in mammalian ceils is first treated with sialidase to trim back sialic acid residues,
then PEGylated using ST3Gal3 and a donor of PEGylated sialic acid, and then sialylated
using ST3Gal3 and a sialic acid donor. Optionaiiy, urokinase expressed in a mammalian
system is first treated with sialidase and galactosidase to trim back glycosyl chains, then
galactosylated using a galactose donor and an a-galactosyltransferase, and then PEGylated
using ST3Gal3 or sialyltransferase and a donor of PEG-sialic acid. In Figure 43D, urokinase
expressed in mammalian cells is first treated with sialidase to bim back sialic acid residues,
then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and then further sialylated
using ST3Gal3 and a sialic acid donor. In Figure 43E, urokinaeast expressed M-antigen is first treated with
endoglycanase to trim back its glycosyl chains, and tlien conjugated to a Neisseria protein
using a galactosyltransferase md a proper donor of galactose HiJced to the Neisseria protein.
Figure 46K is another example of modification of M-antigen expressed in yeast. The
polj^peptide is first treated with mannosidases to trim back terminal mannosyl residues, and
then has N-acetylglucosamine added using GnT-I and/or II. Subsequently, the polypeptide is
galactosylated using a galactose donor and a galactosyltransferase, and then capped with
sialic acid residues using a sialyltransferase and a sialic acid donor.
In another exemplary embodiment, the invention provides methods for modifying
imman grcwtli hormone (N, V, :md varitmts tliereol), as shown in Figures 47A to 47K. In
Figure 47B, human growth hormone either mutated to contain a N-linked she, or a naturally
occurring isoform that has an N-linked side (i.e., the placental enzyme) produced by
mammalian cells is first treated wdth sialidase to trim back tenninal sialic acid residues and
subsequently PEGylated witli ST3Gal3 and using a donor of PEGylated sialic acid. In Figure
47C, human growth hormone expressed in insect cells is mo been conjugated via a Imker to ApoE. In Figure 56G, alpha-
galactosidase A expressed in mammalian, insect, yeast or fungal systems is remodeled by the
addition of galactose-linker-alpha2-macroglobulin moieties. The alpha-galactosidase A
molecule is contacted with Endo-H to trim back glycosyl groups. The molecule is then
contacted with galactosyltransferase and an appropriate galactose donor that has been
conjugated via a linker to aIpha2-macrogIobulin. hi Figure 56H, alpha-galactosidase A
expressed in insect, yeast and fungal systems, is remodeled by the addition of one or more N-
acetyIglucosamine-PEG-mannose-6-phosphate moieties. The alpha-galactosidase molecule
is contacted with GnT-I and an appropriate N-acetyl-glucosamine donor that has been
derivatized with PEG and mannose-6-phosphat6. In Figure 561, alpha-galactosidase A
expressed in insect, yeast or fungal systems, is remodeled b].^ the addition of one or more
terminal galactose-PEG-transferrin moieties. The alpha-galactosidase A molecule is
contacted with GnT-I and an appropriate N-acetyl-glucosamine donor. The molecule is then
contacted with galactos>'ltransferase and an appropriate galactose donor that has been
derivatized with PEG and transferrin. In Figure 56J, alpha-galactosidase A expressed in
insect, yeast or fungi systems is remodeled by the addition of one or more terminal sialic
acid-PEG-melanotransferrin moieties. The alpha-galactosidase A molecule is contacted with
GnT-I and GnT-II and an appropriate N-acetyl-glucosamine donor. The molecule is then
contacted with galactosyltiansferase and an appropriate galactose donor. The molecule is
then contacted with sialyl transferase and an appropriate sialic acid donor that has been
derivatized with PEG and melanotransferrin.
In another exemplary embodiment, the invention provides methods for modifying
alpha-iduronidase (Aldurazyme'^*^). Figures 57A to 57J present some examples. In l-ignre
57B, alpha-iduronidasc expressed in and secreted from various mammalian and insect
systems is remodeled by the addition of one or more terminal galactose-PEG-transferrin
moieties. The alpha-iduronidase molecule is contacted with Endo-H to trim back glycosyl
groups. The molecule is then contacted with galactosyltransferase and an appropriate
galactose donor that h^as been derivatized with PEG and trcan, thus removing one glycan from the mature peptide. For
example, a native recogiiition site with the sequence of asparagine-serine-serine can be
genetically engineered to have the sequence leucine-serine-serine, tlaus eliminating a N-
linked glycosylation site at this position.
Fuitlier, an N-linked glycosylation site can be removed by altering the residues in ihe
recognition site so that even though the asparagine residue is present, one or more of the
additional recognition residues are absent. For example, a native sequence of asparagine-
serine-serine can be mutated to asparagine-serine-lysine, thus eliminating an N-glycosylation
site at that position. In the case of N-linked glycosylation sites comprising residues other
than the typical recognition sites described above, the skilled artisan can determine the
sequence and residues required for recognition by the appropriate glycosyltransferase, and
then mutate at least one residue so the appropriate glycosyltransferase no longer recognizes
that site. In other words, it is well within the skill of the artisan to manipulate the primary
sequence of a peptide such that glycosylation sites are either created or are removed, or both,
thereby generating a peptide having an altered glycosylation pattern. The invention should
therefore not be construed to be limited to any primary peptide sequence provided herein as
the sole sequence for glycan remodeling, but rather should be construed to include any and all
peptide sequences suital^le for glycan remodeling.
To create a mut£int peptide, the nucleic acid sequence encoding the primary sequence
of the peptide is altered so that native codons encoding native amino acid residues are
mutated to generate a codon encoding another amino acid residue. Techniques for altering
nucleic acid sequence are common in the art and are described for example in any well-
known molecular biology manual.
In addition, the nucleic acid encoding a primary peptide structure can be synthesized
in vitro, using standard techniques. For example, a nucleic acid molecule can be synthesi2;ed
in a "gene machine" using protocols such as the phosphoramidite method. If chemically-
synthesized double straided DNA is required for an application such as the synthesis of a
nucleic acid or a fragment tliereof, then each complementai-y strand is synthesized
separately. The production of short nucleic acids (60 to 80 base pairs) is technically
straightforwai'd and can be accomplished by synthesizing the complementary strands and
then aimealing them. For the production of longer nucleic acids (>300 base pairs), special
strategies may be requb-ed, because the coupling efficiency of each cycle during chemical
DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-
strmided) are assembled in modular form from single-stranded fragments tliat are from 20 to
100 nucleotides in length. For reviev/s on polynucleotide synthesis, see, for example, Ulick
and Pasternak (Molecu'ar Biotechnology, Principles and Applications of Recombinant DNA,
1994, ASM Press), Itakura et al. (1984, Annu. Rev. Biochem. 53:323), and Climie et al.
(1990, Proc. Nafl Acad. Sci. USA 87:633).
Additionally, changes in the nucleic acid sequence encoding the peptide can be miade
by site-directed mutagenesis. As will be appreciated, this technique typically employs a
phage vector which exists in both a single stranded and double stranded form. Typical
vectors useful in site-directed mutagenesis include vectors such as the Ml3 phage. These
phage are readily available and their use is generally well known to those skilled in the art.
Double stranded plasmids are also routinely employed in site-directed mutagenesis which
eliminates the step of transferring the nucleic acid of interest from a plasmid to a phage.
In general, site-directed mutagenesis is performed by first obtaining a single-stranded
vector or melting the two strands of a double stranded vector which includes within its
sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer
bearing the desired mutated sequence is prepared generally synthetically. This primer is then
armealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such
as E. coli polymerase I IGenow fragment, in order to complete the synthesis of the mutation-
beaiing strand. Thus, a lieteroduplex is formed wherein one strand encodes the original non-
mutated sequence and the second strand bears tlie desired mutation. Tliis heteroduplex vector
is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are
m
selected which include recombinant vectors bearing the mutated sequence arrangement. A
genetic selection scheme was devised by Kunkel et al. (1987, Kunkel et al.. Methods
EnzjTnol. 154:367-382) to enrich for clones incorporating the mutagenic oligonucleotide.
Alternatively, the use of PCR'^'^ with commercially available thermostable enzymes such as
Taq polymerase may be used to incorporate a mutagenic oligonucleotide primer into an
amplified DNA fragment that can then be cloned into an appropriate cloning or expression
vector. The PCR^'^'-mediated mutagenesis procediu-es of Tomic et al. (1990, Nucl. Acids
Res., 12:1656) and Upender et al. (1995, Biotechniques, 18:29-31) provide two examples of
such protocols. A PCR^"^' employing a thermostable ligase in addition to a thermostable
polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into
an amplified DNA fragment that may then be cloned into an appropriate cloning or
expression vector. The mutagenesis procedure described by Michael (1994, Bioteclmiques
16:410-412) provides an example of one such protocol.
Not all Asn-X-Ser/Thr sequences are N-glycosylated suggesting the context in wliich
the motif is presented is important. In another approach, libraries of mutant peptides having
novel N-linked consensus sites are created in order to identify novel N-linked sites that are
glycosylated in vivo and are beneficial to the activity, stability or other characteristics of the
peptide.
As noted previously, the consensus sequence for the addition of N-linked glycan
chains in glycoproteins is Asn-X-Ser/Thr where X can be any amino acid. The nucleotide
sequence encoding the amino acid two positions to the carboxyl terminal side of the Asn may
be mutated to encode a Ser and/or Thr residue using standard procedures known to those of
ordinary skill in the art. As stated above not all Asn-X-Ser/Thr sites are modified by tlie
addition of glycans. Therefore, each recombinant mutated glycoprotein must be expressed in
a fungal, yeast or animal or mammalian expression system and analyzed for the addition of
an N-linked glycan chain. The techniques for the characterization of glycosylation sites are
well known to one skilled in the art. Further, the biological function of the mutated
recombinant glycoprotein can be determined using assays standard for the particular protein
being examined. Thus, it becomes a simple matter to manipulate the primary sequence of a
peptide and identify novel glycosylation sites contained therein, and further determine the
effect of the novel site on the biological activity of the peptide,
In an alternative embodiment, the nucleotide sequence encoding the amino acid two
positions to the amiao terminal side of Ser/Thr residues may be mutated to encode an Asn
using standard procedures Icnown to those of ordinary skill in the art. The procedures to
determine whetlier a novel glycosylation site has been created imd the effect of this site on the
biological activity of the peptide are described above.
B. Creation or elimination of O-linked glycosvlation sites
The addition of an O-linked glycosylation site to a peptide is conveniently
accomplished by altering the primary ammo acid sequence of the peptide such that it contains
one or more additional O-linked glycosylation sites compared with the beginning primary
amino acid sequence of the peptide. The addition of an O-linked glycosylation site to the
peptide may also be accomplished by incorporation of one or more amino acid species mto
tlie peptide which comprises an -OH group, preferably serine or threonine residues, within
the sequence of tlie peptide, such that the OH group is accessible and available for O-linked
glycosyJation. Similai- to the discussion of alteration of N-linked glycosylation sites in a
peptide, the primary amino acid sequence of the peptide is preferably altered at the nucleotide
level. Specific nucleoi;ides in the DNA sequence encoding the peptide may be altered such
that a desired amino acid is encoded by the sequence. Mutation(s) in DNA are preferably
made using methods kjiown in the art, such as the techniques of phosphoramidite method
DNA synthesis and sit(;-directed mutagenesis described above.
Alternatively, the nucleotide sequence encoding a putative site for O-lioked glycani
addition can be added to the DNA molecule in one or several copies to either 5' or the 3' end
of the molecule. The altered DNA sequence is then expressed in any one of a fungal, yeast,
or animal or mammalian expression system and analyzed for the addition of the sequence to
the peptide and whethei- or not this sequence is a functional 0-linked glycosylation site.
Briefly, a synthetic peptide acceptor sequence is introduced at either the 5' or 3' end of the
nucleotide molecule. In principle, the addition of this type of sequence is less disruptive to
the resulting glycoprotein when expressed in a suitable expression system. The altered DNA
is then expressed in CHO cells or other suitable expression system and the proteins expressed
thereby are examined for the presence of an O-Iinked glycosylation site. In addition, the
presence or absence of glycan chains can be determined.
In yet another approach, advantageous sites for new 0-linked sites may be found in a
peptide by creating librai-ies of the peptide containing various new 0-linked sites. For
example, the consensus finiino acid sequence for N-acetylgalactosamine addition by an N-
acet}'lgalactosaminyltransferase depends on the specific transferase used. The amino acid
sequence of a peptide may be scaimed to identify contiguous groups of amino acids that can
be mutated to generate potential sites for addition of 0-Iinked glycan chains. These
mutations can be generated using standard procedures known to those of ordinary skill in the
art as described previously. In order to determine if any discovered glycosylation site is
actually glycosylated, each recombinant mutated peptide is tlien expressed in a suitable
expression system and is subsequently analyzed for the addition of the site and/or the
presence of an 0-linked glycan chain.
C. Chemical synthesis of peptides
While the primary structure of peptides useful in the invention can be generated most
efficiently in a cell-based expression system, it is within the scope of the present in\ention
-236-
that the peptides may be generated synthetically. Chemical, synthesis of peptides is well
known in the art and inc:Iude, without limitation, stepwise solid phase synthesis, and fragment
condensation either in solution or on solid phase. A classic stepwise solid phase synthesis of
iuvolves covalently linking an amino acid corresponding to the carboxy-terminal amino acid
of the desired peptide chain to a solid support and extending the peptide chain toward the
amino end by stepwise coupling of activated amino acid derivatives having activated
carboxyl groups. After completion of the assembly of the fully protected solid phase bound
peptide chain, the peptide-solid phase covalent attachment is cleaved by suitable chemistiy
and the protecting groups are removed to yield the product peptide. See, R. Merrifield, Solid
Phase Peptide Synthesis: The Synthesis of a Tetrapeptide, J. Am. Chem. Soc, 85:2149-2154
(1963). The longer the peptide chain, the more challenging it is to obtain high-purity well-
defined products. Due to tlie production of complex mixtures, the stepwise solid phase
synthesis approach has size limitations. In general, well-defmed peptides of 100 contiguous
amino acid residues or more are not routinely prepared via stepwise solid phase synthesis.
The segment condensation method involves preparation of several peptide segments
by the solid phase stepwise method, followed by cleavage from the solid phase and
purification of these maximally protected segments. The protected segments are condensed
one-by-one to the first segment, which is bound to the solid phase.
The peptides useful in the present invention may be synthesized by exclusive solid
phase synthesis, partial solid phase metlaods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill in the art (see, for
example, Merrifield, J. j\m. Chem. Soc. 85:2149 (1963), Stewart et al., "Solid Phase Peptide
Synthesis" (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot. 3:3
(1986), Atlierton et al.. Solid Phase Peptide Synthesis: A Practical Approach (IRL Press
1989), Fields and Colowick, "Solid-Phase Peptide Synthesis," Methods in Enzymology
Volume 289 (Academic Press 1997), and Lloyd-Williams et al., Chemical Approaches to the
Synthesis of Peptides and Peptides (CRC Press, Inc. 1997)). Variations in total chemical
synthesis strategies, such as "native chemical ligation" and "e.xpressed peptide ligation" are
also standard (see, for examp,:e, Dawson et al.. Science 266:776 (1994), Hackeng et al., Proc.
Nat'l Acad. Sci. USA 94:7845 (1997), Dawson, Methods Enzymol. 287: 34 (1997), Muir et
al Proc. Nat'i Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol. Chem.
273:16205 (1998)). Also useful are the solid phase peptide synthesis methods developed by
Gryphon Sciences, South San Francisco, CA. See, U.S. Patent Nos. 6,326,468, 6,217,873,
6,174,530, and 6,001,364, all of wliich are incorporated in their entirety by reference herein.
D. Post-translational modifications
It will be appreciated to one of ordinary skill in the art that peptides may undergo
post-translational modification besides the addition of N-linked and/or O-linked glycans
tliereto. It is contemplated that peptides having post-translational modifications other than
glycosylation can be used as peptides in the invention, as long as the desired biological
activity or function of the peptide is maintained or improved. Such post-translational
modifications may be natural modifications usually carried out in vivo, or engineered
modifications of the peptide carried out in vitro. Contemplated known modifications include,
but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent
attacliment of flavin, co^falent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent crosslinks, formation of cysteine, formation of
pyroglutamate, formylati on, gamma carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing,
phosphorylation, prenyls-tion, racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to peptides such as arginylation, and ubiquitination. Enzymes that
may be used to carry out many of these modifications are well known in the art, and available
commercially from companies such as Boehringer Mannheim (Indianapolis, IN) and Sigma
Chemical Company (St. Louis, MO), among others.
Such modifications are well known to those of skill in the art and have been described
in great detail in the scientific literature. Several particularly common modifications,
glycosylation, lipid attaclmient, sulfation, ganmia-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylaiion, for instance, are described in most basic texts, such as
Peptides—Structure and Moleculai" Properties, 2nd Ed., T. E. Crcighton, W. H. Freeman and
Company, New York (IS'93). Many detailed reviews are available on tins subject, such as by
Wold, F., Post-translational Covalent Modification of Peptides, B. C. Joluison, Ed., Academic
Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymoi. 182: 626-646 (1990)) and
Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
Covalent modifications of a peptide may also be introduced into the molecule in vitro
by reacting targeted ami no-acid residues of the peptide with an organic derivatizing agent
that is capable of reacting with selected side chains or terminal amiao-acid residues. Most
commonly derivatized residues are cysteinyl, histidyl, lysinyl, arginyl, tyrosyl, glutaminyl,
asparaginyl and amino terminal residues. Hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl and threonyl residues, methylation of the alpha-
amino groups of lysine, histidine, and histidine side chains, acetylation of the N-terminal
amine and amidation of the C-terminal carboxylic groups. Such derivatized moieties may
improve the solubility, absorption, biological half life and the like. The moieties may also
eliminate or attenuate any undesirable side effect of the peptide and the like.
In addition, derivatization with bifiinctional agents is useful for cross-linking the
peptide to water insolubli^ support matrices or to other macromolecular carriers. Commonly
used cross-linking agents include giutaraldehyde, N-hydroxysuccinimide esters,
homobifimctional imidoesters, 1,1-bis(-diazoIoacetyl)-2-phenylethane, and bifunctional
maleimides. Derivatizing agents such as methyl-3-[9p-azidophenyl)]dithiopropioimidate
yield photoactivatable intermediates that are capable of forming crosslinks in the presence of
light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide activated
carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287 and 3,691,016
may be employed for peptide immobilization.
E. Fusion peptides/peptides
Peptides useful in the present invention may comprise fusion peptides. Fusion
peptides are particularly advantageous where biological and/or functional characteristics of
two peptides are desired to be combined in one peptide molecule. Such fusion peptides can
present combinations of biological activity and function that £ire not found in nature to create
novel and useful molecules of therapeutic and industrial applications. Biological activities of
interest include, but aie not limited to, enzjonatic activity, receptor and/or ligand activity,
immunogenic motifs, and structural domains.
Such fusion peptides are well known in the art, and the methods of creation will be
well-known to those in the art. For example, a human a-interferon—hiunan albumin fusion
peptide has been made wherein the resulting peptide has the therapeutic benefits of a-
interferon combined with the long circulating life of albumin, thereby creating a therapeutic
composition thiit allows reduced dosing frequency and potentially reduced side effects in
patients. See, Albuferon''^'^ from Himian Genome Sciences, Inc. and U.S. Patent No.
5,766,883. Other fusion peptides include antibody molecules that are described elsewhere
herein.
F. Generation of smaller "biologically active" molecules
The peptides used in the invention may be variants of native peptides, wherein a
fragment of the native peptide is used in place, of the full length native peptide. In addition,
pre-pro-, and pic-peptides are contemplated. Variant peptides may be smaller in size that the
native peptide, and may comprise one or more domains of a larger peptide. Selection of
specific peptide domains can be advantageous when the biological activity of certain domains
in the peptide is desired, but the biological activity of other domains in the peptide is not
desired. Also included are truncations of the peptide and internal deletions which may
enhance the desired therapeutic effect of the peptide. Any such forms of a peptide is
contemplated to be usefiil in the present invention provided that the desired biological
activity of the peptide is preserved.
Shortef versions of peptides may have unique advcintages not found in the native
peptide. In the case of human albumin, it has been found that a truncated fonn comprising as
little as 63% of the native albumin peptide is advantageous as a plasma volume expander.
The truncated albumin peptide is considered to be better than the native peptide for this
therapeutic purpose because an individual peptide dose of only one-half to two-thirds that of
natural-human serum albumin, or recombinant human serum albumin is required for the
equivalent colloid osmotic effect. See U.S. Patent No. 5,380,712, the entirety of which is
incorporated by reference herein.
Smaller ''biologically active" peptides have also been found to have enhanced
therapeutic activity as compared to the native peptide. The therapeutic potential of IL-2 is
linn ted by various side effects dominated by tlie vascular leak syndrome. A shorter
chemically synthesized version of the peptide consisting of residues 1-30 corresponding to
the entire a-helix was found to fold properly and contain the natural IL-2 biological activity
with out the attending side effects.
G. Generation of novel peptides
The peptide of the invention may be derived from a primary sequence of a
native peptide, or may be engineered using any of the many means known to those of skill in
the art. Such engineered peptides can be designed and/or selected because of enhanced or
novel properties as compared with the native peptide. For example, peptides may be
engineered to have increased enzyme reaction rates, incrccised or decreased binding affinity
to a substrate or ligand, increased or decreased binding affinity to a receptor, altered
specificity for a substrate, ligand, receptor or other binding partner, increased or decreased
stability in vitro and/or in vivo, or increased or decreased immunogenicity in an animal.
H. Mutations
1 ¦ Rational design mutation
The peptides useful in the methods of the invention may be mutated to enhance a
desired biological activity or function, to diminish an undesirable property of the peptide,
and/or to add novel activities or functions to the peptide. "Rational peptide design" may be
used to generate such altered peptides. Once the amino acid sequence and structure of the
peptide is known and a desired mutation planned, the mutations can be made most
conveniently to the corresponding nucleic acid codon which encodes the amino acid residue
that is desired to be mutated. One of skill in the art can easily determine how the nucleic
acid sequence should be altered based on the universal genetic code, and knowledge of codon
preferences in tlie expression system of choice. A mutation in a codon may be made to
change the amino acid residue that will be polymerized into the peptide during translation.
Alternatively, a codon may be mutated so that the corresponding encoded amino acid residue
is the same, but the codon choice is better suited to the desired peptide expression system.
For example, cys-residues may be replaced with other amino acids to remove disulfide bonds
from the mature peptide, catalytic domains may be mutated to alter biological activity, and m
general, isofonns of the peptide can be engineered. Such mutations can be point mutations,
deletions, insertions and Ixuncations, among others.
Techniques to mutate specific amino acids in a peptide are well known in the art. The
technique of site-directed mutagenesis, discussed above, is well suited for the durected
mutation of codons. The oligonucleotide-mediated mutagenesis method is also discussed in
detail in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, Near York, starting at page 15.51). Systematic deletions, insertions and
truncations can be made using linker insertion mutagenesis, digestion with nuclease Bal31,
and linker-scanning mutagenesis, among other method well known to those in the art
(Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York).
Rational peptide design has been successfully used to increase the stability of
enzymes with respect to theimoinactivation and oxidation. For example, the stability of an
enzyme was improved by removal of asparagine residues in a-amylase (Declerck et al., 2000,
J. Mol. Biol. 301:1041-1057), tlie introduction of more rigid structural elements such as
proline into a-amylase (Igarashi et al., 1999, Biosci, Biotechnol. Biochem. 63:1535-1540)
and D-xylose isomerase (Zhu et al., 1999, Peptide Eng. 12:635-638). Further, the
introduction of additional hydrophobic contacts stabilized 3-isopropylmalate dehydrogenase
(Akanuma et al., 1999, Eur. J. Biochem. 260:499-504) and formate dehydrogenase obtained
from Pseudomonas sp. (Rojkova et al., 1999, FEBS Lett. 445:183-188). The mechanisms
behind the stabilizing effect of these mutations is generally applicable to many peptides.
These and similar mutations tire contemplated to be useful with respect to the peptides
remodeled in the methods of the present invention.
2. Random mutagenesis techniques
Novel peptides useful in the methods of the invention may be generated using
techniques that introduce random mutations in the coding sequence of the nucleic acid. The
nucleic acid is then expressed in a desired expression system, and the resulting peptide is
assessed for properties of interest. Techniques to introduce random mutations into DNA
sequences are well known in tiie art, and include PCR mutagenesis, saturation mutagenesis,
and degenerate oligonucleotide approaches. See Sambrook and B.ussell (2001, Molecular
Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY) and
Ausubel et al. (2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY).
In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random
mutations into a cloned fragment of DNA (Leung et al, 1989, Technique 1:11-15). This is a
very powerful and relatively rapid method of introducing random mutations into a DNA
sequence. The DNA region to be mutagenized is amplified using the polymerase chain
reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA
polymerase, e.g., by using an altered dGTP/dATP ratio and by adding Mn^"" to the PCR
reaction. The pool of amplified DNA fi-agments are inserted into appropriate cloning vectors
to provide random mutant libraries.
Saturation mutagenesis allows for the rapid introduction of a large number of single
base substitutions into cloned DNA fi'agments (Mayers et al., 1985, Science 229:242). This
technique includes generation of mutations, e.g., by chemical treatment or uradiation of
single-stranded DNA in vitro, and synthesis of a complementary DNA strand. The mutation
frequency can be modulated by modulating the severity of the treatment, and essentially all
possible base substitutioni; can be obtained. Because this procedure does not involve a
genetic selection for mutant fragments, both neutral substitutions as well as those that alter
function, are obtained. The distribution of point mutations is not biased toward conserved
sequence elements.
A library of nucleic acid homologs can also be generated from a set of degenerate
oligonucleotide sequences. Chemical synthesis of a degenerate oligonucleotide sequences
can be carried out in an automatic DNA synthesizer, and the synthetic genes may then be
ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is
known in the art (see for ex;miple, Narang, SA (1983) Tetrahedron 39:3; Itakuraetal. (1981)
Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,
Amsterdam: Elsevier pp. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura
et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques
have been employed in the directed evolution of other peptides (see, for example, Scott et al.
(1990) Science 249:386-390: Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990)
Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos.
5,223,409, 5,198,346, and 5,096,815).
a. Directed evolution
Peptides useful in the methods of the invention may also be generated using "directed
evolution" techniques, hi contrast to site directed mutagenesis techniques where knowledge
of the structure of the peptide is required, there now exist strategies to generate libraries of
mutations firom which to obtain peptides with improved properties without knowledge of l[he
structural features of the peptide. These strategies are generally known as "directed
evolution" technologies and are different from traditional random mutagenesis procedures in
that they involve subjecjting the nucleic acid sequence encoding the peptide of interest to
recursive rounds of mutation, screening and amplification.
In some "directed evolution" techniques, the diversity in the nucleic acids obtained is
generated by mutation methods that randomly create point mutations in the nucleic acid
sequence. The point mutation techniques include, but are not limited to, "error-prone
PCR™" (Caldwell and Joyce, 1994; PCR Methods Appl. 2: 28-33; and Ke and Madison,
1997, Nucleic Acids Res. 25: 3371-3372), repeated oligonucleotide-directed mutagenesis
(Reidhaar-Olson et al., 1991, Methods Enzymol. 208:564-586), and any of the
aforementioned methods of random mutagenesis.
Another metho'(.-osyltransferases and related genes, Springer, Tokyo.
Glycosyltransferase amino acid sequences and nucleotide sequences encoding
glycosyltransferases from which tlie amino acid sequences can be deduced are also found in
various publicly available databases, including GenBank, Swiss-Pror, EMBL, and others.
Glycosyltransferases that can be employed in the methods of the invention include,
but are not limited to, galactosyltransferases, fucosyltransferases, glucosyltransferases, N-
acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases, glucuronyltransferases,
sialyltransferases, mannosyltransferases, glucuronic acid transferases, galacturonic acid
transferases, and oligosaccharyltransferases. Suitable glycosyltransferases include those
obtained from eukaryotes, as well as from prokaryotes.
DNA encoding glycosyltransferases may be obtained by chemical synthesis, by
screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by
screening genomic libraries from appropriate cells, or by combinations of these procedures.
Screening of mRNA or genomic DNA may be carried out using oligonucleotide probes
generated from the glycosylfransferases nucleic acid sequence. Probes may be labeled with a
detectable label, such as, but not limited to, a fluorescent group, a radioactive atom or a
chemiluminescent group in accordance with known procedures and used in conventional
hybridization assays. In the alternative, glycosyltransferases nucleic acid sequences may be
obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR
oligonucleotide primers being produced from the glycosyltransferases nucleic acid sequence.
See, U.S. Pat. No. 4,683,195 to MuUis et al. and U.S. Pat. No. 4,683,202 to Mullis.
A glycosyltransferases enzyme may be synthesized in a host cell transformed with a
vector containing DNA encoding the glycosyltransferases enzyme. A vector is a replicable
DNA construct. Vectors are used either to amplify DNA encoding the glycosyltransferases
enzyme and/or to express DNA which encodes tlie glycosyltransferases enzyme. An
expression vector is a replicable DNA construct in which a DNA sequence encoding the
glycosyltransferases enzyme is operably linked to suitable control sequences capable of
effectmg the expression of the glycosyltransferases enzyme in a suitable host. The need for
such control sequences will vary depending upon tlie host selected and the transformation
method chosen. Generall>, control sequences include a transcriptional promoter, an optional
operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal
binding sites, and sequences wliich control the termination of transcription and translation.
Ampl:fication vectors do not require expression control domains. All thai is needed is the
abilit}' to replicate in a host, usually conferred by an origin of replication, and a selection
gene to facilitate recognition of ti'ansformants.
1. Fucosyltransferases
In some embodiments, a glycosyltransferase used in the method of the invention is a
fucosyltransferase, Fucosyltransferases are known to those of skill in the art. Exemplary
fucosyltransferases include enzymes, which transfer L-fticose from GDP-fticose to a hydroxy
position of an acceptor sugar. Fucosyltransferases that transfer from non-nucleotide sugars to
an acceptor are also of use in the present invention.
In some embodiments, the acceptor sugar is, for example, the GlcNAc in a
Gaip(l~>3,4)GicNAcp- group in an oligosaccharide glycoside. Suitable fucosyltransferases
for this reaction include the Gal{i(l-^3,4)GlcNAcpi-a(l-»3,4)fiicosyltransferase (FTIII E.G.
No. 2.4.1.65), which was first characterized from human milk (see, Palcic, et al.,
Carbohydrate Res. 190: 1-11 (1989); Prieels, et al, J. Biol. Chem. 256: 10456-10463 (1981);
and Nunez, et ai., Can. J. Chem. 59: 2086-2095 (1981)) and the Gaip(l-9>4)GlcNAcp-
afucosyltransferases (FTP/, FTV, FTVI) which are found in human serum. FTVII (E.G. No.
2.4.1.65), a sialyl a(2-^3)Galp((l-»3)GIcNAcp fticosyteansferase, has also been
characterized. A recombinjuit form of the GaIp(}->3,4) GIcNAcp-
a(1^3,4)fLicosyItransferase; has also been characterized (see, Dumas, et al., Bioovg. Med.
Letters I: 425-428 (1991) and Kukowska-Latallo, ct al. Genes and Development 4: 1288-
1303 (2990)), Other exemplary fncosyltransferases include, for example, al,2
fucosyltiansferase (B.C. No. 2.4.1.69). Enzymatic fucosylation can be carried out by tfie
methods described in MoUicone, et al, Eur. J. Biochem. 191: 169-176 (1990) or U.S. Patent
No. 5,374,655.
2. Galactosyltransferases
In ^mother group of embodiments, the glycosyltransferase is a galactosyitransferasc.
Exemplary galactosyltransferases include a(l,3) galactosyltransferases (E.G. No. 2.4.1.151,
see, e.g., Dabkowski et al. Transplant Proc. 25:2921 (1993) and Yamamoto et al Nature 345:
229-233 (1990), bovine (GeaB,3nk J04989, Joziasse et al, J. Biol Chem. 264: 14290-14297
(1989)), murine (GenBank m26925; Larsen ct al, Proc. Nat'l. Acad. Sci. USA 86: 8227-8231
(1989)), porcine (GenBank L36152; Slrahan et al, Immunogenetics 4}: 101-105 (1995)).
Another suitable a 1,3 galaciosyltransferase is that wliich is involved in synthesis of the blood
group B antigen (EC 2.4.1.37, Yamamoto et al, J. Biol. Chem. 265: 1146-1151 (1990)
(human)).
Also suitable for use in the methods of the invention are P(l,4) galactosyltransferases,
which include, for example, EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose
synthetase) (bovine (D'Agostaro et al., Eur. J. Biochem. 183: 211-217 (1989)), human (Masri
et al., Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa et ai., J.
Biochem. 104: 165-168 (1988)), as well as E.C. 2.4.1.38 and the ceramide
galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci. Res. 38: 234-242 (1994)). Other
suitable galactosyltransferases include, for example, al,2 galactosyltransferases (from e.g.,
Schizosaccharomyces pombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)). For further
suitable galactosyltransferases, see Taniguchi et al. (2002, Handbook of Glycosyltransferases
and Related Genes, Sprmger, Tokyo), Quo et al. (2001, Glycobiology, 11(10):813-820), and
Breton et al. (1998, J Biochem. 123:1000-1009).
The production of proteins such as the enzyme GalNAc Tj.xiv from cloned genes by
genetic engineering is well known. See, e.g., U.S. Pat. No. 4,761,371. One method involves
collection of sufficient saraples, then the amino acid sequence of the enzyme is determined
by N-tenninal sequencing This information is then used to isolate a cDNA clone encoding a
full-length (membrane bound) transferase which upon expression in the insect cell line Sf9
resulted in the synthesis of a fiilly active enzyme. The acceptor specificity of the enzyme is
then detennined using a semiquantitative analysis of the amino acids surrounding known
glycosylation sites in 16 different proteins followed by in vitro glycosylation studies of
synthetic peptides. This work has demonstrated that certain amino acid residues are
overrepresented in glycos} lated peptide segments and that residues in specific positions
surrounding glycosylated serine and threonine residues may have a more marked influence on
acceptor efficiency than other amino acid moieties.
3. Sialyl transferases
Sialyltransferases are another type of glycosyltransferase tliat is useful m the
recombinant cells and reacdon mixtures of the invention. Examples of sialyltransferases that
are suitable for use in the present invention include ST3Gal III (e.g., a rat or human ST3Gal
III), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal 11, ST6GalNAc I. ST6GalNAc I],
and SI'6GalNAc III (the sialyltransferase nomenclature used herein is as described in Tsuji et
al., Glycobiology 6: v-xiv (1996)). An exemplary a(2,3)sialyltransferase referred to as
a(2,3)sialyltraiisferase (EC 2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of
a GaIpl->3GIc disaccharide or glycoside. See, Van den Eijnden et al., J. Biol. Chem. 256:
3159 (1981), Weinstein et al., J. Biol. Chem. 257: 13845 (1982) and Wen et al., J. Biol.
Chem. 267: 21011 (1992). Another exemplary a2,3-sialyltransferase (EC 2.4.99.4) transfers
sialic acid to the non-reducing terminal Gal of the disaccharide or glycoside, see, Rearick et
al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J. Biol. Chem. 267: 21004 (1992).
Further exemplary enzymes include Gal-p-l,4-GIcNAc a-2,6 sialyltransferase (See,
Kurosawa et al. Eur. J. Biochem. 219: 375-381 (1994)).
Preferably, for glycosylation of carbohydrates of glycopeptides the sialyltransferase
will be able to transfer sialic acid to the sequence Gaipi,4GlcNAc-, Gaipi,3GlcNAc-, or
Galpl,3GalNAc-, the most common penultimate sequences underlying the terminal sialic
acid on fully sialylated carbohydrate structiu-es {see, Table 8). a2,8-Sialyltransferases
capable of transfering sialic acid to a2,3Gaipi,4GIcNAc are also useful in the methods of the
invention.
An example of a sialyltransferase that is useful in the claimed methods is ST3GaI III,
which is also referred to as a(2,3)sialyltransferase (EC 2.4.99.6). This enzyme catalyzes the
transfer of sialic acid to the Gal of a Gaipi,3GIcNAc or Gaipi,4GlcNAc glycoside (see, e.g..
Wen et al., J. Biol. Chem. 267; 210] ] (1992); Van den Eijnden et al., J. Biol. Chera. 256:
3159 (1991)) and is responsible for sialylation of asparagine-linked oligosaccharides in
glycopeptides. The sialic acid is linked to a Gal with the formation of an a-Imkage between
the two saccharides. Bonding (linkage) between the saccharides is between the 2-position of
NeuAc and the 3-position of Gal. This particular enzyme can be isolated from rat liver
(Weinstein et al, J. Biol. Chem. 257: 13845 (1982)); the human cDNA (Sasaki et al. (1993)
J. Biol. Chem. 268: 22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-
I40J) and genomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNA sequences are
known, facilitating production of this enzyme by recombinant expression, hi a preferred
embodiment, the clauned sialylation metliods use a rat ST3Gal III.
An example of a sialyltransferase that is useful in the claimed methods is CST-I from
Campylobacter (see ,for example, U.S. Pat. No. 6,503744, 6,096,529, and 6,210933 and
WO99/490S1, and published U.S. Pat. Application 2002/2,042,369). This enzyme catalyzes
the transfer of sialic acid to the Gal of a Gaipi,4Glc or Gaipi,3GalNAc. Other exemplary
sialyltransferases of use in the present invention include those isolated from Campylobacter
jejuni, including the a(2,3) sialyltransferase. See, e.g, WO99/49051.
Other sialyltransferases, including those listed in Table 8, are also useful in an
economic and efficient large-scale process for sialylation of commercially important
glycopeptides. As a simple test to find out the utility of these other enzymes, various
amounts of each enzyme (1-100 mU/mg protein) are reacted with asialo-a, AGP (at 1-10
mg/ml) to compai-e the ability of Ae sialyltransferase of interest to sialylate glycopeptides
relative to either bovine ST6Gal I, ST3Gal HI or both sialyltransferases. Alternatively, otlier
glycopeptides or glycopeptides, or N-imked oligosaccharides enz>'matica}ly released from the
peptide backbone can be used m place of asialo-a, AGP for this evaluation,
Sialyltransferases with the ability to sialylate N-litiked oUgosacch^mdes of glycopeptides
more efficiently than ST6Gal I are usetnl in a practical large-scale process for peptide
sialylation (as illustrated for ST3Gal III in this disclosure).
4. Other glvcosyltransferases
One of skill in tlie art will understand that other glycosyltraasferases can be
substituted into similar transferase cycles as have been described in detail for the
sialyltransferase. In particular, the glycosyltransferase can also be, for instance,
glucosyltransferases, e.g., Alg8 (Stagljov et al., Proc. Natl. Acad. Sci. USA 91: 5977 (1994))
or AIg5 (Heesen et al., Eur. J. Biochem. 224: 71 (1994)).
N-acetylgalactosaminyltransferases are also of use in practicing the present invention.
Suitable N-acetylgalactosaminyltransferases include, but are not limited to, a(l,3) N-
acetylgalactosaminyltransferase, p(l,4) N-acetylgalactosaminyitransferases (Nagata et al., J.
Biol. Chem. 267: 12082-12089 (1992) and Smith et al., J. Biol Chem. 269: 15162 (1994))
and peptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol. Chem. 268: 12609
(1993)). Suitable N-acetylglucosaminyltransferases include GnT-I (2.4.1.101, Hull et al.,
BBRC 176: 608 (1991)), GnT-II, GnT-III (Ihara et al., J. Biochem. 113: 692 (1993)), GnT-
IV, GnT-V (Shoreibah et al., J. Biol. Chem. 268: 15381 (1993)) and GnT-VI, 0-linked N-
acetylglucosaminyltransfeiase (Bierhuizen et al., Proc. Natl. Acad. Sci. USA 89: 9326
(1992)), N-acetylgIucosamine-1-phosphate transferase (Rajput et al., Biochem J. 285: 985
(1992), and hyaluronan synthase.
Mannosyltransferases are of use to transfer modified marmose moieties. Suitable
mannosyltransferases include a(l,2) mannosyltransferase, a(l,3) mannosyltransferase, a(l,6)
mannosyltransferase, P(l,4) maimosyltransferase, Dol-P-Man synthase, OChl, and Pmtl
(see, Kornfeld et al., Annu. Rev. Biochem. 54: 631-664 (1985)).
Xylosyltransferases are also useful in the present invention. See, for example,
Rodgers, et al., Biochem. J., 288:817-822 (1992); and Elbain, et al., U.S. Patent No.,
6,168,937.
Other suitable glycosyltransferase cycles are described in Ichikawa et al, JACS 114.
9283 (1992), Wong et al., J. Org, Chem. 57: 4343 (1992), and Ichikawa et al. in
CARBOHYDRATES AND CARBOHYDRATE POLYMERS. Yaltami, ed. (ATL Press, 1993),
Prokaryotic glycosyltnmsferases are also useful in practicing the invention. Such
glycosyltransfcrases include enzymes involved in synthesis of lipooligosaccharides (LOSj,
which are produced by many gjam negative bacteria. The LOS t>pically have terminal
glycan sequences that mimic glycoconjugates found on the surface of human epithelial cells
or in host secretions (Preston et al.. Critical Reviews in Microbiology 23(3): 139-180 (1996)).
Such enzymes include, but are not limited to, the proteins of the rfa operons of species such
as E. coli and Salmonella typhimurium, which include a p 1,6 galactosyltransferase and a p 1,3
galactosyltransferase {see, e.g., EMBL Accession Nos. M80599 and M86935 {E. coli);
EIvlBL Accession No. S56361 (iS*. typhimurium)), a glucosyltransferase (Swiss-Prot
Accession No. P25740 (E. coli), an pi,2-glucosyltransferase (r^J)(Swiss-Prot Accession No.
P27129 (E. coli) and Swiss-Prot Accession No. PI9817 (5. typhimurium)), and an pi,2-N-
acetylglucosaminyltransferase {rfdK)(JEMSL Accession No. U00039 {E. coli). Other
glycosyltransferases for wliich amino acid sequences are known include those that are
encoded by operons such as rfaB, which have been characterized in organisms such as
Klebsiella pneumoniae, E. coli. Salmonella typhimurium. Salmonella enterica. Yersinia
enterocolitica, Mycobacterium leprosum, and the rhl operon of Pseudomonas aeruginosa.
Also suitable for use in the present invention are glycosyltransferases that are
involved in producing structures containing lacto-N-neotetraose, D-gaIactosyI-p-I,4-N-
acetyl-D-glucosaminy]-p-1.3-D-galactosyl-p-l,4-D-glucose, and the P'' blood group
trisaccharide sequence, D-galactosyl-a-l,4-D-galactosyl-P-l,4-D-glucose, which have been
identified in the LOS of the mucosal pathogens Neisseria gonnorhoeae and A^, meningitidis
(Scholten et al., J. Med. Microbiol. 41: 236-243 (1994)). The genes from A'^ meningitidis and
N. gonorrhoeae that encode the glycosyltransferases involved in the biosynthesis of these
structures have been identified from N. meningitidis immunotyj)es L3 and LI (Jennings et al.,
Mol. Microbiol. 18: 729-740 (1995)) and the A^ gonorrhoeae mutant F62 (Gotshlich, J. Exp.
Med. 180: 2181-2190 (1994)). In A", meningitidis, a locus consisting of three genes, IgtA,
IgtB and Ig E, encodes the glycosyltransferase enzymes required for addition of the last three
of the sugars in the lacto-A^-neotctraose chain (Wakarchuk et al, J. Biol. Chem. 271: 19166-
73 (1996)). Recently the eni:ymatic activity of the IgtB and IgtA gene product was
demonstrated, providing the first direct evidence for their proposed glycosyltransferase
function (Wakarchuk et al., J. Biol. Chem. 271(45): 28271-276 (1996)). In A^. gonorrhoeae,
there are two additional genes, IgtD which adds P-D-GalNAc to the 3 position of tlie terminal
galactose of the lacto-A^-neotetraose structure and IgtC which adds a terminal a-D-Gai to the
lactose element of a truncated LOS, thus creating the P*^ blood group antigen structure
(Gotshlich (1994), supra). In N. meningitidis, a separate immunotype LI also expresses tbe
P blood group antigen and has been shown to carry an IgtC gene (Jennings et al, (1995),
supra.). Neisseria glycosyltransferases and associated genes are also described in USPN
5,545,553 (Gotschlich). Genes for al,2-fucosyItransferase and al,3-fucosyltransferase from
Helicobacter pylori has also been characterized (Martin et al, J. Biol. Chem. 272: 21349-
21356 (1997)). Also of use in the present invention are the glycosyltransferases of
Campylobacter Jejuni {see, Taniguchi et al., 2002, Handbook of glycosyltransferases and
related genes. Springer, Tokyo).
B. Sulfotransferases
The invention also provides methods for producing peptides that include sulfated
molecules, including, for example sulfated polysaccharides such as heparin, heparan sulfate,
carragenen, and related corapounds. Suitable sulfotransferases include, for example,
chondroitin-6-sulphotransferase (chicken cDNA described by Fukuta et al., J. Biol. Chem.
270: 18575-18580 (1995); GenBank Accession No. D49915), glycosaminoglycan N-
acetylglucosamine N-deacetylase/N-sulphotransferase 1 (Dixon et al.. Genomics 26: 239-241
(1995); UL18918), and glycosaminoglycan N-acetylglucosamine N-deacetylase/N-
sulphotransferase 2 (murine cDNA described in Orellana et al., J. Biol. Chem. 269: 2270-
2276 (1994) and Eriksson el ai., J. Biol. Chem. 269: 10438-10443 (1994); human cDNA
described in GenBank Accession No. U2304).
C. Cell-Bound Glycosyltransferases
In another embodiment, the enzymes utilized in the method of the in\'ention are cell-
bound glycosyltransferases. Although many soluble glycosyltransferases are kno\v:i (see, for
example, U.S. Pat. No. 5,032,519), glycosyltransferases are generally in membrane-bound
form when associated with ceils. Many of the membrane-bound enzymes studied thus far are
considered to be intrinsic proteins; that is, they are not released from the membranes by
soiiication and require detergents for solubilization. Surface glycosyltransferases have been
identified on the surfaces of vertebrate and invertebrate cells, and it lias also been recognized
tliat tliesc surface transferases maintain catalytic activity luider physiological conditions.
However, die more recognized frmction of cell surface glycosyltransferases is for intercellular
recognition (Roth, 1990, Molecular Approaches to Supracellular Phenomena,).
Methods have been developed to alter the glycosyltransferases expressed by cells.
For example, Larsen et ai., Proc. Natl. Acad. Sci. USA 86: 8227-8231 (1989), report a
genetic approach to isolate cloned cDNA sequences that determine expression of cell surface
oligosaccharide structures and their cognate glycosyltransferases. A cDNA library generated
from mRNA isolated from a murine cell line known to express UDP-galactose:.p.-D-
galactosyI-l,4-N-acetyi-D-gIucosaminide a-l,3-gaIactosyltransferase was transfected into
COS-1 cells. The transfected cells ^yere then cultured and assayed for a 1-3
galactosyltransferase activity.
Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717 (1992), disclose a method
of anchoring p-lactamase to the external surface of Escherichia coli. A tripartite fusion
consisting of (i) a signal sequence of an outer membrane protein, (ii) a membrane-spanning
section of an outer membrane protein, and (iii) a complete mature P-lactamase sequence is
produced resulting in an active surface bound P-lactamase molecule. However, the Francisco
method is limited only to prokaryotic cell systems and as recognized by the authors, requires
the complete tripartite fusion for proper functioning.
D. Fusion Enzymes
In other exemplary embodiments, the methods of the invention utilize fusion peptides
that have more than one enzymatic activity that is involved in synthesis of a desired
glycopeptide conjugate. The fusion peptides can be composed of, for example, a catalytically
active domain of a glycosyltransferase that is joined to a catal.ytically active domain of an
accessory enzyme. The accessory enzyme catalytic domain c£in, for example, catalyze a step
in the formation of a nucleotide sugar that is a donor for the glycosyltransferase, or catalyze a
reaction involved in a glycosyltransferase cycle. For example, a polynucleotide that encodes
a glycosyltransferase can be joined, in-frame, to a polynucleotide that encodes an enzyme
involved in nucleotide sugar synthesis. The resulting fusion peptide can then catalyze not
only the synthesis of the nucleotide sugar, but also the transfer of the sugar moiety to the
acceptor molecule. The fusion peptide can be two or more cycle enzymes linked into one
expressible nucleotide sequence. In other embodiments the fusion ppeptide includes the
catalytically active domains of two or more glycosyltransferases. See, for example, U.S.
Patent No. 5,641,668. The modified glycopeptides of the present invention can be readily
designed and manufactured utilizing various suitable fusion peptides {see, for example, PCT
Patent Application PC17CA98/01180, which was published as WO 99/31224 on June 24,
\999)
E. hnmobilized Enzymes
In addition to cell-bound enzymes, the present invention also provides for the use of
enz>'mes that are immobilized on a solid and/or soluble support. In an exemplar>'
embodiment, diere is provided a glycosyltransferase that is conjugated to a PEG via an intact
glycosyl linker according to the methods of the invention. The PEG-Iinker-enzyme conjugate
is optionally attached to S(5lid support. The use of solid supported enzymes in the methods of
the invention simplifies the work up of the reaction mixture and purification of the reaction
product, and also enables the facile recovery of the enzyme. The glycosyltransferase
conjugate is utilized in the methods of the invention. Other combinations of enzymes and
supports will be apparent to those of skill in the art.
F. Mutagenesis of Glycosyltransferases
The novel forms of the glycosyltransferases, sialyltransferases, sulfotransferases, and
any other enzymes used in the method of the invention can be created using any of the
methods described previously, as well as others well known to those in the art. Of particular
interest are transferases with altered acceptor specificity and/or donor specificity. Also of
interest are enzymes with higher conversion rates and higher stability among others.
The tecliniques of rational design mutagenesis can be used when the sequence of the
peptide is known. Since the sequences as well as many of tlie tertiary structures of the
transferases and glucosidases used in the invention are known, these enzymes are ideal for
rational design of mutants. For example, the catalytic site of the enzyme can be mutated to
alter the donor and/or acceptor specificity of the enzj-me.
I'he extensive teniai-y structural data on the glycosyltransferases and glycosidase
hydrolases also make tliese enzyme idea for mutations involving domain exchanges.
Glycosyltransferases and glycosidase hydrolases are modular enzymes (see. Bourne and
Henrissat, 2001, Current Opinion in Structural Biology 11:593-600). Glycosyltransferases
are divided into two families bases on their structure: GT-A and GT-B. The
glycosyltransferases of the GT-.A family comprise two dissimilar domains, one involved in
nucleotide binding and the other in acceptor binding. Thus, one could conveniently fuse tiie
DNA sequence encoding the domain from one gene in franne with a domain from a second
gene to create a new gene that encodes a protein with a new acceptor/donor specificity. Such
exchanges of domains couid additionally include the carbohydrate modules and other
accessory domains.
The techniques of random mutation and/or directed evolution, as described above,
may also be used to create novel forms of the glycosyltransferases and giycosidases used in
the invention.
IV. In vitro and in vivo expression systems
A. Cells for the production of glycopeptides
The action of glycosyltransferases is key to the glycosylation of peptides, thus, the
difference in the expression of a set of glycosyltransferases in any given cell type affects the
pattern of glycosylation on any given peptide produced in that cell. For a review of host ceil
dependent glycosylation of peptides, see Kabata and Takasaki, "Structure and Biosynthesis of
Ceil Surface Carbohydrates,"' in Cell Surface Carbohydrates and Cell Development, 1991, pp-
1-24 Eds Minoru Fukuda, CRC Press, Boca Raton, FL.
' According to the present disclosure, the type of cell in which the peptide is produced
is relevant only with respect to the degree of remodeling required to generate a pept.de
having desired glycosylation. For example, the number and sequence of enzymatic digestion
reactions and the number and sequence of enzymatic synthetic reactions that are required in
vitro to generate a peptide havmg desired glycosylation will vary depending on the structure
of tiie glycan on the peptide produced by a particular cell type. While the invention should in
no way be construed to be lirmted to the production of peptides from any one particular cell
type including any cell type disclosed herein, a discussion of several cell systems .s now
presented which establishes the power of the present mvention and its independence of the
cell type in which the peptides .are generated.
In general, and to express a peptide from a nucleic acid encoding it, the nucleic acid
must be incorporated into an expression cassette, comprising a promoter element, a
tennmator element, ar.d the coding sequence of the peptide operably linked between the two.
The expression casseUe is then operably linked into a vector. Toward this end, adapters or
lurkers mav be employed to join the nucleotide fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of superfluous nucleotides,
removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair,
restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. A
shuttle vector has the genetic elements necessary for replication in a cell. Some vectors may
be replicated only in prokaryotes, or may be replicated in both prokaryotes and eukaryotes.
Such a plasmid expression vector will be maintained in one or more replication systems,
preferably two replication systems, that allow for stable maintenance within a yeast host cell
for expression purposes, and within a prokaryotic host for cloning purposes. Many vectors
with diverse characteristics are now available commercially. Vectors are usually plasmids or
phages, but may also be cosmids or mini-chromosomes. Conveniently, many commercially
available vectors will have the promoter and terminator of the expression cassette already
present, and a multi-linker site where the coding sequence for the peptide of interest can be
inserted. The shuttle vector containing the expression cassette is then transformed m E. coli
where it is replicated during cell division to generate a preparation of vector that is sufficient
to transform the host cells of the chosen expression system. The above methodology is well
know to those in the art, and protocols by wliich to accomplish can be foimd Sambrook et al.
(2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York).
The vector, once purified from the cells in which it is amplified, is then transformed
into the cells of the expression system. The protocol for transformation depended on the kind
of the cell and the nature of the vector. Transformants are gcowxx in an appropriate nutrient
medium, and, where appropriate, maintained under selective pressure to insure retention of
endogenous DNA. Where expression is inducible, gro\vth can be permitted of the yeast host
to yield a high density of cells, and then expression is induced. The secreted, mature
heterologous peptide can be harvested by any conventional means, and purified by
chromatography, electrophoresi;;, dialysis, solvent-solvent extraction, and the like.
The techniques of molecular cloning are well-known in the art. Further, techniques
for the procedures of aioiecular cloning can be found in Sambrook et al. (2001, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
M.Y.); Glover et al, (1985, DNA Cloning: A Practical Approach, Volumes I and II); Gait et
(1985, Oligonucleotide Syntliesis); Hames and Higgins (1985, Nucleic Acid
Hybridization ); Hames and Higgins (1984, Transcription And Translation); Freshney et al.,
(1986, Animal Cell Culture); Perbal, (1986, Immobilized Cells And Enzymes, IRL Press);
Perbal,(1984, A Practical Guide To Molecular Cloning); Ausubel et al. (2002, Current
Protocols in Molecular Biology, John Wiley & Sons, Inc.).
B. Fungi and yeast
Peptides produced in yeast are glycosylated and the glycan structures present thereon
are primarily high mannose structures. In the case of N-glycans, the glycan structures
produced in yeast may contain as many as nine or more maiuiose residues which may or may
not contain additional sugars added thereto. An example of the type of glycan on peptides
produced by yeast cells is shown in Figure 4, left side. Irrespective of the number of
mannose residues and the type and complexity of additional sugars added thereto, N-glycans
as components of peptides produced in yeast cells comprise a trimannosyl core structure as
shown in Figure 4. When the glycan structure on a peptide produced by a yeast cell is a high
marmose structure, it is a sunple matter for the ordinary skilled artisan to remove, in vitro
using available mannosidase enzymes, ail of the mannose residues from the molecule except
for those that comprise tlie trimannosyl core of the glycan, thereby generating a peptide
having an elemental trimaruiosyl core structure attached thereto. Now, using the techniques
available in the art and armed with the present disclosure, it is a simple matter to
enzymatically add, in vitro, additional sugar moieties to the elemental trimannosyl core
structure to generate a peptide having a desired glycan structure attached thereto. Similarly,
when the peptide produced by tlie yeast cell comprises a high mannose structure in addition
to other complex sugars attached thereto, it is a simple matter to enzymatically cleave off all
of the additional sugars, including exti-a mannose residues, to arrive at the elemental
trimannosyl core structure. Once the elemental trimannosyl core stmcture is produced,
generation of a peptide having desired glycosylation is possible following the directions
provided herein.
By "yeast" is intended ascosporogenous yeasts (Endomycetales), basidiosporogenous
yeasts, and yeast belonging to the Fungi Imperfect! (Blastomycetes). The ascosporogenous
yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceae. The later
is comprised of four subfamilies, Schizusaccharomycoideae (e.g., genus
Schizosaccharoinyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera
Pichia, Kluyveromyces, and Saccharomyces). The basidiosporogenous yeasts include the
genera Leucosporidium, Rhodosporidium, Sporidioholus, Filobasidium, and Filobasidiella.
Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae
(e.g., genera Sporobolomyces, Bullera) and Cryptococcaceae (e.g., genus Candida). Of
particular interest to the present invention are species within the genera Saccharomyces,
Pichia, Aspergillus, Trichoderma, Kluyveromyces, especially K. lactis and K. drosophilum,
Candida, Hansenula, Schizpsaccaromyces, Yarrowia, and Chrysoporium. Since the
classification of yeast may change in the fliture, for the purposes of this invention, yeast shall
be defined as described in Skinner et al., eds. 1980) Biology and Activities of Yeast (Soc.
App. Bacteriol. Symp. Series No. 9).
In addition to the foregoing, those of ordinary skill in the art are presumably familiar
with the biology of yeast and the manipulation of yeast genetics. See, for example, Bacila et
al,, eds. (1978, Biochemistry and Genetics of Yeast, Academic Press, New York); and Rose
and Harrison. (1987, The Yeasts (2"'' ed.) Academic Press, London). Methods of introducing
exogenous DNA into yeast hosts are well known in the art. There are a wide variety of
methods for transformation of yeast. Spheroplast transformation is taught by Hinnen et al
. (1978, Proc. Natl. Acad. Sci. USA 75:1919-1933); Beggs, (1978, Nature 275(5676): 104-
109); and Stinchcomb et al., (EPO Publication No. 45,573; herein incorporated by reference),
Electroporation is taught by Becker and Gaurante, (1991, Methods Enzymol. 194:182-187),
Lithium acetate is taught by Gietz et al. (2002, Methods Enzymol. 350:87-96) and Mount et
al. (1996, Metliods Mol Biol. 53:139-145). For a review of transformation systems of non-
Saccharomyces yeasts, see Wang et al. (Crit Rev Biotechnol. 2001;21(3): 177-218). For
general procedures on yeast genetic engineering, see Barr et al.., (1989, Yeast genetic
engineering , Butterwortlis, Boston).
In addition to wild-type yeast and fungal cells, there are also strains of yeast and fungi
that have been mutated and/or selected to enhance the level of expression of tlie exogenous
gene, and the purity, the post-translational processing of the resulting peptide, and tlie
recovery and purity of the mature peptide. Expression of an exogenous peptide may also be
direct to the cell secretory pathway, as illustrated by the expression of insulin (see (Kjeldsen,
2000, Appl. Microbiol. Bioteclmol. 54:277-286, aiid references cited therein). In general, to
cause the exogenous peptide to be secreted from the yeast cell, secretion signals derived from
yeast genes may be used, such as those of the genes of the killer toxin (Stark and Boyd, 1986,
EMBO J. 5:1995-2002) or of the alpha pheromone (Kurjauand Herskowitz, 1982, Cell 30:933;
Brake et al., 1988, Yeast 4:S436).
Regarding the filamentous fungi in general, methods for genetic manipulation can be
found in Kinghom and Turner (1992, Applied Molecular Genetics of Filamentous Fungi,
Biackie Academic and Professional, New York). Guidance on appropriate vectors can be
found in Martinelli and Kinghom (1994, Aspergillus : 50 years, Elsevier, Amsterdam),
1. Saccharomyces
In Saccharomyces, suitable yeast vectors for use producing a peptide include YRp7
(Stnihl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039,1978), YEpl3 (Broach et al.. Gene
8: 121-133, 1979), POT vectors (Kawasaki et al, U.S. Pat. No. 4,931,373, which is
incorporated by reference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108,
1978) and derivatives thereof. Preferred promoters for use in yeast include promoters for
yeast glycol}^ic gene expression (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980;
Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No.
4,599,311) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of
Microorganisms for Chemicals, Hollaender et al., (eds.), p. 355, Plenum, New York, 1982;
.Ammerer, Meth. Enzymol. 101: 192-201, 1983), and the ADH2-4' promoter (Russell et al..
Nature 304: 652-654, 1983; Irani and Kilgore, U.S. patent application Ser. No. 07/784,653,
CA 1,304,020 and HP 284 044, which are incorporated herein by reference). The expression
units may also include a transcriptional terminator. A preferred transcriptional temiinator is
the TPII tenninator (Alber and Kawasaki, ibid.).
Examples of such ycast-bacteria shuttle vectors include Yep24 (Botstein et al. (1979)
Gene 8:17-24; pCl (Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642-4646), and
Yipl7 (Stnichomb et al. (1982) J. Mol. Biol. 158:157). Additionally, a plasmid expression
vector may be a high or low copy number plasmid, the copy number generally ranging from
about I to about 200. In the case of high copy number yeast vectors, there uill generally be
at least 10, preferably at least 20, and usually not exceeding about 150 copies of the vector in
a single host. Depending upon Uie heterologous peptide selected, either a high or low copy
number \'ector may be desirable, depending upon the effect of tiie vector and the recombinant
peptide on the host. See, for example, Brake et al. (1984) Proc. Natl. Acad. Sci. USA
81 .-4642-4646. DNA constructs of the present invention can also be integrated into the yeast
genome by an integrating vector. Examples of such vectors are known in the art. See, for
example, Botstein et al. (1979) Gene 8:17-24.
The selection of suitable yeast and other microorganism hosts for the practice of the
present invention is withm the skill of the art. Of particular uiterest are the Saccharomyces
species S. cerevisiae, S. carlsbergemis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis,
and iS". oviformis. When selecting yeast host cells for expression of a desired peptide, suitable
host cells may include those shovra to have, inter alia, good secretion capacity, low
proteolj^ic activity, and overall vigor. Yeast and other microorganisms are generally
available from a variety of sources, including the Yeast Genetic Stock Center, Department of
Biophysics and Medical Physics, University of California, Berkeley, Calif; and the American
Type Culture Collection, Manassas VA. For a review, see Strathern et al., eds. (1981, The
Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.)
Methods of introducing CKOgenous DNA into yeast hosts are well known in the art.
2. Pichia
The use oi Pichia methanolica as a host cell for the production of recombinant
peptides is disclosed in PCT Applications WO 97/17450, WO 97/17451, WO 98/02536, and
WO 98/02565. DNA molecules for use in transforming P. methanolica are commonly
prepared as double-stranded, circular plasmids, wliich are preferably linearized prior to
transformation. For peptide production in P. methanolica, it is preferred that the promoter
and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica
alcohol utilization gene (AUGl or AUG2). Other usefiil promoters include those of tlic
dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT)
genes, as well as those disclosed in U.S. Patent No. 5,252,726. To facilitate integration of the
DNA into the host chromosome, it is preferred to have the entire expression segment of the
plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use
in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-
aminoimidazole carboxylase (AIRC; EC 4.1.1.21), wliich ailow.s ade2 host cells to grow in
the absence of adenine. For large-scale, industrial processes where it is desirable to minimize
the use of methanol, host cells in which both methanol utilization genes (AUGl and AUG2)
are deleted are preferred. For production of secreted peptides, host cells deficient in vacuoliir
protease genes (PEP4 and PRBl) are preferred. Electroporation is used to facilitate the
introduction of a plasmid containing DNA encoding a peptide of interest into P. methanolica
cells. It is preferred to transform P. methanolica cells by electroporation using an
exponentially decaying, pulsed electric field having a field strength of fi-om 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most
preferably about 20 milliseconds. For a review of the use of Pichia pastor is for large-scale
production of antibody fragments, see Fischer et al., (1999, Biotechnol Appl Biochem. 30 (P*t
2): 117-120).
3. Asperstilus
Methods to express peptides in Aspergillus spp. are well known ki the art, including
but not limited to those described in Carrez et al., 1990, Gene 94:147-154; Contreras, 1991,
Bio/Technology 9:378-381; Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474;
Tilbum et al., 1983, Gene 26:205-221; Kelly and. Hynes, 1985, EMBO J. 4:475-479;
Ballance et al., 1983, Biochem. Biophys. Res. Conrni. 112:284-289; Buxton et al., 1985,
Gene 37:207-214, and U.S. Pat. No. 4,935,349, incorporated by reference herein in its
entirety. Examples of promoters useful in Aspergillus are fomid in U.S. Patent No.
5,252,726. Strains of Aspergillus useful for peptide expression are found in U.S. Patent No.
4,935,349. Commercial production of exogenous peptides is available from Novoenzymes
for Aspergillus niger and Aspergillus oryzae.
4. Trichoderma
Trichoderma has certain advEintages over other species of recombinant host cells for
expression of desired peptides. This organism is easy to grow in large quantities and it has
the ability to glycosylate and efficiently secrete high yields of recombinant mammalian
peptides into the medium, making isolation of the peptide relatively easy. In addition, the
glycosylation pattern on expressed peptides is more similar to that on human peptides than
peptides expressed in many other systems. However, there are still differences in the glycan
structures on expressed peptides from these cells. For example, terminal sialic acid residues
are important to the therapeutic function of a peptide in a mammalian system, since the
presence of these moieties at the end of the glycan structure impedes peptide clearance from
the mammalian bloodstream. The mechanism behind the increased biologic half-life of
sialylated molecules is believed to lie in their decreased recognition by lectins (Drickamer,
1988, J. Biol. Chem. 26.3:9557-9560). However, in general fUngal cells do not add terminal
sialic acid residues to glycans on peptides, and peptides synthesized in fimgal cells are
therefore asialic. According to the present invention, this deficiency can be remedied using
tlie in vitro glycan remodeling methods of the invention described in detail elsewhere herein.
Trichoderma species useful as hosts for the production of peptides to be remodeled
include T. reesei, such as QM6a, ALK02442 or CBS383.78 (Centraalbureau voor
Schimmelcultures, Oosterstraat 1, PO Box 273, 3740 AG Baam, "The Netherlands, or,
ATCC13 631 (American Type Culture Collection, Manassas VA, 10852, USA, type); T.
viride (such as CBS189.79 (det. W. Gams); T. longibrachiatum, such as CBS816.68 (type);
T. pseiidokoningii (such as MUCL19358; Mycotheque de I'Universite Catholique de
Louvain); T. saturnisporum CBS330.70 (type); T. harzianum CBS316.31 (det. W. Gams); T.
virgatum (T. pseudokoningii) ATCC2496I. Most preferably, the host is T. reesei and more
preferably, it is T. reesei sixains QM9414 (ATCC 26921), RUT-C-30 (ATCC 56765), and
highly productive mutants such as VTT-D-79125, which is derived from QM9414
(Nevalainen, Teclmicai Research Centre of Finland Publications 26, (1985), Espoo, Finland).
The transformation of Trichoderma witli DNA is performed using any teclinique
known in the art, including that taught in European patent No. EP0244234, Harkki (1989,
Bio/Technology 7:596-601) and Uusitalo (1991, J. Biotech. 17:35-50). Culture of
Trichoderma is supported by previous extensive experience in industrial scale fermentation
techniques; for example, see Finkelstein, 1992, Biotechnology of Filamentous Fungi:
Technology and Products, Butterworth-Heinemann, publishers, Stoneham, Mass.
5. Kluyveroniyces
yeast belonging to tiie genus Kluyveromyces have been used as host organisms for the
production of recombinant peptides. Peptides produced by this genus of yeast are, in
particular, chymosin (European Patent 96 430), tiiaumatin (European Patent 96 910),
albimiin, interieukin-1 (3, TPA, TIMP (European Patent 361 991) and albumin derivatives
having a therapeutic limction (European Patent 413 622). Species of particular interest in the
genus Kluyveromyces includi? K. lactis.
Methods of expressing recombinant peptides in Kluyvermyces spp. are well kno'Aoi in
the art. Vectors for the expression and secretion of human recombinant peptides in
Kluyvermyces are known in the art (Yeh, J. Cell. Biochem. Suppl. I4C;68, Abst. H402; Fleer,
1990, Yeast 6 (Special Issue):S449) as are procedures for transformation and expression of
recombinant peptides (Ito et al., 1983, J. Bacteriol. 153:163-168; van den Berg, 1990,
BioATechnology 8:135-139; U.S. Patent No. 5,633,146, WO8304050A1, EP0096910,
EP0241435, EP0301670, EP0361991, all of which are incorporated by reference herein in
their entirety). For a review of genetic manipulation of Kluyveromyces lactis linear DNA
plasmids by gene targeting and plasmid shuffles, see Schaffrath et al. (1999, FEMS Microbiol
Lett. 178(2):201-210).
6. Chrysoporium
The fungal genua Chrysoporium has recently been used to expression of foreign
recombinant peptides. A description of the proceedures by wliich one of skill in the art can
use Chrysoporium can be used to express foreign peptides is found in WO 00/20555
(incorporated by reference herein in its entirety). Species particularly suitable for expression
system include, but are not limited to, C. botryoides, C. carmichaelii, C. crassitunicatum, C.
europae, C. evolceannui, F. fastidium, C. filiforme, C. gerogiae, C. globiferum, C. globiferum
var. articulatum, C. globiferum var. niveum, C. hirundo, C. hispanicum, C. holmii, C.
indicum, C. inops, C. keratinophiliim, C. kreiselii, C. kiizurovianum, C. lignoriim, C.
lobatum, C. lucknowense, C. lucknowense Garg 27K, C. medium, C. medium var. spissescens,
C. mephiticum, C. merdarium, C. merdarium var. roseum, C. minor, C. pannicola, C.
parvum, C. parvum var. crescens, C. pilosum, C. peodomerderium, C. pyriformis, C.
queenslandicum. C. sigleri, C. sidfureum, C. synchronum, C. tropicum, C. undulatum, C.
vallenarense, C. vesperlilium, and C. zonatum.
7. Others
Methods for transforming Schwanniomyces are disclosed in European Patent 394
538. Metiiods for transforming ^cremon/ww chiysogenum are disclosed by VS. Par. No.
5,162,22 8. Methods for transforming Neurospora are disclosed by U.S. Pat. No. 4,486,533.
Also know is an expression s>'stem specifically for Schizosaccharomyces pornhe (l:uropean
Patent 385 391). General methods for expressing peptides in fission yeast,
Schizosaccharomyces pombe can be found in Giga-Hama and Kumagai (1997, Foreign gene
expression in fission yeast: Schizosaccharomyces pombe. Springer, Berlin).
C. Mammalian systems
As discussed above, mammalian cells typically produce a heterogeneous mixture of
N-glycan structures which vary with respect to the number and arrangement of additional
sugars attached to the trimannosyl core. Typically, mammalian cells produce peptides having
a complex glycan structure, such as that shown in Figure 3, right side. Usmg the methods of
the present invention, a peptide produced m a mammalian cell may be remodeled in vitro to
generate a peptide having desired glycosylation by first identifying the primary glycan
structure and then determining which sugars must be removed in order to remodel the glycan
structure. As discussed herein, the sugars to be removed will determine which cleavage
enzymes will be used and thus, the precise steps of the remodeling process will vary
depending on the primary glycan structure used as the initial substrate. A sample scheme for
remodeling a glycan structure commonly produced in roarmnalian cells is shown in Figure 2.
The N-glycan biosynthetic pathway in mammalian cells has been well characterized
(reviewed in Moremen, 1994, Glycobioiogy 4:113-125). Many of the enzymes necessary for
glycan synthesis have been identified, and mutant cell lines defective in this enzymatic
pathway have been isolated including the Chinese hamster ovju'y (CHO) cell lines Lec23
(defective in alpha-glucosidase I) and Led 8 (novel GlcNAc-TVlIl). The glycosylation
pattern of peptides produced by these mutant cells is altered relative to normal CHO cells.
As discussed herein, the glycosylation defects in these and other mutant cells can be
exploited for tlie purposes of producing a peptide that lacks a complex glycan structure. For
example, peptides produced by Lec23 cells lack sialic acid residues, and thus require less
enzymatic manipulation in order to reduce tlie glycan structure to an elemental trimarmosyl
core or to Man3GlcNAc4. Thus, peptides produced in these ceils can serve as preferred
substrates for glycan remodeling. One of ordinary skill in the art could isolate or identify
other gljcosylatiou-dcfective ceil lines based on knoxsii methods, for example the metliod
de.-scribed m Stanley et af, 1990, Somatic Cell Mol. Genet., 16: 211-223. Use of
alycosj'lation-defectivc cell lines, those identified and as yet unidentified, is mcluded in tlie
invention for the purpose of generating preferred peptide substrates for the remodeling
processes described herein.
Expression vectors useful for expressing exogenous peptides in mmtmaliaa cells are
numerous, and are well known to those in the art. Many marDmalian expression vectors are
now connnercially available from compmies, including Novagen, Inc (Madison, WI), Gene
Therapy Systems (San Diego, CA), Promega (Madison, WI), CJonTech Inc. (Palo Alto, CA),
aad Stratagene (La JoUa, CA), among others.
There are several mammalian cell lines that are particularly adept at expressing
exogenous peptides. Typically mammalian cell lines originate from tumor cells extracted
&om mammals that have become immortalized, that is to say, they can replicate in culture
essentially indefinitely. These cell lines mclude, but are not limited to, CHO (Chinese
hamster ovary, e.g. CHO-Kl; ATCC No. CCL 61) and variants thereof, NSO (mouse
myeloma), BNK, BliK 570 (ATCC No. CRL 10314), BHK (ATCC No. CRL 1632),
Per.C6™ (immortalized human cells, Crucell N.V., Leiden, The Netherlands), COS-1 (ATCC
No. CRJ. 1650), COS-7 (ATCC No. CRL 1651), HEK 293, mouse L cells, T lymphoid cell
lines, BW5147 cells and MDCK (Madin-Darby canine kidney), HeLa (human), A549 (human
lung carcinoma), 293 (ATCC No. CRL 1573; Graham et al., 1977, Gen. Virol. 36:59-72),
BGMK (Buffalo Green Monkey kidney), Hep-2 (human epidermoid larynx carcmoma), LLC-
MK2 (African Green Monke>- Kidney), McCoy, NCI-H292 (human pulmonary
miicoepidermoid carcinoma tabe), RD (rhabdomyosarcoma), Vero (African Green Monkey
kidney), ITEL (human embryonic lung). Human Fetal Lung-Chang, MRC5 (human
embryonic lung), MRHF (human foreskin), and Wl-38 (human embryonic lung). In some
cases, the cells in which the tiierapeutic peptide is expressed may be cells derived from the
patient to be treated, or they may be derived from another related or unrelated mammal. For
example, fibroblast cells may be isolated from the mamLmal's skin tissue, and cultured and
transformed in vitro. Tliis technology is commercially available from Transkaryotic
Therapies, Inc. (Cambridge, MA). Almost all currently used cell lines are available from the
Am adjusting the recombinant DNA sequences (see, Kusnadi et al.,
1997, Biotechnol. Bioeng. 56:473-484; Khoudi et al., 1999, Biotechnol. Bioeng. 135-143;
Hood et al, 1999, Adv. Exp. Med. Biol. 464:127-147). In addition, peptide synthesis,
secretion and post-translational modification are very similar in plants and animals, with only
minor differences in plant glycosylation (see, Fischer et al., 2000, J. Biol. Regul. Homest.
Agents 14: 83-92). Then, products from transgenic plants are also less likely to be
contaminated by animal pathogens, microbial toxins and oncogenic sequences.
The expression of recombinant peptides in plant cells is well knovtoi in the art. In
addition to transgenic plants, peptides can also produced in transgenic plant cell cultures (Lee
et al., 1997, Mol. Cell. 7:783-787), and non-transgenic plants inoculated with recombinant
plant \'iruses. Several books have been published that describe protocols for the genetic
transformation of plant cells: Potrykus (1995, Gene transfer to plants. Springer, New York).
Nickoloff (1995, Plant cell electroporation and electro fusion protocols, Humana Press,
Totowa, New York) and Draper (1988, Plant genetic transformation, Oxford Press. Boston'
Several methods are currently used to stably transform plant cells with recombinant
genetic material. These methods include, but are not limited to, Agrobacterium
transformation (Bechtold and Pelletier, 1998; Escudero and Hohn, 1997; Hansen and Chilton,
1999; Touraev et al., 1997), biolistics (microprojectiles) (Finer et al., 1999; Hansen and
Chilton, 1999; Shilito, 1999), electroporation of protoplasts (Fromm et al., 1985, Ou-Lee et
al., 1986; Rhodes et al., 1988; Saunders et al., 1989; Trick et al., 1997), polyethylene glycol
treatment (Shilito, 1999; Trick et al., 1997), inplanta mircroinjection (Leduc et al., 1996;
Zhou et al., 1983), seed imbibition (Trick et al., 1997), laser be;am (1996), and silicon carbide
whiskers (Thompson et al., 1995; U.S. Patent Appln. No. 20020100077, incorporated by
reference herein in its entirety).
Many kinds of plants are amenable to transformation and expression of exogenous
peptides. Plants of particular interest to express the peptides to be used in the remodeling
method of the invention include, but are not limited to, Arabidopsis thalliana, rapeseed
(Brassica spp.; Ruiz and Blumwald, 2002, Planta 214:965-969)), soybean (Glycine max),
sunflower (Helianthus unnuus), oil palm (Elaeis guineeis), groundnut (peanut, Arachis
hypogaea; Deng et al., 2001, Cell. Res. 11:156-160), coconut (Cocus nuciferd), castor
{Ricinus communis), safflovv'er (Carthamus tinctorius), mustard (Brassica spp. and Sinapis
alba), coriander, {Coriandrum sativum), squash (Cucurbita maxima; Spencer and Snow,
2001, Heredity 86(Pt 6):694-702), linseed/flax (Linum usitatissimum; Lamblin et al., 2001,
Physiol Plant 112:223-232), Brazil nut (Bertholletia excels a), ]0]ohQ. {Simmondsia chinensis),
maize (Zea mays; Hood et al., 1999, Adv. Exp. Med. Biol. 464:127-147; Hood et al., 1997,
Mol. Breed. 3:291-306; Petolino et al., 2000, Transgenic Research 9:1-9), alfalfa (Khoudi et
al., 1999, Biotechnol. Bioeng. 64:135-143), tobacco {Nicotiana tabacum; Wright et al..
Transgenic Res. 10:177-181; Frigerio et al., 2000, Plant Physiol. 123:1483-1493; Cramer et
al., 1996, Ann. New York Acad. Sci. 792:62-8-71; Cabanes-Macheteau et al., 1999,
Glycobiology 9:365-372; Ruggiero et al., 2000, FEBS Lett. 469:132-136), canola (Bai et al
2001, Bioteclmol. Prog. 17:168-174; Zhang et a!., 2000, J. Anim. Sci. 78:2868-2878)), potato
(Tacket et al., 1998, J. Infect. Dis. 182:302-305; Richteret al., 2000, Nat. Biotechnol.
18:1167-1171; Chong et al., 2000, Transgenic Res. 9:71-78), alfalfa (Wigdorovitz et al..
1999, Virology 255:347-353), Pea (Pisum sativum; Perrin et al., 2000, Mol. Breed. 6:345-
352), rice {Oiyza saliva ; Stoger et al, 2000, Plant Mol. Biol. 42:583-590), cotton
{Gossypium hirsutum; Komyeyev et al., 2001, Physiol Plant 113:323-331), barley {Hordeum
vulgare; Petersen et al., 2002, Plant Mol Biol 49:45-58); wheat {Triticum spp.; Pellegrineschi
et al., 2002, Genome 45:421-430) and bean {Vicia spp.; Saalbach et al., 1994, Mol Gen Genet
242:226-236).
If expression of the recombinant nucleic acid is desired in a whole plant rather than in
cultured cells, plant cells are first transformed with DNA encoding the peptide, following
which, the plant is regenerated. This involves tissue culture procedures that are typically
optimized for each plant species. Protocols to regenerate plants are already well known in the
art for many species. Furthermore, protocols for other species can be developed by one of
skill in the art using routine experimentation. Numerous laboratory manuals are available
that describe procedures for plant regeneration, including but not limited to. Smith (2000,
Plant tissue culture : techniques and experiments. Academic Press, San Diego), Bhojwani £ind
Razdan (1996, Plant tissue culture : theory and practice, Elsevier Science Pub., Amsterdam),
Islam (1996, Plant tissue culture, Oxford & EBH Pub. Co., New Delhi, India), Dodds and.
Roberts ( 1995, Experiments in plant tissue culture. New York : Cambridge University Press,
Cambridge England), Bhojwani (Plant tissue culture : applications and limitations, Elsevier,
Amsterdam, 1990), Trigiano and Gray (2000, Plant tissue culture concepts and laboratory
exercises,. CRC Press, Boca Raton, Fla), and Lindsey (1991, Plant tissue culture manual;
fundamentals and applications, Kluwer Academic, Boston).
W^le purifying recombinant peptides from plants may potentially be costly, several
systems have been deN'cloped to minimize these costs. One method directs the synthesized
peptide to tlie seed endosperm from where it can easily extracted (Wright et al., 2001,
Transgenic Res. 10:177-181, Guda et a., 2000, Plant Cell Res. 19:257-262; and U.S. Patent
No. 5,767,379, which is incoiporated by reference herein in its entiret\'). An alternative
ajDproach is the co-extraction of the recombinant peptide with conventional plant products
such as starch, meal or oil. In oil-seed rape, a fusion peptide of oleosin-hurudin when
expressed in the plant, attaches to the oil body of the seed, and can be extracted from the
plant seed along with the oil (Parmenter, 1995, Plant Mol. Biol. 29:1167-1180; U.S. Patent
Nos. 5,650,554, 5,792,922, 5,948,682 and 6,288,304, and US application 2002/0037303, all
cf which are incorporated in their entirely by reference herein). In a variation on tins
approach, the oleosin is fused to a peptide having affmit)' for the exogenous co-expressed
peptide of interest (U.S. Patent No. 5,856,452, incorporated by reference herein in its
entirety).
Expression of recombinant peptides in plant plastids, such as the chloroplast,
generates peptides having no glycan structures attached thereto, similar to the situation in
prokaryotes. However, tlie yield of such peptides is vastly greater when expressed in these
plant cell organelles, and thus this type of expression system may have advantages over other
systems. For a general review on the technology for plastid expression of exogenous peptides
in higher plants, see Hager and Beck (2000, Appl. Microbiol. Biotechnol. 54:302-310, and
references cited therein). Plastid expression has been particularly successful in tobacco (see,
for example, Staub et al., 2000, Nat. Biotechnol. 18:333-338).
F. Transgenic aniinals
Introduction of a recombinant DNA into the fertilized egg of an animal (e.g., a
mammal) may be accomplished using any number of standard techniques in transgenic
animal teclmology. See, e.g., Hogan et al.. Manipulating the Mouse Embr>'o: A Laboratory'
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986; and U.S.
Pat. No. 5,811,634, which is incorporated by reference herein in its entirety. Most
commonly, the recombinant DNA is introduced into the embryo by way of pronuclear
microinjection (Gordon et al., 1980, PNAS 77:7380-7384; Gordon and Ruddle, 1981, Science
214:1244-1246; Brinster et al., 1981, Cell 27:223-231; Costantini and Lacy, 1981, Nature
294:92-94). Microinjection has the advantage of being applicable to a wide variety of
species. Preimplantation embryos may also be transformed with retroviruses (Jaenisch and
Mintz, 1974, Proc. Natl. Acad. Sci. U.S.A. 71:1250-1254; Jaenisch et al., 1976, Hamatol
Bluttransfus. 19:341-356; Stulihnajin et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:7151-
7155). Retroviral mediated transformation has the advantage of adding single copies of the
recombinant nucleic acid to the cell, but it produces a high degree of mosaicism. Most
recently, embryonic stem cell-mediated tecliniques have been used (Gossler et al., 1986,
Proc. Natl. Acad. Sci. U.S.A.. 83:9065-9069), transfer of entire chromosomal segments
(Lavitnmo et al., 1989, Cell 57:717-723), and gamete transfection in conjunction with in vilru
fertilization (Lavitrano et al., 1989, Cell 57:717-723) have also been used. Several books of
laboratory procedures have been published disclosing these tecliniques: Cid-.Ajregui and
Garcia-Carranca (1998, Microinjection and Transgenesis : Strategies and Protocols, Spriiigei.
Berlin), Clarke (2002, Transgenesis Techniques : Principles and Protocols, Humana Press,
Totowa, NJ), and Pinkert (1994, Transgenic Animal Technology : A Laboratory Handbook.,
Academic Press, San Diego).
Once the recombinant DNA is introduced into the egg, the egg is incubated for a short
period of time and is then transferred into a pseudopregnant animal of the same species from
which the egg was obtained (Hogan et al., supra). In the case of mammals, typically 125
eggs are injected per exj^eriment, approximately two-thirds of which will survive the
procedure. Twenty viable eggs are transferred into a pseudopregnant mammal, four to ten of
which will develop into live progeny. Typically, 10-30% of the progeny (in the case of mice)
carry the recombinant DNA.
While the entire animal can be used as an expression system for the peptides of the
invention, in a preferred embodiment, the exogenous peptide accumulates in products of the
animal, from which it can be harvested without injury to the animal. In preferred
embodiments, the exogenous peptide accumulates in milk, eggs, hair, blood, and urine.
If the recombinant peptide is to be accumulated in the milk of the animal, suitable
manunals are ruminants, ungulates, domesticated mammals, and dairy animals. Particularl}'
preferred animals are goats, sheep, camels, cows, pigs, horses, oxen, and llamas. Methods lor
generating transgenic cows that accumulate a recombinant peptide in their miUc are well
known: see, Newton (1999, J. Inununol. Metliods 231:159-167), Ebert et al. (1991,
Bioteclmology 9: 835-838), and U.S. Patent Nos. 6,210,736, 5,849,992, 5,843,705,
5,827,690, 6,222,094, all of which are incorporated herein by reference in their entirety. The
generation of transgenic mammals that produce a desired recombinant peptide is
commercially available from GTC Biotherapeutics, FramLngham, MA.
If the recombinant peptide is to be accumulated in eggs, suitable birds include, but aie
not limited to, chickens, geese, and turkeys. Other animals of interest include, but are not
limited to, other species of avians, fish, reptiles and amphibi^ms. The introduction of
recombinant DNA to a chicken by retroviral transformation is well known in the art:
Thoraval et al. (1995, Transgenic Research 4:369-376), Bosselman et al., (1989, Science 243;
533-535), Petropoulos el al. (1992, J. Virol. 66: 3391-3397), U.S. Patent No. 5,162,215.
incorporated by reference herein in its entirety. Successful transformation of chickens with
recombinant DNA also been achieved wherein DNA is introduced into blasiodennai ceils
and blastodermal cells so transfected are introduced into the embryo: Brazolot et al. (1991,
Mol. Reprod. Dev. 30: 304-312), Fraser, et al. (1993, Int. J. Dev. Biol. 37: 381-385), and
Petitte et al. (1990, Development 108: 185-189). High throughput technology has been
developed to assess whether a transgenic chicken expresses the desired peptide (Harvey et al.,
2002, Poult. Sci. 81:202-212, U.S. Patent No. 6,423,488, incorporated by reference herein in
its entirety). Using retroviral transformation of chicken with a recombinant DNA, exogenous
beta-lactamase was accumulated in the egg white of the chicken (Harvey et al., 2002, Nat.
Biotechnol. 20(4):396-399). The production of chickens producing exogenous peptides in
egg is commercially available from AviGenics, Inc., Athens GA.
G. Bacteria
Recombinantl)' expressed peptides produced in bacteria are not generally
glycosylated. However, bacteria systems capable of glycosylating peptides are becoming
evident and therefore it is likely that glycosylated recombinant peptides may be produced in
bacteria in the future.
Numerous bacterial expression systems are known in the art. Preferred bacterial
species include, but are not limited to, E. coli. and Bacillus species.
The expression of recombinant peptides in E. coli is well laiown in the art. Protocols for E.
co/z-based expression systems are found in U.S. Appln No. 20020064835, U.S. Patent Nos.
6,245,539, 5,606,031, 5,420,027, 5,151,511, andRE33,653, among others. Methods to
transform bacteria include, but are not limited to, calcium chloride (Cohen et al., 1972, Proc.
Natl. Acad. Sci. U.S.A.. 69:2110-2114; Hanahan, 1983, J. Mol. Biol. 166:557-580; Mandel
and Higa, 1970, J. Mol, Biol. 53:159-162) and electroporadon (Shigekawa and Dower, 1988,
Bioteclmiques 6:742-751), and those described in Sambrook et al., 2001 (supra). For a
review of laboratory protocols on microbial transformation and expression systems, see
Saunders and Saunders (1987, Microbial Genetics Applied to Biotechnology : Principles and
Tecliniques of Gene Trcinsfer and Manipulation, Croom Helm, London), Piihler (1993,
Genetic Engineering of Microorganisms, Weinheim, New York), Lee et al., (1999, Metabolic
Engineering, Marcel Dekker, New York), Adolph (1996, Microbial Genome Methods, CRC
Press, Boca Raton), and Birren and Lai (1996, Nonmanunalian Genomic Analysis : A
Practical Guide, Academic Press, San Diego),
For a general review on the literature for peptide expression in E. coli see Balbas
(2001, Mol. Biotechnol. 19:251-267). Several companies now offer bacterial strains selected
for the expression of mammalian peptides, such as the Rosetta™ strains of £¦. coli (Novagen,
inc., Madison, WI; with enlianced expression of eukaryotic codons not normally used in
bacteria cells, and enhanced disulfide bond formation),
H. Cell engineering
It will be apparent from the present disclosure that the more uniform the starting
material produced by a cell, the more efficient will be the generation in vitro of large
quantities of peptides having desired glycosylation. Thus, the genetic engineering of host
cells to produce uniformly glycosylated peptides as starting material for the in vitro
enzymatic reactions disclosed herein, provides a significant advantage over using a peptide
starting material having a heterogeneous set of glyean structures attached thereto. One
preferred peptide starting material for use in the present invention is a peptide having
primarily glycan molecules wliich consist solely of an elemental trimannosyl core structure.
Anotlier preferred starting material is Man3GlcNAc4. Following the remodeling process, the
preferred peptides will give rise to the greatest amount of peptides having desired
glycosylation, and tlius improved clinical efficacy. However, other glycan starting material is
also suitable for use in the methods described herein, in that for example, high mannose
gl yeans may be easily reduced, in vitro, to elemental trimamiosyl core structures using a
series of raannosidases. As described elsewhere herein, other glycan starting material may
also be used, provided it is possible to cleave off all extraneous sugar moieties so that the
elemental trimannosyl core structure or Man3GlcNAc4 is generated. Thus, the purpose of
using genetically engineered cells for the production of the peptides of the present inventio .
is to generate peptides having as uniform as possible a glycan structure attached thereto,
wherein the glycan structure caii be remodeled in vitro to generate a peptide having desired
glycosylation. This will result in a dramatic reduction in production costs of these peptides.
Since the glycopeptides produced using this methodology will predominantly have the same
N-linked glycan structure, tlie post-production modification protocol can be standardized and
optimized to produce a greater batch-to-batch consistency of final product. As a result, the
final completed-chain products may be less heterogeneous than those presently available.
The products will have an improved biological half-life and bioactivity as compared to the
products of the prior art. Alternatively, if desired, the invention can be used to introduce
limited and specific heterogeneity, e.g., by choosing reaction conditions that result in
differential addition of sugar moieties.
Preferably, though not as a rigid requirement, the genetically engineered cell is one
which produces peptides having glycan structures comprised primarily of an elemental
trimannosyl core structure or Man3GlcNAc4. At a minimum, the proportion of these
preferred structures produced by the genetically engineered cell must be enough to yield a
peptide having desired glycosylation following the remodeling protocol.
In general, any eukaryotic cell type can be modified to become a host cell of the
present invention. First, the glycosylation pattern of both endogenous and recombinant
glycopeptides produced by the organism are determined in order to identify suitable
additions/deletions of enzymatic activities that result in the production of elemental
trimannosyl core glycopeptides or Man3GlcNAc4 glycopeptides. This will typically entail
deleting activities that use trimannosyl glycopeptides as substrates for a glycosyltransferase
reaction and inserting enzymatic activities that degrade more complex N-linked glycans to
produce shorter chains. In addition, genetically engineered cells may produce high mannose
glycans, which may be cdeaved by mannosidase to produce desired starting glycan structures.
The mannosidase may bs active in vivo in the cell (i.e., the cell may be genetically engineered
to produce them), or the;/ may be used in in vitro post production reactions.
Teclmiques for genetically modifying host cells to alter the glycosylation profile of
expressed peptides are well-known. See, e.g., Altmann et al. (1999, Glycoconjugate J. 16:
109-123), Ailor et al. (2000, Glycobiology 10(8): 837-847), Jarvis et al., {In vitrogen
Conference, March, 1999, abstract), Hollister and Jarvis, (2001, Glycobiology 11(1): 1-9),
and Palacpac et al., (1999, PNAS USA 96: 4697), Jarvis et al., (1998. Curr. Opin. Biotechnol.
9:528-533), Gemgross (U.S. Patent Publication No. 20020137134), all of which disclose
techniques to "mammalianize" insect or plant cell expression systems by transfecting insect
or plant cells with glycosyltransferase genes.
Teclmiques also exist to genetically alter the glycosylation profile of peptides
expressed in £. coli. E. coli has been engineered witli various glycosyltransferases from the
bacteria Neisseria meningitidis and Azorhizobium to produce oligosaccharides in vivo (Bettle/
et al.. 1999, Glycoconj. J. 16:205-212). E. coli which has been genetically engineered to
over-express Neisseria meningitidis pl,3 N acetyl glucosaminyltransferase IgtA gene will
efficiently glycosylate exogenous lactose (Priem et al., 2002, Glycobiology 12:235-240).
Fungal cells have also been genetically modified to produce exogenous
glycosyltransferases (Yoshida et al., 1999, Glycobiology, 9(l):53-58; Kalsner et al., 1995,
Glycoconj. J. 12:360-370; Schwientek and Ernst, 1994, Gene I45(2):299-303; Chiba et al,
1995, Biochem J. 308:405-409).
Thus, in one aspect, the present invention provides a cell that glycosylates a
glycopeptide population such that a proportion of glycopeptides produced thereby have an
elemental trimannosyl core or a Man3GlcNAc4 structure. Preferably, the cell produces a
peptide having a glycan structure comprised solely of an elemental trimannosyl core. At a
minimum, the proportion of peptides having an elemental trimannosyl core or a
Man3GlcNAc4 structure is enough to yield peptides havmg desked glycosylation following
tlie remodeling process. The cell has hitroduced into it one or more heterologous nucleic acid
expression units, each of which may comprise one or more nucleic acid sequences encoding
one or more peptides of interest. The natural form of the glycopeptide of interest may
comprise one or more complex N-linked glycans or may simply be a high mannose glycan.
The cell may be any type of cell and is preferably a eukaryotic cell. The cell may be a
mammalian cell such as human, mouse, rat, rabbit, hamster or other type of mammalian cell.
When the cell is a mammalian cell, tlie mammalian cell may be derived from or contained
within a non-human transgenic mammcd where the cell in the mammal encodes the desired
glycopeptide and a variety of glycosylating and glycosidase enzymes as necessary for the
production of desired glycopeptide molecules. In addition, the cell may be a fungal cell,
preferably, a yeast cell, or the cell may be an insect or a plant cell. Similarly, when the cell is
a plant cell, the plant cell may be derived from or contamed within a transgenic plant,
wherein the plant encodes tiie desired glycopeptide and a variety of glycosylating and
glycosidase enzymes as are necessary for the production of desired glycopeptide molecules.
In some embodiments the host cell may be a eukaryotic cell expressing one or more
heterologous glycosyltransferase enzymes and/or one or more heterologous glN'cosidase
enzymes, wherein expression of a recombinant glycopeptide m the host cell results in the
production of a recombinant glycopeptide having an elemental trimannosyl core as the
primary glycan structure attached thereto.
In some embodiments the heterologous glycosyltransferase enzyme useful in the cell
may be selected from a group consisting of any known glycosyltransferase enzyme included
for example, in the list of Glycosyltransferase Families available in Taniguchi et al. (2002,
Handbook of Glycosyltransferases and Related Genes, Springer, New York).
In other embodiments, the heterologous glycosylase enzyme may be selected from a
group consisting of mannosidase 1, mannosidase 2, mannosidase 3, and other mannosidases,
including, but not limited to, microbial mannosidases. Additional disclosure regarding
enzymes useful in the present invention is provided elsewhere herein.
In yet other embodiments, the host cell may be a eukaryotic cell wherein one or more
endogenous glycosyltransferase enzymes and/or one or more endogenous glycosidase
enzymes have been inactivated such that expression of a recombinant glycopeptide in the
host cell results in the production of a recombinant glycopeptide having an elemental
trimannosyl core as the primary glycan structure attached thereto.
In additional embodiments, the host cell may express heterologous
glycosyltransferase enzymes and/or glycosidase enzymes while at the same time one or more
endogenous glycosyltransferase enzymes and/or glycosidase enzymes are inactivated.
Endogenous glycosyltransferase enzymes and/or glycosidase enzymes may be inactivated
using any teclmique known to those skilled in the art includmg, but not limited to, antisense
techniques and techniques involving insertion of nucleic acids into the genome of the host
cell. In some embodiments, the endogenous enzymes may be selected from a group
consisting of GnT-I, a selection of mannosidases, xylosyitransferase, core a.1,3
fticosyltransferase, serine/threonine O-mannosyitransferases, and tlie like.
Alternative 1}', an expression system that naturally glycosylates peptides such that the
N-linked glycans are predominantly the trimannosyl core type, or the Man3GlcNAc4 type,
can be exploited. An example of a cell type that produces the trunannosyl core is Sf9 cells.
Other such expression systems can be identified by analyzing glycopeptides that are naturally
or recombinantiy expressed in cells and selecting those which exhibit the desired
glycosylation characteristics. The mvention should be construed to include any and all such
cells for the production of the peptides of the present invention.
V. Purification of glvcan remodeled and/or glvcoconiugated peptides
If tlie modified glycoprotein is produced intracellularly or secreted, as a first step, the
particulate debris, either host cells, lysed fi-agments, is removed, for example, by
centrifugation or ultrafiltration; optionally, the protein may be concentrated with a
commercially available protein concentration filter, followed by separating the peptide
variant fi:om other impuiities by one or more steps selected from immunoaffinity
chromatography, ion-exchange column fi-actionation (e.g., on diethylaminoethyl (DEAE) or
matrices containing carboxymethyl or sulfopropyl groups), chromatography on Blue-
Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose, WGA-
Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, or
protein A Sepharose, SDS-PAGE chromatography, silica chromatography,
chromatofocusing, reverse phase HPLC (RP-HPLC), gel filtration using, e.g., Sephadex
molecular sieve or size-exclusion chromatography, chromatography on columns that
selectively bind the peptide, and ethanol, pH or ammonium sulfate precipitation, membrane
filtration and various techniques.
Modified peptides produced in culture are usually isolated by initial extraction from
cells, enzymes, etc., followed by one or rqore concentration, salting-out, aqueous ion-
exchange, or size-exclusion chromatography steps. Additionally, the modified glycoprotein
may be purified by affinity chromatography. Then, HPLC may be employed for final
purification steps.
A protease inhibitor, e.g., phenylmethylsulfonylfluoride (PMSF) may be included in
any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent
the growlh of adventitious contaminants.
Within another embodiment, supematants fi-om systems which produce the modified
peptide of the invention are first concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following tlie concentration step, the concentrate may be applied to a suitable purification
matrix. For example, a suitable affinity matrix may comprise a ligand for the peptide, a lectin
or antibody molecule bound to a suitable support. Alternatively, an anion-exchange resin
may be employed, for example, a matrix or substrate having pendant DE^VE groups. Suitable
matrices include acrylamide, agarose, dextran, cellulose, or other types cormiionly employed
in protein purification. Alternatively, a cation-exchange step may be employed. Suit^ible
cation exchangers include various insoluble matrices comprising sulfopropyl or
carboxymethyl groups. Sulfopropyl groups are particularly preferred.
Then, one or more RP-HPLC steps employing hydrophobic RP-HPLC media, e.g.,
silica gel having pendant methyl or other aliphatic groups, may be employed to further purify
a peptide variant composition. Some or all of the foregoing purification steps, in various
combinations, can also be employed to provide a homogeneous modified glycoprotein.
The modified peptide of the invention resulting from a large-scale fermentation may
be purified by methods analogous to those disclosed by Urdal et al, J. Chromatog. 296: 171
(1984). This reference describes two sequential, RP-HI'LC steps for purification of
recombinant human IL-2 on a preparative HPLC column. Alternatively, techniques such as
affinity chromatography may be utilized to purify the modified glycoprotein.
VL Preferred Peptides and Nucleic Acids Encoding Preferred Peptides
The present invention includes isolated nucleic acids encoding various peptides and
proteins, and similar molecules or fragments thereof. Tlie invention should not be construed
to be limited in any ^vay solely to the use of these peptides in the methods of tlie invention,
but rather should be construed to include any and all peptides presently available or which
become available to those in the art. In addition, the invention should not be construed to
include only one particular nucleic acid or amino acid sequence for the peptides listed herein,
but rather should be construed to include any and all variants, homologs, mutants, etc. of each
of the peptides. It should be noted tliat when a particular peptide is identified as having a
mutation or other alteration in tlie sequence for that peptide, the numbering of the aminc'
acids which identify the alteration or mutation is set so that the first amino acid in the mature
peptide sequence is amino acid no. 1, unless otherwise stated herein.
Preferred peptides include, but are not limited to human granuloc54e colony
stimulating factor (G-CSF), human interferon alpha (IFN-alpha), human interferon beta (IFN-
beta), human Factor \'II (Factor VII), human Factor IX (Factor DC), human follicle
stimulating hormone (FSH), human erythropoietin (EPO), human granulocyte/macrophage
colony stimulating factor (GM-CSF), human interferon gamma (IFN-gamma), hiunan alpha-
1-protease inliibitor (also known as alpha-]-antitrypsin or alpha-1-trypsin inhibitor; A-l-PI),
glucocerebrosidase, human tissue-type activator (TPA), human interleukin-2 (IL-2), human
Factor VIII (Factor VIII), a 75 kDa tumor necrosis factor receptor fused to a human IgG
immunoglobulin Fc portion, commercially known as ENBREL™ or ETANERCEPT^m
(chimeric TNFR), human urokinase (urokinase), a Fab fragment of the human/mouse
cliimeric monoclonal antibody that specifically binds glycoprotein lib/ Ilia and the
vitronectin alphav beta.3 receptor, known commercially as JREOPRO™ or ABCIXIMAB
(cliimeric anti-glycoprotein Ilb/IIIa), a mouse/human chimeric monoclonal antibody that
specifically binds human HER2, known commercially as HERCEPTINtm (chimeric anti-
HER2), a human/mouse chimeric antibody that specifically binds the A antigenic site or the F
protein of respiratory syncytial virus commercially known as SYNAGIS'^'^ or
PALIVIZUMAB (chimeric anti-RSV), a chimeric himian/mouse monoclonal antibody that
specifically binds CD20 on human B-cells, known commercially as RITUXANtm or
RITUXAMAB (chimeric anti-CD20), human recombinant DNase (DNase), a chimeric
human/mouse monoclonal antibody that specifically binds human tumor necrosis factor,
known commercially as REMICADEtm or INFLIXIMAB (chimeric anti-TNF), hiunan
insulin, the surface antigen of a hepatitis B virus (adw subtype; HBsAg), and human growth
homione (UGH), aJpha-galactosidase A (Fabryzyme^""), a-Iduronidase (AldurazymeTW),
antithrombin (antithrombin III, AT-III), human chorionic gonadotropin (hCG), interferon
omega, and the like.
The isolated nucleic acid of the invention should be construed to include an KNA or a
DNA sequence encoding any of the above-identified peptides of the invention, and any
modified fonns thereof, including chemical modifications of the DNA or RNA which render
the nucleotide sequence more stable when it is cell free or when it is associated with a cell.
As a non-limiting example, oligonucleotides which contain at least one phosphorotliioate
modification are known to confer upon the oligonucleotide enhanced resistance to nucleases.
Specific e.vamples of modified oligonucleotides include those which contain
phosphorothioate, phosphotricster, methyl phosphonaie, short chain alkyl or cycloalkyl
intersugar linkages, or short chain heteroatomic or heterocyclic intersugar ("backbone")
linkages. In addition, oligonucleotides having morpholino backbone structures (U.S. Patent
No; 5,034,506) or polyamidc backbone structures (Nielsen ct al., 1991, Science 254: 1497)
may also be used.
Chemical modifications of nucleotides may also be used to enhance the efficiency
with which a nucleotide sequence is taken up by a cell or the efficiency with which it is
expressed in a cell. Any and all combinations of modifications of the nucleotide sequences
are contemplated in the present invention.
The present invention should not be construed as being limited solely to the nucleic
and amino acid sequences disclosed herein. As described in more detail elsewhere herein,
once armed with the present invention, it is readily apparent to one skilled in the art that other
nucleic acids encoding the peptides of the present invention c£m be obtained by following the
procedures described herein (e.g., site-directed mutagenesis, frame shift mutations, and the
like), and procedures that are well-known in the art.
Also included are isolated nucleic acids encoding firagments of peptides, wherein the
peptide firagments retain titie desired biological activity of the peptide. In addition, although
exemplary nucleic acids encoding preferred peptides are disclosed herein in relation to
specific SEQ ID NOS, the invention should in no way be construed to be limited to any
specific nucleic acid disclosed herein. Rather, the invention should be construed to include
any and all nucleic acid molecules having a sufficient percent identity with the sequences
disclosed herein such that these nucleic acids also encode a peptide having the desired
biological activity disclosed herein. Also contemplated are isolated nucleic acids that are
shorter than full length nucleic acids, wherein the biological activity of the peptide encoded
thereby is retained. Methods to determine the percent identity between one nucleic acid and
another are disclosed elsewhere herein as are assays for the determination of the biological
activity of any specific preferred peptide.
Also as disclosed elsewhere herein, any other number of procedures may be used for
the generation of derivative, mutant, or variant forms of the peptides of the present invention
using recombinant DNA methodology well known in the art such as, for example, that
described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in
Moleculai" Biology, Green & Wiley, New York). Procedures for the introduction of amino
acid changes in a peptide or polypeptide by altering the DNA sequence encoding the peptide
are well known in the art and are also described in Sambrook et al. (1989, supra); Ausubel et
al. (1997, supra).
The invention includes a nucleic acid encoding a G-CSF, IFN-alpha, IFN-beta, Factor
VII, Factor IX, FSH, EPO, GM-CSF, IFN-gamma, A-l-PI, glucocerebrosidase, TPA, IL-2,
Factor VIII, chimeric TNFR, urokinase, chimeric anti-glycoprotein Hb/IIa, chimeric anti-
HER2, chimeric anti-RSV, chimeric anti-CD20, DNase, chimeric anti-TNF , human insulin,
IIBsAg, and HGH, wherein a nucleic acid encoding a tag peptide is covalently linked thereto.
iTiat is, the invention encompasses a chimeric nucleic acid M^herein the nucleic acid sequence
encoding a tag peptide is covalently linked to the nucleic acid encoding a peptide of the
present invention. Such tag peptides are well known in the art and include, for instance,
green fluorescent protein (GFP), myc, myc-pyruvate kinase (myc-PK), Hise, maltose binding
protein (MBP), an influenza virus hemagglutinin tag polypeptide, a flag tag polypeptide
(FLAG), and a glutathione-S-transferase (GST) tag polypeptide. However, the invention
should in no way be construed to be limited to the nucleic acids encoding the above-listed tag
peptides. Rather, any nucleic acid sequence encoding a peptide which may function in a
manner substantially similar to these tag peptides should be construed to be included in the
present invention.
The nucleic acici comprising a nucleic acid encoding a tag peptide can be used to
localize a peptide of the: present invention within a cell, a tissue, and/or a whole organism
(e.g., a mammalian embryo), detect a peptide of the present invention secreted from a cell,
and to study the role(s) of the peptide in a cell. Further, addition of a tag peptide facilitates;
isolation and purification of the "tagged" peptide such that the peptides of the invention can
be produced and purified readily.
The invention includes the following preferred isolated peptides: G-CSF, IFN-alpha,
IFN-beta, Factor VII, Factor IX, FSH, EPO, GM-CSF, IFN-gamma, A-l-PI,
glucocerebrosidase, TPA, IL-2, Factor VllI, chimeric TNFR, urokinase, chimeric anti-
glycoprotein Ilb/llla, chimeric anti-HER2, cliimeric anti-RSV, chimeric anti-CD20, DNase,
chimeric anti-TNF, human insulin, HBsAg, HGH, alpha-galactosidase A. , a-Iduronidase.
antithrombin III, hCG, and interferon omega, and the like.
The present invention should also be construed to encompass "derivatives,"
"mutants", and "variants" of the peptides of the invention (or of the DNA encoding the same)
which derivatives, mutants, and variants are peptides which are altered in one or more amino
acids (or, when referring to the nucleotide sequence encoding the same, are altered in one or
more base pairs) such that the resviiting peptide (or DNA) is not identical to the sequences
recited herein, but has the same biological property as the peptides disclosed herein, in that
the peptide has biologicalTjiochemical properties of G-CSF, IFN-alpha, IFN-beta, Factor VII,
Factor DC, FSH, EPO, GM-CSF, IFN-ganuna, A-l-PI, glucocerebrosidase, TEA, IL-2, Factor
VIII, chimeric TNFR, urokinase, chimeric anti-glycoprotein Ilb/IEIa, chimeric anti-HER2:,
chimeric anti-RSV, cliimeric anti-CD20, DNase, chimeric anti-TNF, human insulin, HBsAg,
and HGH.
Further included are fragments of peptides that retain the desired biological activity of
tlie peptide irrespective of the length of the peptide. It is well within the skill of the artisan to
isolate smaller than full length forms of any of the peptides usefiil in the invention, and to
determine, using the assays provided herein, which isolated firagments retain a desired
biological activity and are therefore useful peptides in the uivention.
A biological property of a protein of the present invention should be construed to
include, but not be limited to include the ability of the peptide to function in the biological
assay and environments described herein, such as reduction of inflammation, elicitation of an
immune response, blood-clotting, increased hematopoietic output, protease inhibition,
immune system modulation, binding an antigen, growth, alleviation of treatment of a disease,
DNA cleavage, and tlie like.
A. G-CSF
The present invention encompasses a method for the modification of the glycaii
structure on G-CSF. G-CSF is well known in the art as a cytokine produced by activated T-
cells, macrophages, endothelial cells, and stromal fibroblasts. G-CSF primarily acts on the
bone marrow to increase the production of inflammatory leukocytes, and further ftinctions as
an endocrine hormone to initiate the replenishment of neutrophils consumed during
inflammatory functions. G-CSF also has clinical applications in bone marrow replacement
Ibllowing chemotherapy.
A remodeled G-CSF peptide may be administered to a patient selected from the group
consisting of a non-myeloid cancer patient receiving myelosuppressive chemotherapy, a
patient having Acute Myeloid Leukemia (AML) receiving induction or consolidation
chemotherapy, a non-myeloid cancer patient receiving a bone marrow transplant, a patient
undergoing peripheral blood progenitor cell collection, a patient having severe chronic
neutropenia, and a patient having persistent neutropenia and also having advanced HIV
infection. Preferably, the patient is a human patient.
While G-CSF has been shown to be an important and useful compound for
therapeutic applications in mammals, especially hiunans, present methods for the production
of G-CSF from recombinant cells results in a product having a relatively short biological life,
an inaccurate glycosylation pattern that could potentially lead to immunogenicity, loss of
function, and an increased need for both larger and more frequent doses in order to achieve
the same effect, and the like.
G-CSF has been isolated and cloned, the nucleic acid and amino acid sequences of
which are presented as SEQ ID N0:1 and SEQ ID N0:2, respectively (Figure 58A and 58B,
respectively). The present invention encompasses a method for modifying G-CSF,
particularly as it relates to the ability of G-CSF to function as a potent and fiinctional
biological molecule. The skilled artisan, when equipped with the present disclosure and the
teachings herein, will readily understand that the present invention provides compositions aiid
methods for the modification of G-CSF.
The present invention further encompasses G-CSF variants, as well known in the art.
As an example, but in no way meant to be limiting to the present invention, a G-CSF variant
has been described in U.S. Patent No. 6,166,183, in which a G-CSF comprising the natural
complement of lysine residues and further linked to one or two polyethylene glycol
molecules is described. Additionally, U.S. Patent Nos. 6,004,548, 5,580,755, 5,582,823, and
5,676,941 describe a G-CSF variant in which one or more of tlie cysteine residues at position
17, 36, 42, 64, and 74 are replaced by alanine or alternatively serine. U.S. Patent No.
5,416,195 describes a G-CSF molecule in which the cysteine at position 17, tlie aspartic acid
at position 27, and the serines at positions 65 and 66 are substituted with serine, serine,
proline, and proline, respectively. Other variants are well known in the art, and are described
in, for example, U.S. Patent No. 5,399,345.
The expression and activity of a modified G-CSF molecule of the present invention
can be assayed using methods well known in the art, and as described in, for example, U.S.
Patent No. 4,810,643. As an example, activity can be measured using radio-labeled
thymidine uptake assays. Briefly, human bone marrow from healthy donors is subjected to a
density cut with FicoU-Hypaque (1.077 g/ml, Pharmacia, Piscataway, NJ) and low density
cells are suspended in Iscove's medium (GIBCO, La JoUa, CA) containing 10% fetal bovine
senmi, glutamine and antibiotics. About 2 X lO** human bone marrow cells are incubated
with either control medium or the G-CSF or the present invention in 96-well flat bottom
plates at about 37° C in 5% (3O2 in air for about 2 days. Cultures are then pulsed for about 4
hours with 0.5 p,Ci/well of ^H-thymidine (New England Nuclear, Boston, Mass.) and uptake
is measured as described in, for example, Ventua, et al.(1983. Blood 61:781). An increase in
^H-thymidine incorporation into human bone marrow cells as compared to bone marrow cells
treated with a control compound is an indication of a active and viable G-CSF compound.
B. IFN alpha, IFN beta and IFN omega
The present invention further encompasses a method for the remodeling and
modification of IFN alpha, IFN beta and IFN omega. IFN alpha is part of a family of
approximately twenty peptides of approximately 18kDa in weight. IFN omega is very similar
in structure and function to IFN alpha. IFN omega is useful for treatment of hepatitis C virus
infection when an immune response to IFN alpha is mounted in the host rendering that
treatment ineffective. Antibodies raised against IFN alpha do not cross-react with IFN
omega. Thus, treatment of hepatitis C may continue using IFN omega when IFN alpha
therapy is no longer possible.
IFN alpha, omega, and IFN beta, collectively known as the Type I interferons, bind to
the same cellular receptor and elicit similar responses. Type I IFTvls inhibit viral replication,
increase the lytic potential of NK cells, modulate MHC molecule expression, and inhibit
cellular proliferation, among other things. Type I IFN has been used as a therapy for viral
infections, particularly hepatitis viruses, and as a therapy for multiple sclerosis.
Current compositions of Type I IFN are, as described above, useful compounds for
both the modulation of abeirant immunological responses and as a therapy for a variety of
diseases. However, they are hampered by decreased potency and function, and a limited half-
life in tlie body as compared to natural cj^okines comprising the natural complement of
glycosylation.
A remodeled interferon-alpha peptide may be administered to a patient selected from
the group consisting of a patient having hairy cell leukemia, a patient having malignant
melanoma, a patient having follicular lymphoma, a patient having condylomata acuminata, a
patient having AIDS-related Kaposi's sarcoma, a patient having Hepatitis C, a patient having
Hepatitis B, a patient having a human papilloma virus infection, a patient having Chronic
Myeloid Leukemia (CML), a patient having chronic phase Philadelphia chromosome (Ph)
positive Chronic Myelogenous Leukemia, a patient having non-Hodgkin's lymphoma (NHL),
a patient having lymphoma, a patient having bladder cancer, and a patient having renal
cancer. Preferably, the patient is a human patient.
A remodeled interferon-beta peptide may be administered to a patient selected from
the group consisting of a patient having multiple sclerosis (MS), a patient having Hepatitis B,
a patient having Hepatitis C, a patient having human papilloma virus infection, a patient
having breast cancer, a patient having brain cancer, a patient having colorectal cancer, a
patient having pulmonary fibrosis, and a patient having rheumatoid arthritis. Preferably, the
patient is a human patient.
A remodeled interferon-omega peptide may be administered to a patient selected from
the group consisting of a patient having hairy cell leukemia, a patient having malignant
melanoma, a patient having follicular lymphoma, a patient having condylomata acuminata, a
patient having AIDS-related Kaposi's sarcoma, a patient having Hepatitis C, a patient having
Hepatitis B, a patient having a human papilloma virus infection, a patient having Chronic
Myeloid Leukemia (CML), a patient having chronic phase Pliiladelphia chromosome (Ph)
positive Chronic Myelogenous Leukemia, a patient having non-Hodgkin's lymphoma (NHL),
a patient having lymphoma, a patient having bladder cancer, and a patient having renal
cancer. Preferably, the patient is a human patient.
The prototype nucleotide and amino acid sequence for IFN alpha is set forth herein as
SEQ ID N0:3 and SEQ ID N0:4, respectively (Figure 59A and 59B, respectively). The
prototype nucleotide and amino acid sequence for IFN omega is set forth herein as SEQ ID
NO:74 and SEQ ID NO:75, respectively (Figures 84A and 84B, respectively). IFN beta
comprises a single gene product of approximately 20 kDa, the nucleic acid and amino acid
sequence of which are presented herein as SEQ ID N0:5 and SEQ ID NO:6 (Figure 60A and
60B, respectively). The present invention is not limited to the nucleotide and amino acid
sequences herein. One of skill in the art will readily appreciate that many variants of IFN
alpha exist both naturally and as engineered derivatives. Similarly, IFN beta has been
modified in attempts to achieve a more beneficial therapeutic profile. Examples of modified
Type I IFNs are well known in the art (see Table 9), and are described in, for example U.S.
Patent No. 6,323,006, in which cysteine-60 is substituted for tyrosine, U. S. Patent Nos.
4,737,462, 4,588,585, 5,545,723, and 6,127,332 where an IFN beta with a substitution of a
variety of amino acids is described. Additionally, U.S. Patent Nos. 4,966,843, 5,376,567,
5,795,779 describe IFN alpha-61 and IFN-alpha-76. U.S. Patent Nos .4,748,233 and
4,695,543 describe IFN alpha gx-1, whereas U.S. Patent No. 4,975,276 describes IFN alpha-
54. In addition, U.S. Patent Nos. 4,695,623, 4,897,471, 5,661,009, and 5,541,293 all describe
a consensus IFN alpha sequence to represent all variants known at the date of filing. While
this list of Type I IFNs and variants thereof is in no way meant to be exhaustive, one of skill
in the art will readily understand that the present invention encompasses IFN beta and IFN
alpha molecules, derivatives, and variants known or to be discovered in the future.
Methods of expressing IFN in recombinant cells are well known in the art, and is
easily accomplished using techniques described in, for example U.S. Patent No. 4,966,843,
and in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in Molecular
Biology, Green & Wiley, New York). Assays to determine the biological activity of a Type I
IFN modified by the present invention will be well known to the skilled artisan. For
example, the assay descritjed in Rubinstein et al., (1981, Journal of Virology 37:755-758) is
commonly used to determine the effect of an Type I EFN by measuring the cytopathic effects
of viral infection on a population of cells. This method is only one of many known in the art
for assaying the biological function of a Type IFN.
C. Factor Vila
The present invention further encompasses a method for the remodeling and
modification of Factor VII. The blood coagulation pathway is a complex reaction comprismg
many events. An intermediate event in this pathway is Factor VII, a proenzyme that
participates in the extrinsic patliway of blood coagulation by converting (upon its activation
to Factor Vila) Factor X to Xa in the presence of tissue factor and calcium ions. Factor Xa in
turn then converts prothiombin to thrombin in the presence of Factor Va, calcium ions and
phospholipid. The activation of Factor X to Factor Xa is an event shared by both the intrinsic
and extrinsic blood coagulation pathways, and therefore. Factor Vila can be used for the
treatment of patients with deficiencies or inhibitors of Factor VIII. There is also evidence to
suggest that Factor Vila may participate in the intrinsic pathway as well therefore increasing
the prominence and importance of the role of Factor VII in blood coagulation.
Factor VII is a single-chain glycoprotein with a molecular weight of approximately 50
kDa. In this form, the factor circulates in the blood as an inactive zymogen. Activation o:f
Factor VII to Vila may be catalyzed by several different plasma proteases, such as Factor
Xlla. Activation of Factor VII results in the formation of a heavy chain and a light chain held
together by at least one disulfide bond. Further, modified Factor VII molecules that cannot
be converted to Factor Vila have been described, and are useful as anti-coagulation remedies.
such as in the case of blood clots, thrombosis, and the like. Given the importance of Factor
VII in the blood coagulation pathway, and its use as a treatment for both increased and
decreased levels of coagulation, it follows that a molecule that has a longer biological half-
life, increased potency, and in general, a therapeutic profile more similar to wild-type Factor
VII as it is synthesized and secreted in the healthy human would be beneficial and useful as a
treatment for blood coagulation disorders.
A remodeled Factor VII peptide may be administered to a patient selected from the
group consisting of a hemophiliac patient having a bleeding episode, a patient having
Hemophilia A, a patient with Hemophilia B, a patient having Hemophilia A, wherein the
patient also has antibodies to Factor VIII, a patient having Hemophilia B, wherein the patient
also has antibodies to Factor IX, a patient having liver cirrhosis, a cirrhotic patient having an
ortliotopic liver transplant, a cirrhotic patient having upper gastrointestinal bleeding, a patient
having a bone marrow transplant, and a patient having a liver resection. Preferably, the
patient is a human patient.
Factor VII has been cloned and sequenced, and the nucleic acid and amino acid
sequences are presented hereui as SEQ ID N0:7 and SEQ ID N0:8 (Figure 61A and 61B,
respectively). The present invention should in no way be construed as limited to the Factor
VII nucleic acid and amino acid sequences set forth herein. Variants of Factor VII are
described in, for example, U.S. Patent Nos. 4,784,950 and 5,580,560, in which lysine-38,
lysine-32, arginine-290, arginine-341, isoleucine-42, tyrosine-278, and tyrosine-332 is
replaced by a variety of amino acids. Further, U.S. Patent Nos. 5,861,374, 6,039,944,
5,833,982, 5,788,965, 6,183,743, 5,997,864, and 5,817,788 describe Factor VII variants that
are not cleaved to fonn Factor Vila. The skilled artisan will recognize that the blood
coagulation pathway and the role of Factor VII therein are well known, and therefore many
variants, both naturally occurring and engineered, as described above, are included in the
present invention.
Methods for the expression and to determine tlie activity of Factor VII are well known
in the art, and are described in, for example, U.S. Patent No. 4,784,950. Briefly, expression
of Factor VII, or variants thereof, can be accomplished in a variety of both prokaryotic and
eukaryotic systems, including E. coli, CHO cells, BHK cells, insect cells using a baculovirus
expression system, all of which are well known in the art.
Assays for the activity of a modified Factor VII prepared according to the methods of
the present invention can be accomplished using methods well known in the art. As a non-
limiting example. Quick et al. (Hemorragic Disease and Thrombosis, 2nd ed,, Leat Febiger,
Philadelphia, 1966), describes a one-stage clotting assay useful for determining the biological
activity of a Factor VII molecule prepared according to the methods of the present invention.
D. Factor IX
The present invention further encompasses a method for remodeling and/or modifying
Factor IX. As described above, Factor DC is vital in the blood coagulation cascade. A
deficiency of Factor DC in the body characterizes a type of hemophilia (type B). Treatment of
this disease is usually limited to intravenous tranfusion of human plasma protein concentrates
of Factor IX. However, in addition to the practical disadvantages of time and expense,
transfusion of blood concentrates involves the risk of transmission of viral hepatitis, acquired
immune deficiency syndrome or tliromboembolic diseases to the recipient.
Wliile Factor IX has demonstrated itself as an important and useful compound for
therapeutic applications, present methods for the production of Factor IX from recombinant
cells (XJ.S. Patent No. 4,770,999) results in a product with a rather short biological life, an
inaccurate glycosylation pattern that could potentially lead to immunogenicity, loss of
function, an increased need for both larger and more frequent doses in order to achieve the
same effect, and the like.
A remodeled Factor IX peptide may be administered to a patient selected from the
group consisting of a hemophiliac patient having a bleeding episode and also having
Hemopliilia B, a patient having Hemophilia B, a patient having Hemophilia B and having
antibodies to Factor DC, a patient having liver cirrhosis, a cirrhotic patient having an
orthotopic liver transplant, a cirrhotic patient having upper gastrointestinal bleeding, a patient
having a bone marrow triuisplant, and a patient having a liver resection. A remodeled Factor
IX peptide may also be administered to control and/or prevent hemorrhagic episodes in a
patient having Hemophilia B, congenital Factor IX deficiency, or Christmas disease. A
remodeled Factor IX peptide may also be administered to a patient to control and/or prevent
hemorrhagic episodes in the patient during surgery. Preferably, the patient is a human
patient.
The nucleic and amino acid sequences of Factor DC is set forth herein as SEQ ID
N0:9 and SEQ ID NO: 10 (Figure 62A and 62B, respectively). The present invention is in no
way limited to the sequences set forth herein. Factor IX variants are well known in the art, as
described in, for example, U.S. Patent Nos. 4,770,999, 5,521,070 m which a tyrosine is
replaced by an alanine in the first position, U.S. Patent No. 6,037,452, in which Factor XI is
linked to an alkylene oxide group, and U.S. Patent No. 6,046,380, in which the DNA
encoding Factor IX is modified in at least one splice site. As demonstrated herein, variants of
Factor IX are well known :in the art, and the present disclosure encompasses those variants
known or to be developed or discovered in the future.
Methods for determining the activity of a modified Factor IX prepared according to
the methods of the present invention can be carried out using the methods described above, or
additionally, using methods well known in the art, such as a one stage activated partial
thromboplastin time assay as described in, for example. Biggs (1972, Human Blood
Coagulation Haemostasis and Thrombosis (Ed. 1), Oxford, Blackwell, Scientific, pg. 614).
Briefly, to assay the biological activity of a Factor IX molecule developed according to the
methods of the present invention, the assay can be performed \vith equal volumes of
activated partial thromboplastin reagent. Factor IX deficient plasma isolated ft-om a patient
with hemophilia B using sterile phlebotomy techniques well known in the art, and normal
pooled plasma as standard, or the sample. In this assay, one unit of activity is defined as that
amount present in one milliliter of normal pooled plasma. Further, an assay for biological
activity based on the ability of Factor IX to reduce the clotting time of plasma from Factor
IX-deficient patients to normal can be performed as described in, for example, Proctor and
Rapaport (1961, Amer. J. Clin. Path. 36: 212).
E. FSH
The present invention further includes a method for remodeling and/or modifying
FSH. Human reproductive fiinction is controlled in part by a family of heterodLmeric human
glycoprotein hormones which have a common 92 amino acid glycoprotein alpha subunit, bul
differ in their hormone-specific beta subunits. The family includes follicle-stimulating
homione (FSH), luteinizing hormone (LH), thyrotropin or thyroid-stimulating hormone
(TSH), and human chorionic gonadotropin (liCG). Human FSH ;ind LH are used
therapeutically to regulate various aspects of metabolism pertinent to reproduction in the
human female. For example, FSH partially purified from urine is used clinically to stimulate
follicular maturation in anovtilatory women with anovulatory syndrome or luteal phase
deficiency. Luteinizing hormone (LH) and FSH are used in combination to stimulate the
development of ovarian follicles for in vitro fertilization. The role of FSH in the reproductive
cycle is sufficiently well-known to permit therapeutic use, but difficulties have been
encountered due, in part, to the heterogeneity and impurity of the preparation from native
sources. This heterogeneity is due to variations in glycosylation pattern.
FSH is a valuable tool in both in vitro fertilization and stimulation of fertilization in
vivo, but as stated above, its clinical efficacy has been hampered by inconsistency in
glycosylation of the protein. It therefore seems apparent that a method for remodeling FSH
will be of great benefit to the reproductive sciences.
A remodeled FSH peptide may be administered to a patient selected from the group
consisting of a patient undergoing intrauterine insemination (lUI), a patient undergoing in
vitro fertilization (IVF), and an infertile patient. A remodeled FSH peptide may also be
administered to induce or increase ovulation in a patient, to stimulate development of an
ovarian follicle in a patient, to induce gametogenic follicle growth in a patient, to stimulate,
induce or increase follicle development and subsequent ovulation in a patient, or to treat
infertility in a patient. Preferably, the patient is a human female patient. A remodeled FSH
peptide may also be administered to a patient having a pituitary deficiency or to a patient
during puberty. Preferably this patient is a human male patient.
FSH has been cloned and sequenced, the nucleic and amino acid sequences of which
are presented herein as SEQ ID N0:11, SEQ ID NO: 12, respectively (alpha subunit) and
SEQ ID NO: 13 and SEQ ID NO: 14, respectively (beta subunit) (Figure 63A, 63B, 63C and
63D, respectively). The .skilled artisan will readily appreciate that the present in\'ention is not
limited to the sequences depicted herein, as variants of FSH are well known in the art. As a
non-limiting example, U.S. Patent No. 5,639,640 describes the beta subunit comprising two
different amino acid sequences and U.S. Patent No. 5,338,835 describes a beta subunit
comprising an additional amino acid sequence of approximately twenty-se\'en amino acids
derived from the beta subunit of human chorionic gonadotropin. Therefore, the present
invention comprises FSH variants, both natural and engineered by the human hand, all well
known in the art.
Methods to express FSH in cells, both prokaryotic and eukaryotic, are well known in
the art and abundantly described mthe literature (U.S. Patent Nos. 4,840,896, 4,923,805,
5,156,957). Further, methods for evaluating the biological activity of a remodeled FSH
molecule of the present invention are well known La the art, and are described in, for
example, U.S. Patent No. 4,589, 402, in which methods for determining the effect of FSH on
fertility, egg production, and pregnancy rates is described in both non-human primates and
human subjects.
F. EPO
The present invention further comprises a method of remodeling and/or modifying
EPO. EPO is an acidic glycoprotein of approximately 34 kDa and may occur in three naturjil
forms: alpha, beta, and asialo. The alpha and beta forms differ slightly in carbohydrate
components but have the same potency, biological activity and molecular weight. The asialo
form is an alpha or beta form with the terminal sialic acid removed. EPO is present in very
low concentrations in plasma when the body is in a healthy state wherein tissues receive
sufficient oxygenation from the existing number of erythrocytes. This normal concentratiori
is enough to stimulate replacement of red blood cells which are lost normally through aging.
The amount of erythropoietin in the circulation is increased under conditions of hypoxia
when oxygen transport b}' blood cells in the circulation is reduced. Hypoxia may be caused
by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-
exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged
unconsciousness, or various forms of anemia. Therefore EPO is a useful compound for
replenishing red blood cells after radiation therapy, anemia, and other life-threatening
conditions.
A remodeled EPO peptide may be administered to a patient selected from the group
consisting of a patient having anemia, an anemic patient having chronic renal insufficiency,
an anemic patient having end stage renal disease, an anemic patient undergoing dialysis, an
anemic patient having clironic renal failure, an anemic Zidovudine-treated HIV infected
pcitient, an anemic patient having non-myeloid cancer and undergoing chemotherapy, and ari
anemic patient scheduled to undergo non-cardiac, non-vascular surgery. A remodeled EPO
peptide may also be administered to a patient undergoing surgery to reduce the need for an
allogenic blood transfusion. A remodeled EPO peptide may also be administered to a patient
at increased risk for a perioperative blood transfusion with significant anticipated blood loss.
Preferably, the patient is a human patient.
In light of the importance of EPO in aiding in the recovery from a variety of diseases
and disorders, the present invention is useful for the production of EPO with a natural, and
therefore more effective saccharide component. EPO, as it is currently synthesized, lacks the
full glycosylation complement, and must therefore be administered more frequently and in
higher doses due to its short life in the body. The invention also provides for the production
of PEGylated EPO molecules with greatly improved half-life compared with what might be
achieved by maximizing desirable glycoforms.
EPO has been cloned and sequenced, and the nucleotide and amino acid sequences are
present herein as SEQ ID NO: 15 and SEQ ID NO: 16, respectively (Figure 64A and 64B,
respectively). It will be readily understood by one of skill in the art that the sequences set
forth herein are only an example of the sequences encoding and comprising EPO. As an
example, U.S. Patent No. 6,187,564 describes a fusion protein comprising the amino acid
sequence of two or more EPO peptides, U.S. Patent Nos. 6,048,971 and 5,614,184 describe
mutant EPO molecules having amino acid substitutions at positions 101, 103, 104, and 108.
U.S. Patent No. 5,106,954 describes a truncated EPO molecule, and U.S. Patent No.
5,888,772 describes an EPO analog with substitutions at position 33, 139, and 166.
Therefore, the skilled artisan will realize that the present invention encompasses EPO and
EPO derivatives and variants as are well documented in the literature and art as a whole.
Additionally, methods of expressing EPO in a cell are well known in the art. As
exemplified in U.S. Patent Nos. 4,703,008, 5,688,679, and 6,376,218, among others, EPO can
be expressed in prokaryotic and eukaiyotic expression systems. Methods for assaying the
biological activity of EPO a-e equally well known in the art. As an example, the Krystal
assay (Krystal, 1983, Exp. Hematol. 11:649-660) can be employed to determine the activity
of EPO prepared according lo the methods of the present invention. Briefly, the assay
measures the effect of er>'tl-u-opoietin on intact mouse spleen cells. Mice are treated with
phenylhydrazine to stimulate production of erythropoietin-responsive red blood cell
progenitor cells. After treatment, the spleens are removed, intact spleen cells are isolated and
incubated with various amounts of wild-type erythropoietin or the erythropoietin proteins
described herein. After an overnight incubation, ''H-thymidine is added and its incorporation
into cellular DNA is measured. The amount of ^H-thymidine incorporation is indicative of
erythropoietin-stimulated production of red blood cells via interaction of erythropoietin with
its cellular receptor. The concentration of the erythropoietin protein of the present invention,
as well as the concentration of wild-type erythropoietin, is quiintified by competitive
radioimmunoassay methods well known in the art. Specific activities are calculated as
international units measui'ed in the Krystal assay divided by micrograms as measured as
immunoprecipitable protein by radioimmunoassay.
Several different mutated EPO's with different glycosylation patterns have been
reported. Many have improved stimulation of reticulocytosis activity without effecting the
half-life of the peptide in the blood stream of the animal. It is contemplated that mutated
EPO peptides can be used in place of the native EPO peptides in any of the glycan
remodeling, glycoPEGylation and/or glycoconjugation embodiments described herein.
Preferred mutations of EPO are listed in the following table, but not limited to those listed in
the table (see, for example, Chem et al., 1991, Eur. J. Biochem. 202:225-229; Grodberg et al.,
1993, Eur. J. Biochem. 218:597-601; Bums et al., 2002, Blood 99:4400-4405; U.S. Patent
No. 5,614,184; GenBank Accession No. AAN76993; O'Connell et al., 1992, J. Biol. Chem,
267:25010-25018; Elliolt etal., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:2708-2712; Biossel et
al., 1993, J. Biol. Chem. 268:15983-15993). The most preferred mutations of EPO are Arg^^^
to Ala'^^ Arg"*^ to Ala'''^ and Lys'^'' to Ala'^". The preferred native EPO from which to
make these mutants is die 165 aa form, which is depicted in Fig. 65; however other native
forms of EPO may also be used. Finally, the mutations described in Table 10 may be
combined with each otlier and with other mutations to make EPO peptides that are useful in
the present invention.
G. GM-CSF
The present invention encompasses a method for the modification of GM-CSF. GM-
CSF is well known in the arl; as a cytokine produced by activated T-cells, macrophages,
endothelial cells, and stromal fibroblasts. GM-CSF primarily acts on the bone marrow to
increase the production of inflammatory leukocytes, and further functions as an endocrine
hormone to initiate the replenishment of neutrophils consumed during inflammatory
functions. Further GM-CSF is a macrophage-activating factor and promotes the
differentiation of Lagerhans cells into dendritic cells. Like G-CSF, GM-CSF also has clinical
applications in bone marrow replacement following chemotherapy.
While G-CSF has demonstrated itself as an important and useful compound for
therapeutic applications, present methods for the production of G-CSF from recombinant
cells results in a product with a rather short biological life, an inaccurate glycosylation pattern
that could potentially lead to immunogenicity, loss of function, an increased need for both
larger and more frequent doses in order to achieve the same effect, and the like.
A remodeled GM-CSF peptide may be administered to a patient selected from the
group consisting of a patient having Acute Myelogenous Leukemia (AML) or acute non-
lymphocytic leukemia (A>JLL), a patient undergoing leukapheresis to collect hematopoietic
progenitor cells from the peripheral blood, a patient undergoing transplantation of autologous
peripheral blood progenitor cells, a non-Hodgkin's lymphoma (NHL) patient undergoing an
autologous bone marrow transplant, a Hodgkin's disease patient undergoing an autologous
bone marrow transplant, and an acute lymphoblastic leukemia (ALL) patient undergoing an
autologous bone marrow transplant. A remodeled GM-CSF peptide may also be
administered to a patient to accelerate myeloid engraftment, to shorten time to neutrophil
recovery following chemotherapy, to mobilize hematopoietic progenitor cells into the
peripheral blood for collection by leukapheresis, or to promote myeloid reconstitution after
autologous or allogeneic bone marrow transplantation (BMT). A remodeled GM-CSF
peptide may also be administered to a patient in which bone marrow transplantation has
failed or in which myeloid engraftment is delayed. Preferably, the patient is a human patient.
GM-CSF has been isolated and cloned, the nucleic acid imd amino acid sequences of
which are presented as SEQ ID NO: 17 and SEQ ID NO: 18, respectively (Figure 66A and
66B, respecti'S}ely). The present invention encompasses a method for modifying GM-CSF,
particularly as it relates to the ability of GM-CSF to function as a potent and functional
biological molecule. The skilled artisan, when equipped with the present disclosure and the
teachings herein, will readily understand that the present invention provides compositions and
methods for the modification of GM-CSF.
The present invention further encompasses GM-CSF variants, as well known in the
art. As an example, but in no way meant to be limiting to the present invention, a GM-CSF
variant has been described in WO 86/06358, where the protein is modified for an alternative
quaternary structure. Further, U.S. Patent No. 6,287,557 describes a GM-CSF nucleic acid
sequence ligated into the genome of a herpesvirus for gene therapy applications.
Additionally, European Patent Publication No. 0288809 (corresponding to PCT Patent
Publication No. WO 87/02060) reports a fusion protein comprising IL-2 and GM-CSF. The
IL-2 sequence can be at either the N- or C-terrainal end of the GM-CSF such that after acid
cleavage of the fusion protein, GM-CSF having either N- or C-tenninal sequence
modifications can be generated. Therefore, GM-CSF derivatives, mutants, and variants are
well known in the art, and are encompassed within the methods of the present invention.
The expression and activity of a modified GM-CSF molecule of the present invention
can be assayed using methods well known in the art, and as described in, for example, U.S.
Patent No. 4,810,643. As an example, activity can be measured using radio-labeled
thymidine uptake assays. Briefly, human bone marrow from healthy donors is subjected to a
density cut with FicoU-Hypaque (1.077 g/ml, Pharmacia, Piscataway, NJ) and low density
cells are suspended in Iscove's medium (GIBCO, La JoUa, CA) containing 10% fetal bovine
serum, glutamine and antibiotics. About 2 X 10"* human bone marrow cells are incubated
with either control mediiun or the GM-CSF or the present invention in 96-well flat bottom
plates at about 37° C in 5% CO2 in air for about 2 days. Cultures are then pulsed for about 4
hours with 0.5 fiCi/well of ¦'H-thymidine (New England Nuclear, Boston, Mass.) and uptake
is measured as described in, for example, Ventua, et al.(1983, Blood 61:781). An increase in
¦^H-thymidine incorporation into human bone marrow cells as compared to bone marrow cells
treated with a control compound is an indication of a active and viable GM-CSF compound.
H. IFN-gamma
It is an object of the present invention to encompass a method of modifying and/or
remodeling IFN-gamma. IFN-gamma, otherwise knovm as Type II interferon, in contrast to
IFN alpha and IFN beta, is a homodimeric glycoprotein comprising two subunits of about 21 -
24 kDa. The size variation is due to variable glycosylation patterns, usually not replicated
when reproduced recombinantly in various expression systems known in tlie art. IFN-gamma
is a potent activator of macrophages, increases MHC class I molecule expression, and to a
lesser extent, a MHC class II molecule stimulatory agent. Further, IFN-ganmia promotes T-
cell differentiation and isotype switching in B-cells. IFN-gamma is also well documented as
a stimulator of neutrophils, NK ceils, and antibody responses leading to phagocyte-mediated
clearance. IFN-gamma has been proposed as a treatment to be used in conjunction with
infection by intracelluUir pathogens, such as tuberculosis and leishmania, and also as an anti-
proliferative therapeutic, useful in conditions with abnormal cell proliferation as a hallraaik,
such as various cancers and other neoplasias.
IFN-gamma has demonstrated potent immunological activity, but due to variations in
glycosylation jfrom systems currently used to express IFN-gamma, the potency, efficacy,
biological half-life, and other important factors of a therapeutic have been variable at best.
The present invention encompasses methods to correct this crucial defect.
A remodeled interferon-gamma peptide may be administered to a patient selected
from the group consisting of a patient having chronic granulomatous disease, a patient having
malignant osteopetrosis, a patient having pulmonary fibrosis, a patient having tuberculosis, a
patient having Cryptococcal meningitis, and a patient having pulmonary Mycobacterium
avium complex (MAC) infection. Preferably, the patient is a human patient.
The nucleotide and amino acid sequences of EFN-gamma are presented herein as SEQ
ID NO: 19 and SEQ ID NO :20, respectively (Figure 67A and 67B, respectively). It will be
readily understood that the sequences set forth herein are in no way limiting to the present
invention. Ln contrast, varicuits, derivatives, and mutants of IFN-gamma are well known to
the skilled artisan. As an example, U.S. Patent No. 6,083,724 describes a recombinant avian
IFN-gamma and U.S. Patent No. 5,770,191 describes C-terminus variants of human IFN-
gamma. In addition, U.S. Patent No. 4,758,656 describes novel IFN-gamma derivatives, and
methods of synthesizing them in various expression systems. ITierefore, the present
invention is not limited to tlie sequences of IFN-gamma disclosed elsewhere herein, but
encompasses all derivatives, variants, muteins, and the like well known in the art.
Expression systems for IFN-gamma are equally well known in the art, and include
prokaryotic and eukaryotic systems, as well as plant and insect cell preparations, methods of
which are known to the skilled artisan. As an example, U.S. Patent No. 4,758,656 describes a
system for expressing IFN-gamma derivatives in E. coli, whereas U.S. Patent No. 4,889,803
describes an expression system employing Chinese hamster ovary cells and an SV40
promoter.
Assays for the biological activity of a remodeled IFN-gamma prepared according to
the methods disclosed herein will be well known to one of skill in the art. Biological assays
for IFN-gamma expression can be found in, for example, U.S. Patent No. 5,807,744. Briefly,
IFN-ganmia is added to cultures of CD34"^CD38'ceIls (100 cells per well) stimulated by
cytokine combinations to induce proliferation of CD34 CD38" cells, such as IL-3, c-kit
ligand and either IL-1, lL-6 or G-CSF. Cell proliferation, and generation of secondary
colony forming cells will be profoundly inhibited in a dose dependent way, with near
complete inhibition occurring at 5000 U/milliliter of IFN-gamma. As a confirmatory test to
the inhibitory effect of IFN-gamma, addition of IFN-gamma antibodies can be performed as a
control.
I. alpha-Protease inhibitor ("g-antitrvpsin')
The present invention further includes a method for the remodeling of alpha-protease
inhibitor (A-l-PI, a-1-antitrypsin or a-1-trypsin inhibitor), also known as alpha-antitrypsin.
A-l-PI is a glycoprotein having molecular weight of 53 kDa. A-l-PI plays a role in
controlling tissue destruction by endogenous serine proteases, and is the most pronounced
serine protease inhibitor in blood plasma. In particular, A-l-PI inhibits various elastases
including neutrophil elastase. Elastase is a protease which breaks down tissues, and can be
particularly problematic when its activity is unregulated in lung tissue. This protease
functions by breaking down foreign proteins. However, when API is not present in sufficient
quantities to regulate elastase activity, the elastase breaks down lung tissue. In time, this
imbalance results m chronic lung tissue damage and emphysema. In fact, a genetic
deficiency of A-l-PI has been shown to be associated with premature development of
pulmonary emphysema. A-l-PI replenishment has been successfully used for treatment of
this form of emphysema. Further, a deficiency of A-l-PI may also contribute to the
aggravation of other diseases such as cystic fibrosis and arthritis, where leukocytes move in
to the lungs or joints to fight infection.
Therefore, A-l-PI could conceivably be used to treat diseases where an imbalance
between inhibitor and protease(s), especially neutrophil elastase, is causing progression of a
disease state. Antiviral activity has also been attributed to A-l-PI. In light of this, it logically
follows that the present invention is useful for the production of A-1-PI that is safe, effective,
and potent in the ever changing atmosphere of the lungs.
A remodeled A-1-P1 peptide may be administered to a patient selected from the group
consisting of a patient having congenital alpha-1-antitrypsin deficiency and emphysema, a
patient having cystic fibrosis, and a patient having pulmonary fibrosis. Preferably, the paticni
is a human patient.
A-l-PI has been cloned and sequenced, and is set forth in SEQ ID N0:21 and SEQ ID
NO:22 (Figure 68A and 68B, respectively). As is understood by one of skill in the art,
natural and engineered variants of A-l-PI exist, and are encompassed in the present
invention. As an example, U.S. Patent No. 5,723,316 describes A-l-PI derivatives having
amino acid substitutions at positions 356-361 and further comprises an N-terminal extension
of approximately three amino acids. U. S. Patent No. 5,674,708 describes A-l-PI analogs
with amino acid substitutions at position 358 in the primary amino acid sequence. The
skilled artisan will readily realize that the present invention encompasses A-l-PI variants,
derivatives, and mutants known or to be discovered.
Methods for the expression and determination of activity of a remodeled A-l-PI
produced according to the methods of the present invention are well known in the art, and are
described in, for example, U.S. Patent No. 5,674,708 and U.S. Patent No. 5,723,316. Briefly,
biological activity can be determined using assays for antichymotrypsin activity by
measuring the inhibition of the chymotrypsin-catalyzed hydrolysis of substrate N-suc-Ala~
Ala~Pro~Phe-p-nitroanilide (0.1 ml of a 10 mM solution in 90% DMSO), as described in,
for example, DelMar et al. (1979, Anal, Biochem. 99: 316).. A typical chymotrypsin assay
contains, in 1.0 milliliters: 100 mM Tris-Cl buffer, pH 8.3, 0.005% (v/v) Triton X-100,
bovine pancreatic chymotrypsin (18 kmmol) and A-l-PI of the present invention. The assay
mixture is pre-incubated at room temperature for 5 minutes, substrate (0.01 ml of a 10 mM^
solution in 90% DMSO) is added and remaining chymotryf)sin activity is determined by the
rate of change in absorbance at 410imi caused by the release of p-nitroaniline. Measurements
of optical absorbance are conducted at 25° C using a spectrophotometer fitted with a
temperature controlled sample compartment.
J. Glucocerebrosidase
The in\'ention de scribed herein fiirtlier includes a method for the modification of
glucocerebrosidase. Glucocerebrosidase is a lysosomal glycoprotein enzyme which catalyzes
the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Variants of
glucocerebrosidase are sold cotnmercially as Cerezyme''^''^ and Ceredase'"'^*, and is an
approved tlierapeutic for llie treatment of Gaucher disease. Ceredase™' is a placental derived
form of glucocerebrosidase with complete N-linked stnactures. Cerezyme^''^' is a recombinant
variant of glucocerebrosidase which is 497 amino acids in length and is expressed in CHO
cells. The 4N-linkedglycansofCere2yme have been modified to terminate in the
trimannose core.
Glucocerebrosidase is presently produced m recombinant mammalian cell cultures,
and therefore reflects the glycosylation patterns of those cells, usually rodent cells such as
Chinese hamster ovary cells or baby hamster kidney cells, which differ drastically from those
of human glycosylation patterns, leading to, among other things, immunogenicity and lack of
potency.
A remodeled glucocerebrosidase peptide may be administered to a patient selected
from the group consisting of a patient havmg a lysosomal storage disease, a patient having a
glucocerebrosidase deficiency, and a patient having Gaucher disease. Preferably, the patient
is a human patient.
The nucleic acid and amino acid sequences of glucocerebrosidase are set forth herein
as SEQ ID NO:23 and 24 (Figure 69A and 69B, respectively). However, as will be
appreciated by the skilled artisan, the sequences represented herein are prototypical
sequences, and do not limit the invention. In fact, variants of glucocerebrosidase are well
known, and are described in, for example, U.S. Patent 6,015,703 describes enhanced
production of glucocerebrosidase analogs and variants thereof Further, U.S. Patent No.
6,087,131 describes the cloning and sequencing of yet another glucocerebrosidase variant. It
is the intention of the present invention to encompass these and other derivatives, variants,
and mutants kitown or to be discovered in the future.
Methods for the expression of glucocerebrosidase are well known in the art using
standard techniques, and are described in detail in, for example, U.S. Patent No. 6,015,703.
Assays for the biological efficacy of a glucocerebrosidase molecule prepared according to the
methods of tlie present invention are similarly well known in the art, and a mouse Gaucher
disease model for evaluation arid use of a glucocerebrosidase therapeutic is described in, for
example, Marshall et al. (2002, Mol. Ther. 6:179).
The present invention fiirther encompasses a metliod for tlie remodeling of tissue-typ>
activator (TPA). TPA acti\'ates plasminogen to form plasniin which dissolves fibrin, tlie
main component of the protein substrate of the thrombus. TPA preparations were developed
as a thrombolytic agents having a very high selectivity towaird the thrombus in the
thrombolytic treatment for thrombosis which causes myocardial infarction and cerebral
infarction.
Further, various modified TPA's have been produced by genetic engineering for the
purpose of obtaining higher affinity to fibrin and longer half-life in blood than that of natural
TPA. TPA's are proteins that are generally extremely difficult to solubilize in water. In
particular, the modified TPA's are more difficult to solubilize in water than natural TPA,
making very difficult the preparation of modified TPA's. Modified TPA's are thus difficult to
dissolve in water at the time of the administration to a patient. However, tlie modified TPA's
have various advantages, such as increased affinity for fibrin and longer half-life in blood. It
is the object of the present invention to increase the solubility of modified TPA's.
A remodeled TPA peptide may be administered to a patient selected firom the group
consisting of a patient suffering fi-om an acute myocardial infarction and a patient suffering
from an acute ischemic stroke. A remodeled TPA peptide may also be administered to a
patient to improve ventricular function following an acute myocardial infarction, to reduce
the incidence of congestive heart failure following an acute myocardial infarction, or to
reduce mortality associated with acute myocardial infarction. A remodeled TPA peptide may
also be administered to a patient to improve neurological recovery following an acute
ischemic stroke or to reduce the incidence of disability or paralysis following an acute
ischemic stroke. Preferably, the patient is a human patient.
The nucleic and amino acid sequences of TPA are set forth herein as SEQ ID NO: 25
and SEQ ID NO:26, respectively (Figure 70A and 70B, respectively). As described above,
variants of TPA have been constructed and used in therapeutic applications. For example,
U.S. Patent 5,770,425 described TPA variants in which some of all of the fibrin domain has
been deleted. Further, U.S. Patent 5,736,134 describes TPA in which modifications to tiie
amino acid at position 276 are disclosed. The skilled artisan, v^-hen equipped with tiie present
disclosure and the teacliings herein, will readily realize tiiat the present invention comprises
the TPA sequences set forth herein, as well as those vari;ants well knovm to one versed in the
i iterature.
The expression of TPA from a nucleic acid sequence encoding the same is well
known in the art, and is described, in detail, in, for example, U.S. Patent No. 5,753,486.
Assays for determining the biological properties of a TPA molecule prepared according to the
methods of the present invention are similarly well known in the art. Briefly, a TPA
molecule synthesized as disclosed elsewhere herein can be assayed for their ability to lyse
fibrin in the presence of saturating concentrations of plasminogen, according to the method of
Carlsen et al. (1988, Anal. Biochem. 168: 428 ). The in vitro clot lysis assay measures the
activity of tissue-type activators by turbidimetry using a microcentrifiigal analyzer. A
mixture of thrombin and TPA is centrifuged into a mixture of fibrinogen and plasminogen to
initiate clot formation and subsequent clot dissolution. The resultant profile of absorbance
versus time is analyzed to determine the assay endpoint. Activities of the TPA variants are
compared to a standard curve of TPA. The buffer used throughout the assay is 0.06M sodium
phosphate, pH 7.4 containing 0.01% (v/v) TWEEN 80 and 0.01% (w/v) sodium azide.
Human thrombin is at a concentration of about 33 units/ml. Fibrinogen (at 2.0 mg/ml
clottable protein) is chilled on wet ice to precipitate fibronectin and then gravity filtered.
Glu-plasminogen is at a concentration of 1 mg/ml. The analyzer chamber temperature is set
at 37'^ C. The loader is set to dispense 20 microliters of TPA (about 500 nanograms/milliliter
to about 1.5 micrograms per milliliter) as the sample for the standard curve, or 20 microliters
of variant TPAs at a concentration to cause lysis within the range of the standard curve.
Twenty microliters of tlirombin as the secondary reagent, and 200 microliters of a 50:1 (v/v)
fibrinogen: plasminogen mixture as the primary reagent. The absorbance/time program is
used with a 5 min incubation time, 340-nanometer-filter and 90 second interval readings.
L. lL-2
The present invention further encompasses a method for the remodeling and
modification of IL-2. IL-2 is the main growth factor of T lymphocytes and increases the
humoral and cellular immune responses by stimulating cytotoxic CDS T cells and NK cells.
IL-2 IS therefore crucial in the defense mechanisms against tumors and viral infections. 0,-2
is also used in therapy against metastatic melanoma and renal adenocarcinoma, and has been
used in clinical trials in many fonns of cancer. Further, IL-2 has also been used in HIV
infected patients where it leads to a significant increase in CD4 counts.
Given the success IL-2 has demonstrated in the management and treatment of life-
threatening diseases such as various cancers and AIDS, it follows that the methods of the
present invention would be useful for developing an IL-2 molecule that has a longer
biological half-life, increased potency, and in general, a therapeutic profile more similar to
wild-type IL-2 as it is synthesized secreted in the healthy human.
A remodeled IL-2 peptide may be administered to a patient selected from the group
consisting of a patient having metastatic renal cell carcinoma, a patient having metastatic
melanoma, a patient having ovarian cemcer, a patient having Acute Myelogenous Leukemia
(AML), a patient having non-Hodgkin's lymphoma (NHL), a patient infected with HIV, and
a patient infected with Hepatitis C. A remodeled IL-2 peptide may also be useful for
adrainisteration to a patient as a cancer vaccine adjuvant. Preferably, the patient is a human
patient.
IL-2 has been cloned and sequenced, and the nucleic acid and amino acid sequences
are presented herein as SEQ ID NO:27 and SEQ ID NO:28 (Figure 71A and 71B,
respectively). The present mvention should in no way be construed as limited to the IL-2
nucleic acid and amino acid sequences set forth herein. Variants of IL-2 are described in, for
example, U.S. Patent No. 6,348,193, in which the asparagine at position 88 is substituted for
arginine, and in U.S. Patent No. 5,206,344, in which a polymer comprising IL-2 variants with
various amino acid substitutions is described. The present invention encompasses these IL-2
variants and others well known in the art.
Methods for the expression and to determine the activity of IL-2 are well known in
the art, and are described in, for example, U.S. Patent No. 5,417,970. Briefly, expression of
IL-2, or variants thereof, can be accomplished in a variety of both prokaryotic and eukaryotic
systems, including E. coli, CHO cells, BHK cells, insect cells using a baculovirus expression
system, all of which are well known in tlie art.
Assays for the activity of a modified IL-2 prepared according to the methods of tiie
present invention can proceed as follows. Peripheral blood lymphocytes can be separated
from the erythrocytes and granulocytes by centrifuging on a Ficoil-Hypaque (Phannacia,
Piscataway, NJ) gradient by the method described in, for example, A. Boyum et al. (Methods
in Enzymology, 1984, Vol. 108, page 88, Academic Press, Inc.). Lymphoc>les are
subsequently washed about tluee times in culture medium consisted of RPMI 1640 (Gibco-
BRL, La Jolla, CA) plus 10% AB human serum (CTS Purpan, Toulouse, France) inactivated
by heat (1 hour at 56° C), 2 mM sodium pyruvate, 5 mM HEPES, 4 mM L-glutamine, 100
U/ml penicillin, 100 ^ig/ml streptomycin and 0.25 i^g/ml amphotericm B (complete medium).
Adhesive cells (monocytes and macrophages) are eliminated by adhesion to plastic and the
remainder of the cells are suspended in complete medium at a concentration of about 5 to 10
XI0^ cells per milliliter and seeded in culture flasks at a density of about 1-2 X 10^ cells per
square centimeter. Flasks are then incubated at 37° C in a 5% CO2 atmosphere for about 1
horn-, after which the non-adhesive lymphocytes are recovered by aspiration after gentle
agitation of the culture flasks.
Non-adhesive lymphocytes are washed once and cultivated at a concentration of about
10^ cells per milliliter in complete medium in the presence of the IL-2 of the present
invention for about 48 hours in an incubator as described above. The cells are then washed
once.
The cytotoxic activity of the cells is evaluated after about 4 hours of contact with
target cells of the human T lymphoid line C8166-45/C63 (HTl cells) resistant to NK cell
cytotoxicity, as described by Salahuddin et al. (1983, Virology 129: 51-64; 1984, Science:
223, 703-707). 6X10^ HTl cells are radio-tagged with about 200 |iCi of ^'Cr (sodium
chromate, Amersham, Arlington Heights, IL) at 37° C for about 1 hour in complete medium
without serum, and then washed several times. The target cells and effective cells are
distributed in round-bottomed microtitration plates with varying ratios of effective cells to
target cells (50:1, 10:1, 1:1). The microtitration plates are centrifuged and, after incubation as
described above, the supernatant from each well is recovered and the radioactivity is
measured using a gamma counter. Cytotoxicity is determined from the quantity of Cr
released by dead target cells. Non-specific cytotoxicity is determined from the amount of
radioactivity spontaneously released from the target cells in the absence of effective cells.
The present method is just one of many well known in the art for measuring the
cytotoxicity of effector cells, and is should not be construed as luniting to the present
invention.
M. Factor Vin
The invention further encompasses a method for the remodeling and modification of Factor
VIII. As described earlier for Factor VII and Factor DC, Factor VIU is a critical component of
the blood coagulation pathway. Human Factor VIU, (antihemophilic factor; FVIII:C) is a
human plasma protein consisting of 2 peptides (light chain molecular weight of 80 kDa and
heavy chain molecular weight variable from 90 to 220 kDa, depending on glycosylation
state). It is an essential cofactor in the coagulation pathway and is required for the
conversion of Factor X into its active form (Factor Xa). Factor VIII circulates in plasma as a
non-covalent complex with von Willibrand Factor (aka FVIIIrRP), a dimer of a 2050 aa
peptide (See, U.S. Patent No. 6,307,032). Blood concentrations of Factor VIII below 20% of
normal cause a bleeding disorder designated hemophilia A. Factor VIII blood levels less than
1% result in a severe bleeding disorder, with spontaneous joint bleeding being the most
common symptom.
Similar to other blood coagulation factors. Factor VIII is a therapeutic with a great
deal of potential for the treatment of various bleeding disorders, such as hemophilia A and
hemophilia B. Due to the glycosylation of the heavy chain, current methods for the
preparation of Factor VIII from recombinant cells results in a product that is not as effective
as natural Factor VIII. Purification methods from human plasma result in a crude
composition that is less effective and more difficult to prepare than recombinant Factor VIII.
The current invention seeks to improve this situation.
A remodeled Factor VIII peptide may be administered to a patient selected from the
group consisting of a patient having von Willebrand's disease, a patient having Hemophilia
A, a patient having Factor VJILC deficiency, a patient having fibrinogen deficiency, a patient
having Factor XIII deficienc\-, and a patient having acquired Factor VIII inhibitors (acquired
hemophilia). A remodeled Factor VIII peptide may also be administered to a patient to
prevent, treat or control bleeding or hemorrhagic episodes. Preferably, the patient is a human
patient.
The nucleic acid and iunino acid sequences of Factor VIII are presented herein as
SEQ ID NO:29 and SEQ ID NO:30, respectively (Figiu-e 72A and 72B, respectively). The
art is rife with variants of Factor VIII, as described in, for example, U.S. Patent No.
5,668,108, in which the aspailic acid at position 1241 is replaced by a glutamic acid witli the
accompanying nucleic acid changes as well. U.S. Patent No. 5,149,637 describes a Factor
VIII variants comprising the C-terminal fraction, either glycosylated or unglycosylated, and
U.S. Patent No. 5,661,008 describes a Factor VIII variant comprising amino acids 1-740
linked to amino acids 1649 to 2332 by at least 3 amino acid residues. Therefore, variants,
derivatives, modifications and complexes of Factor VIII are well known in the art, and are
encompassed in the present invention.
Expression systems for the production of Factor VIII are well known in the art, and
include prokaryotic and eukaryotic cells, as exemplified in U.S. Patent Nos. 5,633,150,
5,804,420, and 5,422,250.
To determine the biological activity of a Factor VIII molecule synthesized according
the methods of the present invention, the skilled artisan will recognize that the assays
described herein for the evaluation of Factor VII and Factor IX .are applicable to Factor VIII.
N. Urokinase
The present invention also includes a method for the remodeling and/or modification
of urokinase. Urokinase is a serine protease which activates plasminogen to plasmin. The
protein is synthesized in a variety of tissues including endothelium and kidney, and is
excreted in trace amounts into urine. Purified urokinase exists in two active forms, a high
molecular weight fonn (HUll; approximately 50 kDa) and a low molecular weight form
(LUK; approximately 30 kDa). LUK has been shown to be derived from HUK by a
proteolysis after lysine 135, releasing the first 135 amuio acids fiom HUK. Conventional
wisdom has held that HUK or LUK must be converted to proteolytically active forms by the
proteolytic hydrolysis of a single chain precursor, also termed prourokinase, between lysine
158 and isoleucine 159 to generate a two-chain activated form (which continues to
correspond to either ITUK or LUK). The proteolytically active urokinase species resulting
from this hydrol>^ic clip contains two amino acid chains held together by a single disulfide
bond. The two chains formed by tlie activation clip are termed the A or Ai chains (HUK or
LUK, respectively), and the B chain comprising the protease domain of the molecule.
Ui'okinase has been shown to be an effective thrombol>'1ic agent. Ho\\e\'er, since ii is
produced naturally in trace quantities the cost of the enzyme is high for an effective dosage.
Urokinase has been produced in recombinant cell culture, and DNA encoding urokinase is
known together with suitable vectors and host microorganisms. Present compositions
comprising urokinase and methods for producing urokinase recombinantly are hampered by a
product that has deficient glycosylation patterns, and given the complex proteolytic cleavage
events surrounding the activation of urokinase, this aberrant glycosylation leads to a less
effective and less potent product.
A remodeled urokinase peptide may be administered to a patient selected from the
group consisting of a patient having an embolism, a patient having an acute massive
pulmonary embolism, and a patient having coronary artery thrombosis. Preferably, the
patient is a human patient. A remodeled urokinase peptide may also be used to restore
patency to an intravenous catheter, including a central venous catheter obstructed by clotted
blood or fibrin.
The sequence of the nucleotides encoding the primary amino acid chain of urokinase
are depicted in SEQ ID NO:33 and SEQ ID NO:34 (Figure 73A and 73B, respectively).
Variants of urokinase are well known in the art, and therefore tlie present invention is not
limited to the sequences set forth herein. In fact, the skilled artisan will readily realize that
lu-okinase variants described in, for example U.S. Patent Nos. 5,219,569, 5,648,253, and
4,892,826, exist as functional moieties, and are therefore encompassed in the present
invention.
The expression and e\'aluation of a urokinase molecule prepared according to the
methods of the present invention are similarly well known m the ;art. As a non-limiting
example, the expression of urokinase in various systems is detailed in U.S. Patent No.
5,219,569. An assay for determining the activity and functionality of a urokinase prepared in
accordance to the methods set forth herein are described throughout the literature, and are
similar to assays for other plasminogen and fibrm related assays described elsewhere
throughout. One example of an assay to determine the activity of an urokinase molecule
S3'nthesized as described herein can be as described in, for example, Ploug, et al. (1957,
Biochim. Biophys. Acta 24: 278-282), using fibrin plates comprising 1.25% agarose, 4.1
mg/ml human fibrinogen, 0.3 uiits/ml of thrombin and 0.5 ^ig/ml of soybean trypsin
inliibitor.
O. Human DNase
The present invention further encompasses a method for the remodeling and/or
modification of recombinant human DNase. Human DNase I has been tested as a therapeutic
agent and was shown to diminish the viscosity of cystic fibrosis mucus in vitro. It has been
determined that purulent mucus contains about 10-13 mg/ml of DNA, an ionic polymer
predicted to affect the rheologic properties of airway fluids. Accordingly, bovine pancreatic
DNase I, an enzyme that degrades DNA, was tested as a mucolytic agent many years ago but
did not enter clinical practice, because of side effects induced by antigenicity and/or
contaminating proteases. Recombinant human DNase is currently used as a therapeutic agent
to alleviate the symptoms of diseases such as cystic fibrosis.
A remodeled rDNase peptide may be administered to a patient having cystic fibrosis.
A remodeled rDNase peptide may also be administered to a cystic fibrosis patient to improve
pulmonary function. Preferably, the patient is a human patient.
Similar to DNase derived from bovine sources, recombinant human DNase poses
some problems, mostly due to lowered efficacy due to improper glycosylation imparted by
mammalian expression systems currently in use. The present invention describes a method
for remodeling DNase, leading to increased efficacy and better therapeutic results.
The nucleotide and amino acid sequences of human DNAse are presented herein as
SEQ ID NO:39 and SEQ ID NO:40 (Figure 74A and 74B, respectively). Variants of the
peptide comprising DNase are well known in the art. As an example, U.S. Patent No.
6,348,343 describes a human DNase with multiple amino acid substitutions throughout the
primary structure. Additionally, U.S. Patent No. 6,391,607 describes a hyperactive variant of
DNase with multiple amino acid substitutions at positions 9, 14, 74, 75, and 205. The present
examples, and others well known in the art or to be discovered in the future are encompassed
in the present invention.
E.xpression systems for producing a DNase peptide are well known to the skilled
artisan, and have been described in prokaryotic and eukaryotic systems. For example, PCT
Patent Publication No. WO 90/07572 describes these methods in considerable detail.
Assays to determine the biological activity of a DNase molecule developed according
to the methods of the present invention are well known in the art. As an example, but in no
way meant to be limiting to the present invention, an assay to determine the DNA-hydrol>tic
-317-
activity of human DNase I is presented herein. Briefly, two different plasmid digestion
assays are used. The first assay ("supercoiled DNA digestion assay") measures the
conversion of supercoiled double-stranded plasmid DNA to relaxed (nicked), linear, and
degraded forms. The second assay ("linear DNA digestion assay") measured the conversion
of linear double-stranded plasmid DNA to degraded forms. Specifically, DNase prepared
according to the methods of the present invention is added to 160 microUters of a solution
comprising 25 micrograms per milliliter of either supercoiled plasmid DNA or EcoRI-
digested linearized plasmid DNA m 25 mM HEPES, pH 7.1, 100 jag/ml bovine serum
albumin, 1 mM MgCl2, 2.5 mM CaCh, 150 mMNaCl, and the samples are incubated at room
temperature. At various times, aliquots of the reaction mixtures are removed and quenched
by the addition of 25 mM EDTA, together with xylene cyanol, bromophenol blue, and
glycerol. The integrity of the plasmid DNA in the quenched samples is analyzed by
electrophoresis of the samples on agarose gels. After electrophoresis, the gels are stained
with a solution of ethidium bromide and the DNA in the gels is visualized by ultraviolet light.
The relative amounts of supercoUed, relaxed, and linear forms of plasmid DNA are
determined by scanning the gels with a fluorescent imager (such as the Molecular Dynamics
Model 575 Fluorlmager) and quantitating the amount of DNA in the bands of the gel that
correspond to the different forms.
P. Insulin
The invention further includes a method for remodeling insulin. Insulin is well
known as the most effective treatment for type I diabetes, m which the beta islet cells of the
pancreas do not produce insulin for the regulation of blood glucose levels. The ramifications
of diabetes and micontrolled blood glucose include circulatory and foot problems, and
blindness, not to mention a variety of otlier complications that either result from or are
exacerbated by diabetes.
Prior to the cloning and sequencing of human insulin, porcine insulin was used as a
treatment for diabetes, hisulin is now produced recombinantly, but the short, 51 amino acid
sequence of the mature molecule is a complex structure comprising multiple sulfide bonds.
Current methods to recombinantly produce insulin result in a product that lacks sirailaritv' to
the native protein as produced in healthy non-diabetic subjects. The present invention seeks
to repair this flaw.
A remodeled insulin peptide may be administered to a patient selected from the group
consisting of a patient having Type I Diabetes (diabetes mellitus) and a patient having Type 2
diabetes mellitus who requires basal (long-acting) insulin for the control of hyperglycemia.
A remodeled insulin peptide may also be administered to a diabetic patient to control
hyperglycemia. Preferably, the patient is a human patient.
The nucleotide and amino acid sequence of human msulin is portrayed in SEQ ID
NO:43 and SEQ IDNO:44, respectively (Figure 75A and 75B, respectively). Variants of
insulin are abundant throughout the art. U.S. Patent No. 6,337,194 describes insulin fusion
protein analogs, U.S. Patent No. 6^323,311 describes insulin derivatives comprising a cyclic
anhydride of a dicarboxylic acid, and U.S. Patent No. 6,251,856 describes an insulin
derivative comprising multiple amino acid substitutions and a lipophilic group. The skilled
artisan will recognize that the following examples of insulin derivatives are in no way
exhaustive, but simply represent a small sample of those well known in the art. Therefore,
the present invention comprises insulin derivatives known or to be discovered.
Expression systems for the production of insulin are well kaown in the art, and can be
accomplished using molecular l^iology techniques as described in, for example, Sambrook et
al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
New York).
Assays to determine the functionality of an insulin molecule prepared according to the
methods of the present invention are similarly well known in tlie art. For example, an in vivo
model of glucose depression can be used to evaluate the biological activity of insulin
synthesized using the methods of the present invention. Useful for this purpose is a rat
model. Th3 animals are fasted overnight (16 hours) prior to the experiment, and then
anesthetized with intraperitoneally administered sodium pentobarbital or another suitable
anesthetic such as ketamine. Each animal receives an i.v. injection (tail vein) of the particular
insulin derivative (20 p,g/ml/kg). Blood samples are taken from the jugular vein 15 and 5
minutes before injection and 15, 30, 60, 90, 120, 180, and 240 minutes after injection. Blood
glucose le\els are measured wiiii a blood glucose monitor, available from a variety of
commercial suppliers.
0. Hepatitis B Vaccines (HBsAg)
The present invention further comprises a method for the remodeling the antigen used
in hepatitis B vaccines (HbsAg or Hepatitis B sAg). HBsAg is a recombinantly produced
surface antigen of the hepatitis B S-protein, and is used to illicit an immune response to the
hepatitis B virus, an increasing dangerous virus that results in, anriong other things, liver
disease including cirrhosis and carcinoma, and results in over 1 nriiliion deaths worldwide
annually. Currently the HBsAg vaccine is administered three times over a six month interval
to illicit a protective and neutralizing immune response.
HBsAg is currently produced in yeast strains, and therefore reflects the glycosylation
patterns native to a fungus. The present invention provides a method to remodel HBsAg,
resulting in among other things, improved immunogenicity, antibodies with improved affinity
for the vims, and the like.
A remodeled HBsAg peptide may be administered to a patient to immunize the
patient against disease caused by a Hepatitis B virus. A remodeled HBsAg peptide may also
be administered to a predialysis patient or a dialysis patient to immunize the patient against
disease caused by a Hepatitis B virus. Preferably, the patient is a human patient.
The sequences of the S-protein from a Hepatitis B virus (IfBsAg) nucleic acid and
primary amino acid chain are set forth herein as SEQ ID NO:45 and SEQ ID NO:46 (Figiure
76A and 76B, respectively). The nucleotide is 1203 bases in length. The amino acid is 400
residues long. The last 226 amino acid residues are the small S-aatigen, which is used in the
GlaxoSmithKline vaccine and the Merck vaccine. Fifty-five amino acids upstream from the
small S-antigen is the Pre-S start codon. The Pre-S + S regions aire tlie middle S antigen,
which is used in the Aventis Pasteur vaccine. From the first start codon to the Pre-S start
codon comprises tlie rest of the S-protein, and is called the large S-protein. Tliis is but one
example of the HBsAg used in vaccines, and other subtypes are well known, as exemplified
in GenBank AccNos.: AF415222, AF41522I, AF415220, and AF4152I9. The sequences
presented herein are simply examples of HBsAg knov^7i in the art. Similar antigens have
been isolated from other strains of hepatitis B virus, and may or may not have been e\aluaL-
for antigenicity and potential as vaccine candidates. The present invention diereforc
encompasses hepatitis B vaccme S-protein suriace antigens known or to be discovered.
-170-
Expression of an HBsAg in an expression system is a routine procedure for one of
skill in the art, and is described in, for example, U.S. Patent No. 5,851,823. Assays for the
immunogenicity of a vaccine are well known in the art, and comprise various tests for the
production of neutralizing antibodies, and employ techniques such as ELISA, neutralization
assays. Western blots, immimoprecipitation, and the like. Briefly, a sandwich ELISA for the
detection of effective anti-HBsAg antibodies is described. The Enzygnost HBsAg assay
(Aventis Behring, King of Prussia, PA) is used for such methods. Wells are coated with anti-
HBs. Serum plasma or purified protein and appropriate controls are added to the wells and
incubated. After washing, peroxidase-labeled antibodies to HBsAg are reacted with the
remaining antigenic determinants. The unbound enzyme-linked antibodies are removed by
washing and the enzyme activity on the solid phase is determined by methods well known in
the art. The enzymatically catalyzed reaction of hydrogen peroxide and chromogen is
stopped b)' adding diluted sulftiric acid. The color intensity is proportional to the HBsAg
concentration of the sample and is obtained by photometric comparison of the color intensity
of the unknown samples with the color intensities of the accompanying negative and positive
control sera.
R. Human Growth HoiTnone
The present invention further encompasses a method for the remodeling of human
growth hormone (HGH). The isoform of HGH which is secreted in the human pituitary',
consists of 191 amino acids and has a molecular weight of about 21,500. The isoform of
HGH which is made in the placenta is a glycosylated form. HGH participates in much of the
regulation of normal human growtli and development, including liinear growth
(soraatogcuesis), lactation, activation of macrophages, and insulin-like and diabetogenic
effects, among others.
HGH is a complex hormone, and its effects are varied as a result of interactions with
various ccllulai- receptors. Wliile compositions comprising HGH have been used in the
clinical setting, especially to tieat dwarfism, the efficacy is limited by the absence of
glycosylation of the HGH produced recombinantly.
A remodeled HGH peptide may be administered to a patient selected from the group
consisting of a patient having a grovvtli hormone deficiency, a patient having Turner
TOT
syndrome, a patient having growth failure due to a lack of adequate endogenouse growth
hormone secretion, a patient having growth failvire due to Prader-Willi syndrome (PWS), a
patient having growth failure associated with chronic renal insufficiency, and a patient having
AIDS associated wasting or cachexia. A remodeled HGH peptide may also be administered
to a patient having short stature. Preferably, the patient is a human patient.
The nucleic and amino acid sequence of HGH are set forth elsewhere herein as SEQ
Ii:) NO:47 and SEQ ID NO:48 (Figure 77A and 77B, respectively). The skilled artisan v^U
recognize that variants, derivatives, and mutants of HGH are well known. Examples can be
found in U.S. Patent No. 6,143,523 where amino acid residues at positions 10, 14, 18, 21,
167, 171, 174, 176 and 179 are substituted, and in U.S. Patent No. 5,962,411 describes splice
\ariants of HGH. The present invention encompasses these HGH variants known in the art of
to be discovered.
Methods for the expression of HGH in recombin;int cells is described in, for example,
U.S. Patent No. 5,795,745. Methods for expression of HGH in, inter alia, prokaryotes,
cukaryotes, insect cell systems, plants, and in vitro translation systems are well known in the
art.
An HGH molecule produced using the methods of the current invention can be
assayed for activity using a variety of methods known to the skilled artisan. For example,
U.S. Patent 5,734,024 describes a method to determine the biological functionality of em
expressed HGH.
S. Anti-Tlirombin III
Antithrombiji (antithrombin III, AT-III) is a potent inhibitor of the coagulation
cascade in blood. It is a non-vitamin K-dependent protease tliat inliibits the action of
tlirombin as well as other procoagulant factors (e.g., Factor Xa). Congenital antithrombin III
deficiency is an autosomal dominant disorder in which an individual inherits one copy of a
defective gene. This condition leads to increased risk of venous and arterial thrombosis, with
onset of clinical manifestations typically presenting in young adulthood. Severe congenital
antithrombin HI detlciency, in which the individual inlierits two defective genes, is ani
autosomal reccssiw; condition associated with increased thrombogcnesis, typically noted in
infcincy. Acquired antitlrrombin III deficiency most ccaiumorily is seen in situations where
there is inappropriate activation of the coagulation system. Common conditions that result in
acquired antithrombin in deficiency include disseminated intravascular coagulation,
microangiopathic hemolytic anemias due to endothelial damage (i.e., Hemolytic-uremic
syndrome), and veno-occlusive disease (VOD) seen in patients undergoing bone marrow
transplant. AT-III deficiency may be corrected acutely by infusions of AT-III concentrates.
A remodeled AT-III peptide may be administered to a pafient selected from the group
consisting of a patient having a hereditary AT-III deficiency m coimection with a surgical or
obstetrical procedure and a hereditary AT-III deficient patient having a thromboembolism.
Preferably, the patient is a human patient.
Antithrombin III (AT-III) is an a2 -glycoprotein of molecular weight 58,000. It is
sold commercially as Thrombate III™ (Bayer Corp., West Haven, CT). The nucleic acid
and amino acid sequences of human antithrombin HI are displayed in Figure 78 A (SEQ ID
NO:63) and 78B (SEQ ID NO:64), respectively.
Methods to make anti-throrabin III are well know to those in the art. For example,
published nucleic acid and amino acid sequences are available for human antithrombin III
(see, U.S. Pateat No. 4,517,294) and mutants of human antithrombin III (see, U.S. Patent
Nos. 5,420,252, 5,618,713, 5700,663). The methods of the invention may be used with any
of these amino acid sequences and any nucleic acid sequences that encode them, but are not
limited to these sequences. Exemplary methods to produce recombinant antithrombu) III arc
well ioiONvn in the art, and several are described in U.S. Patent Nos. 5,420,252, 5,843,705,
6,441,145 and 5,994,628. Exemplary methods to purify recombinant antitlirombin III are
described in U.S. Patent Nos. 5,989,593, 6,268,487, 6,395,888, 6,395,881, 6,451,978 and
6,518,406.
There are many blown uses for recombinant antitlirombin III. Antitlirombin III can
be used a? a anticoagulant during surgery (U.S. Patent Nos. 5,252,557, 5,182,259), as part of
a pharmaceutical preparation or method to inliibit tlirombosis (U.S. Patent Nos. 5,565,471,
6,001,820), and to reduce the adverse side effects of ceUuiar transplantation (X'.S. Patent No.
6,387,366). Additionally, antiihrombin III preparations can be used to incre^ise placental
biood flov/ (U.S. Patent No. 5,888,964), mhibit fertilization (U.S. Patent No. 5,545,615), treai
asthma (U.S. Patent No. 6,355,626) and treat artliritis (U.S. Patent No. 5,252,557) and other
iatlamroatory processes (U.S. Patent No. 6,399,572). .Antithrombin HI can also be used to
manufacture replacement blood plasma (U.S. Patent Nos. 4,900,720) or prepare a stabilized
cellular blood product (U.S. Patent No. 6,139,878) for transfiisions. Antithrombin III may be
administered as a pharmaceutical preparation (U.S. Patent Nos. 5,084,273, 5,866,122,
6,399,572, 6,156,731 and 6,514,940) or using gene therapy methodology (U.S. Patent No.
6,410,015). Compositions comprising antithrombin III can be used as tissue adhesives (U.S.
Patent No. 6,500,427) or lubricants for medical devices that are introduced to the patient
(U.S. Patent No. 6,391,832). Antithrombin III can also be used to coat endovascular stents
(U.S. Patent Nos. 6,355,055, 6,240,616, 5,985,307, 5,685,847 and 5,222,971), ocular
implants (U.S. Patent No. 5,944,753) and prostheses in general (TJ.S. Patent Nos. 6,503,556,
6,491,965 and 6,451,373). Antithrombin III can also be used in methods to locate an internal
bleeding site in apatient (U.S. Patent No. 6,314,314) and to determine hemostatic
dysfunction in a patient (U.S. Patent No. 6,429,017).
T. Human Chorionic Gonadotropin
Human Chorionic Gonadotropin (hCG) is a glycoprotein composed of an alpha
subunit and a beta subimit. HCG is closely related to two other gonadotropins, luteinizing
hormone (LH) and follicle stmiulating hormone (FSH), as well as thyroid stimulating
hormom; (TSH), all tliree of wliich are glycoprotein hormones. The alpha subunits of these
various glycoprotein hormones are structurally very similar, but the beta subunits differ in
amino acid sequence.
7'he nucleic acid and ;imino acid sequences of the human chorionic gonadotropin a-
subunit cire displayed in FiguJ-es 79A (SEQ ID NO:69) and 79B (SEQ ID NO:70),
respecti\'ely. The nucleic acid and amino acid sequences of the human chorionic
gonadotropin p-subunit are displayed in Figures 79C (SEQ ID N0:7l) and 79D (SEQ ID
NO;72), respectively.
Human chorionic gonadotropin is used in an infertility treatment to promote ovulatiu-¦
or release of an egg from the ovary in women who do not ovulate on their own. Human
chorionic gonadotropin is also given to young males to treat undescended or underdeveloped
testicles. It is used in men to stimulate the production of testosterone. Some physicians also
prescribe hiunan chorionic gonadotropin for men having erictile dysftmctionor lack of sexua!
desire, and for treatment of male "menopause."
A remodeled hCG peptide may be administered to a patient selected from the group
consisting of a patient undergoing assisted reproductive technology (ART), a patient
undergoing in vitro fertili2ation (TVF), a patient undergoing embryo transfer, an infertile
patient, a male patient having prepubertal cryptoorchidism not due to anatomical obstruction,
and a male patient having hypogonadotropic hypogonadism. A remodeled hCG peptide may
also be administered to induce final follicular maturation and early luteinization in an infertile
female patient, wherein the infertile female patient has undergone pituitaiy desensitization
and pretreatment with follicle stimulating hormones. A remodeled hCG peptide may also be
administered to induce ovulation and pregnancy in an anovulatory infertile patient.
Preferably, the patient is a human patient.
Methods to make human chorionic gonadotropin are well known in the art. The
heterodimeric hCG can be recombinantly made in any one of many expression systems
cunently used for industrial manufacture of recombinant proteins. One method of making
recombinant hCG is described in U.S. Patent No. 5,639,639. Methods for making
recombinant heterodimeric proteins by expressing both subunits in the same cell are, in
general, well known in the ait, and several methods are described in the U.S. Patent Nos.
5,643,745 (expression in a filamentous ftmgus), 5,985,611 and 6,087,129 (expression in
secretory cells). Alternatively, each subunit can be expressed individually in cells, and die
two subunits later brought together in vitro for assembly into the heterdimer.
Metliods for using human chorionic gonadotropin are numerous and well known in
the art. Commonly, hCG is used to induce or synchronize ovulation in mammals (see, U.S.
Patent Nos. 6,489,288, 5,589,457, 5,532,155, 4,196,123, 4,062,942 and 4,845,077).
Additionally, hCG can be used in pregnancy tests, and in particular agglutination-based test.s
(see, U.S. Patent Nos, 3,991,175, 4,003,988, 4,071,314 and 4,088,749). hCG can also be
used in a contraceptive vaccine (see, U.S. Patent Nos. 4,161,519 find 4,966,888). In addition.
hCG can be used to treat conditions related to aging and altered hormonal balance such as
benign prostatic hypertrophy (see, U.S. Patent No. 5,610,136) and central ner\'ous system
diseases common in the elderly (see, U.S. Patent No. 4,791,099).
Altemati\'eiy, hCG can be used to detect and treat cancers that express hCG or one ¦)!'
its subunits. hCG-expressing tumors include, but are not limited to, breast, prostate, ovar>
and stomach carcinomas, and neuroblastomas such as Kaqiosi's sarcoma. Antibodies can be
raised to hCG which has been glycoreraodeled so as to have glycan structures similar ro those
found on the tumor-expressed hCG, and these antibodies may be used to detect hCG-
expressing tumors in patients according to methods well known in the art (see, U.S. Patent
Nos. 4,311,688, 4,478,815 and 4,323,546). Additionally, remodeled hCG can be used to
raise an immune response to a tumor that is expressing hCG (see, U.S. Patent Nos. 5,677,275,
5,762,931, 5,877,148, 4,970,071 and 4,966,753).
hCG can also be used in methods to generally immunomodulate an animal, such as
described in U.S. Patent Nos. 5,554,595, 5,851,997 and 5,700,781. In addition, hCG can be
used as an inhibitor of the matrix metalloprotease in conditions benefiting from such
treatment, such as chronic inflammatory diseases, multiple sclerosis and angiogenesis-
dependent diseases (see, U.S. Patent No. 6,444,639).
U. g-Iduronidase
a-Iduronidase is sold commercially as Aldurazyme''^'*^ (BioMarin and Genzyme). It is
useful for replacement therapy for the treatment of MPS I, a lysosomal storage disease. h/fPS
I (also known as Hurler disease) is a genetic disease caused by the deficiency of alpha-L-
iduronidase, an enzyme normally required for the breakdown of certain complex
carbohydrates knowTi eni glycosaminoglycans (GAGs). The normal breakdown of GAGs is
incomplete or blocked if the enzyme is not present in sufficient quantity. The cell is then
unable to excrete the caibohydrate residues and they accumulate in the lysosoraes of the cell
and cause MPS I.
A remodeled alpha-iduronidase peptide may be administered to a patient selected
from the group consisting of a patient having a lysosomal storage disease, a patient having an
alpha-L-iduronidase deficiency, a patient having mucopolysaccaridosis I (MPS 1), and a
patient having Hurler disease. Preferably, the patient is a human patient.
Methods to produce and purify a-iduronidase, as well as methods to treat certain
genetic disorders including a-L-iduronidase deficiency and mucopolysaccharidosis I (MPS 1)
are described in U.S. Patent No. 6,426,208. The nucleic acid and amino acid sequences of
human a-iduronidase are found in Figures 80A (SEQ ID NO:65) and SOB (SEQ ID NO;66),
respectively.
V. g-Galactosidase A
a-Galactosidase A (also known as agalsidase beta) is sold commercially as
Fabrazyme'^'^ (Genzyme). a-Galactosidase A is usefiil for the treatment of Fabry disease.
Fabry disease is a rare, inherited disorder caused by the deficiency of the essential enzyme a-
galactosidase. Without ttiis enzyme, Fabry patients are unable to breakdown a fatty acid
substance in their body called globotriasylceramide (GL-3), vi/hich accumulates in cells in the
blood vessels of the heart, kidney, brain and other vital organs. The progressive buildup of
this substance puts patients a risk for stroke, heart attack, kidney damage and debilitating
pain. Most patients develi^p kidney failure during adulthood, and severe organ complication;?
lead to death around age forty.
A remodeled alpha-galactosidase A peptide may be administered to a patient selected
from the group consisting of a patient having a lysosomal storage disease, a patient having ari
alpha-galactosidase A deficiency, and a patient having Fabry disease. Preferably, the patient
is a human patient.
The a-galactosidase A enzyme is a lysosomal enzyme wliich hydrolyzes
globotriaosylceramide and related giycolipids which have terminal a-galactosidase linkages.
It is a 45 kDa N-glycosylated protein encoded on the long arm of the X chromosome. The
initial glycosylated fonns (Mr=55,000 to 58,000) synthesized in human fibroblasts or Chang
liver cells tire processed to a mature glycosylated form (Mr=50,000). The mature active
enzyme as purified from human tissues and plasma is a homodimer (Bishop et al., 1986,
Proc. Natl. Acad. Sci. USA 83: 4859-4863). The nucleic acid £md amino acid sequences of
a-galactosidase A are found in Figures 81A (SEQ ID NO;67) and 81B (SEQ ID NO:68).
Other useful nucleic acid and amino acid sequences of alpha-galactosidase A are foimd in
U.S. Patent No. 6,329,191.
References teaching how to make alpha-galactosidase A are found in U.S. Patent Nos.
5.179,023 and 5,658,567 (expression in insect cells), U.S. Patent No. 5,356,804 (expression
and secretion from mammalian cells, including CHO cells), U.S. Patent No. 5,401,451
(expression in mammalian cells), U.S. Patent No. 5,580,757 (expression in mammalian cells
as a fusion protein) and U.S. Patent No. 5,929,304 (expression in plant cells). Methods for
purifying recombinant alpha-galactosidase A are found in U.S. Patent No. 6,395,884.
References teaching how to use alpha-galactosidase A to treat patients include, but are
not limited to, U.S. Patent No. 6,066,626 (gene therapy) and U.S. Patent No. 6,461,609
(treatment with the protein). Mutant forms of alpha-galactosidase A that are useful in the
methods of the invention include, but are not limited to, those described in U.S. Patent No.
6,210,666.
W. Antibodies
The present invention further comprises a method for the remodeling of various
antibody preparations including chimeric antibody preparations, including, chimeric TNFR,
chimeric anti-glycoprotein Ilb/IIIa, chimeric anti-HER2, chimeric anti-RSV, chimeric anti-
C!D20, and chimeric anti-TNF. Chimeric antibody preparations comprise a humair Fc portion
fiom an IgG antibody and the variable regions from a monoclonal antibody specific for an
antigen. Other prepaiations comprise a receptor, for example the 75 kDa TNF receptor, fused
to a human IgG Fc portion. These molecules fiirther include Fab fragments comprising light
and heavy chains from human and mice. A chimeric TNFR is useful in the treatment of
inflammatory diseases, such as rheumatoid arthritis. Chimeric anti-glycoprotein Ilb/Illa is
useful in the treatment of cardiac abnomialities, blood clotting, and platelet function
disturbances. A chimeric anti-HER2 is useful as a treatment for breast cancer, chimeric anti-
RSV is useful for the treatment of respiratory syncytial virus, ciiiineric anti-CD20 is uselul
for the treatment of Non-Hodgkin's lymphoma, and cliimeric miti-TNF is used for treatmer;
of Crolin's disease.
While tliese chimeric antibodies have proved useful in the management ol' varied
diseases, administration has to be fairly frequent and at fmrly high doses due lo the rclati\'ei;
short half-life of a recombinant protein produced in rodent cells. Wliile a majority of tlic
chimeric antibody is human, and therefore regarded as "self by the immune system, the>' are
degraded and destroyed due to non-native glycosylation patterns. The present invention
proposes to repair this problem, greatly increasing the efficacy of these novel medicines.
Antibodies and Methods of their Generation
The tenn "antibody," as used herein, refers to an immunoglobulin molecule which is
able to specifically bind to a specific epitope on an antigen. Antibodies can be intact
immunoglobulins derived fiom natural sources or from recombinant sources and can be
immunoreactive portions of intact immunoglobulins. Antibodies are typically tetraniers of
immunoglobulin molecules. The antibodies in the present invention may exist in a variety of
forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and
F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999,
Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow
et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
By the term "synthetic antibody" as used herein, is meant an antibody which is
generated using recombinant DNA technology, such as, for example, an antibody expressed
by a bacteriophage as described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA molecule encoding the
antibody imd which DNA molecule expresses an antibody peptide, or an amino acid sequence
specifying the antibodj', wherein the DNA or amino acid sequence has been obtained using
s>Tithetic DNA or amino acid sequence technology which is available and well known in the
art.
Monoclonal antibodies directed against full length or peptide fragments of a peptide
or peptide may be prepared using any well known monoclonal antibody preparation
procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A
Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynski et al. (1988, Blood, 72:109-
115). Quantities of the desired peptide may also be synthesized using chemical s\Tithesis
teclinology. Alternatively, DNA encoding the desired peptide may be cloned and expressed
from an apjiropriate promoter sequence in cells suitable for the generation of large quantities
of peptide. Monoclonal anUboaics directed against the peptide are generated from mice
iimnunized with the peptide using standai'd procedures as referenced herein.
Nucleic acid encoding the monoclonal antibody obtained using the procedures
described herein may be cloned and sequenced using technology which is available in the art,
and is described, for example, in Wright et al. (1992, Critical Rev. in hnmunol. 12(3,4): 125-
168) and tlie references cited therein. Further, the antibody of the invention may be
"humanized" using the technology described in Wright et al., {supra) and in the references
cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77(4):755-759).
To generate a phage antibody library, a cDNA library is first obtained firom mRNA
which is isolated firom cells, e.g., the hybridoma, which express the desired peptide to be
expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are
produced using reverse transcriptase. cDNA which specifies immunoglobulin fi'agments are
obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to
generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes.
The procedures for making a bacteriophage library comprising heterologous DNA are well
known in tlie art and are described, for example, in Sambrook and Russell (2001, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, NY).
Bacteriophage which encode the desired antibody, may be engineered such that the
peptide is displayed on the surface thereof in such a manner that it is available for binding to
its corresponding binding peptide, e.g., the antigen against which the antibody is directed.
Thus, when bacteriophage which express a specific antibody are incubated in the presence of
a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell.
Bacteriophage which do not express the antibody will not bind to the cell. Such panning
tecluiiques arc well knov^Ti in the art and are described for example, in Wright et al., (supro
Processes such as those described above, have been developed for the production ¦ i
human antibodies using MIS bacteriophage display (Burton et ai., 1994, Adv. Immunol,
57:191-280). Essentially, a cDNA library is generated from mRNA obtained from a
population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin
genes and thus, the cDNA encodes tlie same. Amplified cDNA is cloned into MI3
expression vectors creating a library of phage which express hiunan antibody fragments o!i
their surface. Phage which display the antibody of interest cU-e selected by antigen bindinu
and are propagated in bacteria to produce soluble human immunoglobulin. Thus, in conti:.
to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding
human immunoglobulin rather than cells which express human immunoglobulin.
Remodeling glvcans of antibody molecules
The specific glycosylation of one class of peptides, namely immunoglobulins, has a
particularly important effect on the biological activity of these peptides. The invention should
not be construed to be hmited solely to immunoglobulins of the IgG class, but should also be
construed to include immunoglobulins of the IgA, IgE and IgM classes of antibodies.
Further, the invention should not be construed to be limited solely to any type of
traditional antibody structure. Rather, the invention should be construed to include all types
of antibody molecules, including, for example, fragments of eintibodies, chimeric antibodies,
human antibodies, humanized antibodies, etc.
A typical immunoglobulin molecule comprises an effector portion and an antigen
binding portion. For a review of immunoglobulins, see Harlow etal., 1988, Antibodies: A
Laboratory Manual, Cold Spring Harbor, New York, and Harlow et al., 1999, Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY. The effector
portion of the inununoglobulin molecule resides in the Fc portion of the molecule and is
responsible in part for efficient binding of the immunoglobulin to its cognate cellular
receptor. Improper glycosylation of immunoglobulin molecules particularly in the CH2
domain of the Fc portion of the molecule, affects the biological activity of the
immunoglobulin.
More specifically with respect to the iraniuiioglobulin IgG, IgG effector function is
governed in large part b>' whether or not the IgG contains an N-acetylglucosamine (GIcNAc'
residue attached at the 4-0 position of the branched mannose of the trimannosyl core of the
N-glycan at Asparagine (Asn) 297 in the CH2 domain of the IgG molecule. This residue is
known as a "bisecting GlcNAc." The purpose of adding bisecting GlcNAc to the N-glycau
chains of a natural or recombinant IgG molecule or a IgG-Fc-containing chimeric constru;
to optimize Fc immune effector function of the Fc portion of the molecule. Such effector
ftmctions may include antibody-dependent cellular cytotoxicity (ADCC) and any other
biological effects tliat require cftlcient binding to FcyR receptors, tuid binding to the CI
component of complement. T!ie importance of bisecting GlcNAc for achieving maxim un;
immune effector function of IgG molecules has been described (Lifely et al., 1995,
Glycobiology 5 (8): 813-822; Jeffns et al., 1990, Biochem. J. 268 (3): 529-537).
The glycans found at the N-glycosylation site at Asn 297 in the CH2 domain of IgG
molecules have been structurally characterized for IgG molecules found circulating in human
and animal blood plasma, IgG produced by myeloma cells, hybridoma cells, and a variety of
transfected immortalized mammalian and insect cell lines. In all cases the N-glycan is either
a high mannose chain or a complete ( Man3, GlcNAc4, Gal2, NeuAc2, Fuel) or variably
incomplete biantennary chain with or without bisecting GlcNAc (Raju et al., 2000,
Glycobiology 10 (5): 477-486; Jeffris et al., 1998, Immunological. Rev. 163L59-76; Lerouge
et al., 1998, Plant Mol. Biol. 38: 31-48; James et al., 1995, Biotechnology 13: 592-596).
The present invention provides an in vitro customized glycosylated immunoglobulin
molecule. The immunoglobulin molecule may be any immunoglobulin molecule, including,
but not limited to, a monoclonal antibody, a synthetic antibody, a ciiimeric antibody, a
humanized antibody, and the like. Specific metliods of generating antibody molecules and
their characterization are disclosed elsewhere herein. Preferably, the immunoglobulin is
IgG, and more preferably, the IgG is a humanized or human IgG, most preferably, IgGl.
The present invention specifically contemplates using |31,4-mannosyl-glycopeptide
P1,4-N-acetylglucosaminyltransferase, GnT-III: EC2.4.1.144 as im in vitro reagent to
glycosidically linkN-acetylglucosamine (GlcNAc) onto the 4-0 position of the branched
mannose of the trimannosyl core of the N-glycan at Asn 297 in the CH2 domain of an IgG
molecule. However, as will be appreciated from the disclosure provided herein, the invention
should not be construed to solely include the use of tliis enz>'me to provide a bisecting
GlcNAc to an immunoglobulin molecule. Rather, it has been discovered that it is possible to
modulate the glycosylation pattern of an antibody molecule such that the antibody molecule
has enlianced biological activity, i.e., effector function, in addition to potential enlaancement
of other properties, e.g., stability, and the like.
There is provided in tht? present invention a general method for removing fucose
molecules from die Asn(297) N-linked glycan for the purpose of enliancing binding to Fc-
gfuimiaRIIlA, and enhanced antibody-dependent cellular c>totoxicity (see. Shields et al.,
2002, J. Biol. Chem. 277:26733-26740). llie mediod entails contacting the antibody
molecule with a fucosidase apjiropriate ibr the linkage of liie fucose molecule(s) on the
antibody glycan(s). Alternately, the recombinant antibody can be expressed in cells that do
express flicosyltransferases, such as the Led 3 varient of CHO cells. The removal of fiicose
from the glycan(s) of the antibody can be done alone, or in conjunction with other methods to
remodel the glycans, such as adding a bisecting GlcNAc. Expression of antibodies in cells
lacking GnT-I may also result in Fc glycans lacking core fucose, which can be further
modified by the present invention.
There is provided in the present invention a general method for introducing a
bisecting GlcNAc for the purpose of enhancing Fc immune effector function in any
preparation of IgG molecules containing N-lmked oligosaccharides in the CH2 domain,
typically at Asn 297. The method requires that the population of IgG molecules is brought to
a state of glycosylation such that the glycan chain is an acceptor for GnT-III. This is
accomplished in any one of tliree ways: 1) by selection or genetic manipulation of a host
expression system that secretes IgG with N-glycan chains that are substrates for GnT-III; 2)
by treatment of a population of IgG glycoforms with exoglycosidases such that the glycan
structure(s) remaining after exoglycosidase treatment is an acceptor for GnT-III; 3) some
combination of host selection and exoglycosidase treatment as in 1) and 2) above plus
successive additions of GlcNAc by GnT-I and GnT-II to create an acceptor for GnT-III.
For example, IgG obtained from chicken plasma contains primarily high mannose
chains and would require digestion with one or more a-raannosidases to create a substrate for
addition of GlcNAc to the a 1,3 mannose branch of the trtmannosyl core by GnT-I. This
substrate could be the elemental trimannosyl core, Man3GlcNAc2. Treatment of this core
structure with a combination of GnT-I, GnT-II, and GnT-III uskig UDP-GlcNAc as a sugar
donor creates Man3GlcNAc5 as shown in Figure 1. The order of action of these
giycosyltransferases may be varied to optimize tlie production of the desired product.
Optionally, tliis structure can then be extended by treatment with pi,4 galactosyltransferas'"
If required, the galactosylated oligosaccharide can be further extended using a2,3- or a2/-
sialyltransferase to acliieve a completed biantennary structure. Using tliis method
biantemiary glycan chains can be remodeled as required for the optima! Fc inmaune effccr, ;
function of any therapeutic IgCi under development (Figure 3).
Alternatively, IgG molecules found in the plasma of most animals or IgG which i-
secreted as a recombin;uit product by most animal cells or by transgenic animals typicallinclude a spectrum of biantennary glycoforms including complete (NeuAc2, Gal2, GlcNAc4,
Man3, ±Fucl) (Figure 3) and variably incomplete forms, with or without bisecting GIcNAc
(Raja et al., 2000, Glycobiology 10 (5): 477-486; Jeffris et al., 1998, Immunological Rev.
163: 59-76). To ensure that bisecting GlcNAc is present in the entire population of
immunoglobulin molecules so produced, the mixture of molecules can be treated with the
following exoglycosidases, successively or in a mixture: neurammidase, p-galactosidase, P-
hexosaminidase, a-fucosidase. The resulting trunannosyl core can then be remodeled using
glycosyltransferases as noted above.
In some cases it may be desired to abolish effector function from existing antibody
molecules. The present invention also includes modifying the Fc glycans with appropriate
glycosidases and glycosyltransferases to eliminate effector function. Also anticipated is the
addition of sugars modified with PEG or other polymers that serve to hinder or abolish
binding of Fc receptors or complement to the antibody.
In addition, IgG secreted by transgenic animals or stored as "plantibodies" by
transgenic plants have been characterized. An IgG molecule produced in a transgenic plant
having N-glycans that contain pi,2 linked xylose and/or al,3 linked fucose can be treated
with exoglycosidases to remove those residues, in addition to the above described
exoglycosidases in order to create the trimannosyl core or a Man3GicNAc4 structure, and are
then treated with glycosyltransferases to remodel the N-glycan as described above.
The primary novel aspect of the current invention is the application of appropriate
glycosyltransferases, with or wthout prior exoglycosidase treatment, applied in the correct
sequence to optimize the effector function of the antibody. In one exemplary embodiment, h
bisecting GlcNAc is introduced into the glycans of IgG molecules or or other IgG-Fc-
chiiueric constructs where bisecting GlcNAc is required. In another exemplarv' embodimeni
the core fucose is removed from the glycans of IgG molecules or other IgG-Fc-chimeric
constructs..
X. TNF receptor-IgG Fc fusion protein
The nucleotide and amino acid sequences of the 75 kDa human TNF receptor are set
forth herein as SEQ ID N(3:31 and SEQ ID NO:32, respectively (Figure 82A and 82B,
respectively). The amino acid sequences of tiie light and heavy variable regions of chimeric
¦dim-HERll are set forth as SEQ ID NO:35 and SEQ ID NO:36, respectively (Figure 83A and
83B, respectively). The amino acid sequences of the heavy and light variable regions of
chimeric anti-RSV are set forth as SEQ ID NO:38 and SEQ ID NO:37, respectively (Figure
84A and 84B, respectively). The amino acid sequences of the non-human variable regions of
anti-TNF are set forth herein as SEQ ID N0:41 and SEQ ID NO:42, respectively (Figure 85A
and 85B, respectively). The nucleotide and amino acid sequence of the Fc portion of human
IgG is set forth as SEQ ID NO:49 and SEQ ID NO:50 (Figure 86A and 86B, respectively).
A remodeled cliimeric ENBREL'^'^ may be administered to a patient selected from the;
group consisting of a patient having rheumatoid arthritis and a patient havuig polyarticular-
course juvenile arthritis. A remodeled chimeric ENBREL"""^ may also be administered to an
arthritis patient to reduce signs, symptoms, or structural damage in the patient. Preferably,
the patient is a human patient.
A remodeled Synagis'''" antibody may be administered to a patient to immunize the
patient against infection by respiratory syncytial virus (RSV). A remodeled Synagis"^*^
antibody may also be administered to a patient to prevent or reduce the severit>' of a lower
respiratory tract disease caused by RSV. Preferably, the patient is a human patient.
Y. MAb anti-glycoprotein Ilb/IIIa
The amino acid sequences of a murine anti-glycoprotein Ilb/IIIa antibody variable
regions are set forth in S13Q ID NO:52 (murine mature variable light chain. Figure 87) and
SEQ ID NO: 54 (murine mature variable heavy chain, Figure 88). These murine sequences
can be combined with human IgG amino acid sequences SEQ ID NO:51 (human mature
variable light chain, Figure 89), SEQ ID NO: 53 (human mciture variable heavy chain, Figu;
90), SEQ ID NO: 55 (human light chain. Figure 91) and SEQ ID NO: 56 (human heavy
chain, Figure 92) according to the proceedures found in U.S. Patent No. 5,777,085 to creal ^
chimeric humanized murine anti-glj'-coprotein Ilb/IIIa antibody. Otlier anti-glycoprotein
Ilb/IlIa humanized antibodies are found in U.S. Patent No. 5,877,006. A cell line express
the emti-glycoprotein Ilb/IIIa MAb 7E3 can be conmiercially obtained from the .A.TCC
(Manassas, VA) as accession no. HB-8832.
Indications for selected antibodies
A remodeled Reopro''"" may be administered to a patient selected from the group
consisting of a patient undergoing percutaneous coronary intervention and a patient having
unstable angina, wherein the patient is scheduled for percutaneous coronary intervention
within 24 hours. A remodeled Reopro'^'^ may also be administered to a patient imdergoing
percutaneous coronary intervention to reduce or prevent a cardiac ischemic complication in
the patient. Preferably, the patient is a human patient.
A remodeled Herceptin™ may be administered to a patient having metastatic breast
cancer that overexpresses the HER2 protein. Preferably, the patient is a human patient.
A remodeled Remicade'''" antibody may be administered to a patient selected from the
group consisting of a patient having rheumatoid arthritis, a patient having Crohn's disease,
and a patient having fistulizing Crohn's disease. A remodeled Remicade''"'^ antibody may
also be administered to a rheumatoid arthritis patient to reduce signs and symptoms of
rheumatoid arthritis in the patient. A remodeled Remicade'^'^ eintibody may also be
administered to a Crohn's disease patient to reduce signs and symptoms of Crohn's disease in
the patient. Preferably, the patient is a human patient.
Z. MAbanti-CD20
The nucleic acid and amino acid sequences of a clihneric anti-CD20 antibody are set
forth in SEQ ID NO: 59 (nucleic acid sequence of murine variable region light chain, Figure
93A), SEQ ID NO:60 (amino acid sequence of murine variable region light chain, Figure
93B), SEQ ID N0:61 (nucleic acid sequence of murine variable region heavy chain. Figure
94A) ai\d SEQ ID NO:62 (amino acid sequence of mxirine variable region heavy chain,
Figure 94B). In order to humanize a murine antibody, the TCAE 8 (SEQ ID NO:57, Figuii.^
95A - 95E), which contains the human IgG heavy and light constant domains, may be
conveniently used. By cloning tlie above murine variable region encoding DNA into the
TCAE 8 vector according to instructions given in U.S. Patent No. 5,736,137, a vector is
created (SEQ ID NO: 58, Figure 96A - 96E) which when transformed into a niammaliani cell
line, expresses a chimeric anti-CD20 antibody. Other humanized anti-CD20 cUUibodies are
found in U.S. Patent Nl>. 6,120,767. A cell line expressing the anti-CD20 M^b C273 car b-.
ccunmcrcially obtained from the ATCC (Manassas, VA) as accession no. HB-'^)303.
The skilled artisan will readily appreciate that the sequences set forth herein are not
exhaustive, but are rather examples of the variable regions, receptors, and other binding
moieties of chimeric antibodies. Further, methods to construct chimeric or "humanized"
antibodies are well known in the art, and are described in, for example, U.S. Patent No.
6,329,511 and U.S. Patent No. 6,210,671. Coupled with the present disclosure and methods
well known throughout the art, the skilled artisan will recognize that the present invention is
not limited to the sequences disclosed herein.
The expression of a chimeric antibody is well known in the art, and is described in
detail in, for example, U.S. Patent No. 6,329,511. Expression systems can be prokaryotic,
eukaryotic, and the like. Further, the expression of chimeric antibodies in insect cells using a
baculovirus expression system is described in Putlitz et al. (1990, Bio/Technology 8:651-
654). Additionally, methods of expressing a nucleic acid encoding a fusion or chimeric
protein are well known in the art, and are described in, for example, Sambrook et al. (2001,
Molecular Cloning: A Laboratory Mcinual, Cold Spring Harbor Laboratory Press, New York)
and Ausubel et al. (1997.. Current Protocols in Molecular Biology, Green & Wiley, New
York).
Determining tlie function and biological activity of a chimeric antibody produced
according to the method:? of the present invention is a similarly basic operation for one of
skill in the art. Methods for determining the affinity of an antibody by competition assays are
detailed in Berzofsk>' (J. A. Berzofsky and L J. Berkower, 1984, in Fundamental
Immunology (ed. W. E. Paul), Raven Press (New York), 595). Briefly, the affinity of the
chimeric antibody is compared to that of the monoclonal antibody from which it was deri\'ed
using a radio-iodinated monoclonal antibody.
A remodeled aiati-CD20 antibody may be administered to a patient having relapsed or
refractory low grade or follicular, CD20-positive, B-cell non-Hodgkin's lymphoma.
Preferably, the patient is a human patient.
VII. Pharmaceutical Compositions
lu another aspect, die invention provides a pharmaceutical composition. Tiic
pharmaceutical conipo.sition includes a pharmaceutical!y acceptable diluent and a co\'aiei].t
conjugate between a non-naturally-occurring, water-soluble pohmer, tiierapeutic moiety or
biomoiecule and a glycosylated or non-glycosylated peptide. The polymer, tiierapeutic
moiety or biomolecule is conjugated to the peptide via an intact glycosyl linking group
interposed between and covalently linked to both the peptide and the polymer, therapeutic
moiety or biomolecule.
Phamiaceutical compositions of the invention are suitable for use in a variety of drug
delivery systems. Suitable formulations for use in the present invention are found in
Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, I'Zth
ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1 527-
1533(1990).
The phamiaceutical compositions may be formulated for any appropriate manner of
administration, including for example, topical, oral, nasal, intravenous, intracranial,
intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration,
such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a
wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as
mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose,
sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres {e.g.,
polylactate polyglycolate) may also be employed as carriers for the pharmaceutical
compositions of this mvention. Suitable biodegradable microspheres are disclosed, for
example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
Conmaonly, the phamiaceutical compositions are administered parenterally, e.g.,
intravenously. Thus, the invention provides compositions for parenteral administration which
comprise the compound dissolved or suspended in an acceptable carrier, preferably an
aqueous carrier, e.g., water, buffered water, saline, PBS and the like. The compositions may
contain pharmaceutically acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents,
wetting agents, deterg,ents and the like.
These compositions may be sterilized by conventional sterilization tecimiques, or ma}
be sterile filtered. Tlie resulting aqueous solutions may be packaged for use as is. or
lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to
administration, llie pH of the preparations typically wi!! be beiweeri 3 emd 1 \. more
preferably from 5 to 9 and most preferably from 7 and 8.
In some embodiments the peptides of the invention can be incorporated into
liposomes formed from standard vesicle-forming lipids. A variety of methods are available
for preparing liposomes, as described in, e.g., Szoka et al.,Ann. Rev. Biophys. Bioeng. 9: 467
(1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomes using
a variety of targeting agents (e.g., the sialyl galactosides of the invention) is well knovra in
the art (see, e.g, U.S. Patent Nos. 4,957,773 and 4,603,044).
Standard methods for couplmg targeting agents to liposomes can be used. These
metliods generally mvolve incorporation into liposomes of lipid components, such as
phosphatidylethanolamine, which can be activated for attachment of targeting agents, or
derivatized lipophilic compounds, such as lipid-derivatized peptides of the invention.
Targetmg mechanisms generally require that the targeting agents be positioned on the
surface of the liposome in such a manner that the target moieties are available for interaction
with the target, for example, a cell surface receptor. The carbohydrates of the invention may
be attached to a lipid molecule before the liposome is formed using methods known to those
of skill in the art (e.g., alkylation or acylation of a hydroxyl group present on the
carbohydrate with a long chain alkyl halide or with a fatty acid, respectively). Alternatively,
the liposome may be fashioned in such a way that a connector portion is first incorporated
into the membrane at the time of forming the membrane. The connector portion must have a
lipophilic portion, which is firmly embedded and anchored in the membrane. It must also
have a reactive portion, which is chemically available on the aqueous surface of the liposome.
The reactive portion is selected so that it will be chemically suitable to form a stable chemical
bond with the targeting agent or carbohydrate, which is added later. In some cases it is
possible to attach tlie target agent to the connector molecule directly, but in most instcmces it
is more suitable to use a third molecule to act as a chemical bridge, thus linking the connectoi
molecule which is in the membrane with the target agent or carbohydrate which is extended,
three dimensionally, off of the vesicle surface. The dosage ranges for the administration of
the peptides of the invention are those large enough to produce the desired effect in wioich the
symptoms of the immune response show some degree of suppression. The dosage should not
be so large as to cause adverse side effects. Generally, the dosage will vary with the age,
condition, sex and extent of the disease in tlie animal and can be determined b\ one of skill iv
the art. The dosage can be adjusted by the individual physician in the event of any
counterindications.
Additional pharmaceutical methods may be employed to control the duration of
action. Controlled release preparations may be achieved by the use of polymers to conjugate,
complex or adsorb the peptide. The controlled delivery may be exercised by selecting
appropriate macromolecules (for example, polyesters, polyamino carboxymethylcellulose,
and protamine sulfate) and the concentration of macromolecules as well as the methods of
incorporation in order to control release. Another possible method to control the duration oj^
action by controlled release preparations is to incorporate the peptide into particles of a
polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or
ethylene vinylacetate copolymers.
In order to protect peptides from binding with plasma proteins, it is preferred that the
peptides be entrapped in microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules
and poly (methymetliaciylate) microcapsules, respectively, or in colloidal drug delivery
systems, for example, lijjosomes, albumin microspheres, microemulsions, nanoparticles, and
nanocapsules or in macroemulsions. Such teachings are disclosed in Remington's
Pharmaceutical Sciences (16th Ed., A. Oslo, ed.. Mack, Easton, Pa., 1980).
The peptides of 1he invention are well suited for use in targetable drug delivery
systems such as synthetic or natural polymers in the form of macromolecular complexes,
nanocapsules, microspheres, or beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, liposomes, and resealed erythrocytes. These systems
are known collectively as colloidal drug delivery systems. Typically, such colloidal particle,;
containing the dispersed peptides are about 50 nm-2 |J,m in diameter. The size of the colloidal
pailicles allows them to be administered intravenously such as by injection, or as an aerosol.
Materials used in the preparation of colloidal systems are typically sterilizable via filter
sterilization, nontoxic, c,nd biodegradable, for example albumin, ethylcellulose, casein,
gelatin, lecithin, phospholipids, and soybean oil. Polymeric colloidal systems are prepared '-a process similar to the joacervation of microencapsulation.
In an exemplary embodiment, the peptides are components of a liposome, used as ;'
t;u"geted delivery system. When phospholipids are gently dispersed in aqueous media, they
swell, hydrate, and spontaneously form multilamellar concentric biiayer vesicles with layers
of aqueous media separating the lipid biiayer. Such systems are usually referred to as
multilamellar liposomes or multilamellar vesicles (MLVs) and have diameters ranging from
about 100 mn to about 4 ^m. When MLVs are sonicated, small unilamellar vesicles (SUVS)
with diameters in the range of from about 20 to about 50 rmi are formed, which contain an
aqueous solution in the c;ore of the SUV.
Examples of lipids useful in liposome production include phosphatidyl compounds,
such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, and
phosphatidylethanolami:ie. Particularly useful are diacylphosphatidylglycerols, where the
lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
are saturated. Illustrative phospholipids include egg phosphatidylcholine,
dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
In preparing liposomes containing the peptides of the invention, such variables as ttie
efficiency of peptide encapsulation, lability of the peptide, homogeneity and size of the
resulting population of liposomes, peptide-to-lipid ratio, permeability instability of the
preparation, and pharmaceutical acceptability of the formulation should be considered.
Szoka, et al, Annual Review of Biophysics and Bioengineering, 9: 467 (1980); Deamer, et al.,
in Liposomes, Marcel Dekker, New York, 1983, 27: Hope, et al., Chem. Phys. Lipids, 40: 89
(1986)).
The targeted delivery system containing the peptides of the invention may be
administered in ci variety/ of ways to a host, particularly a mammalian host, such as
intravenously, intramuscularly, subcutaneously, intra-peritoneally, intravascularly, topically,
intracavitarily, transdennally, intranasally, and by inhalation. The concentration of the
peptides will vary upon the particular application, the nature of the disease, the frequency of
administration, or the Hive. The targeted delivery system-encapsulated peptide may be
provided in a formulation comprising other compounds as appropriate and an aqueous
physiologically acceptable medium, for example, saline, phosphate buffered saline, or the
like.
The compounds prepaied by the methods of the invention may also find use as
diagnostic reagents. For example, labeled compounds can be used to locate areas oX
inflammation or tumor metastasis in a patient suspected of having an inflammation. For this
use, the compounds can be labeled with ^^^I, ^''c, or tritium.
EXPERIMENTAL EXAMPLES
The invention is now described with reference to the following Examples. These
Examples are provided for the purpose of illustration only and the invention should in no way
be construed as being limited to these Examples, but rather should be construed to encompass
any and all variations wliich become evident as a result of the teaching provided herein.
The materials and methods used in the experiments presented in this Example are now
described.
A. General Procedures
1. Preparadon of CMP-SA-PEG
Tills example seis forth the preparation of CMP-SA-PEG.
Preparation of 2-(benzjioxycarboxainido)-gIycyIaniido-2-deoxy-D-
mannopyranose. N-benzyloxycarbonyl-glycyl-N-hydroxysuccinimide ester (3.125 g, 10.2,
mmol) was added to a solution containing D-mannosamine-HCI (2 g, 9.3 mmol) and
triethylamine (1.42 mL,10.2 mmol) dissolved in MeOH (10 mL) and H2O (6 mL). The
reaction was stirred at room temperature for 16 hours and concentrated using
rotoevaporation. Chronaatography (silica, 10% MeOH/CH2Cl2) yielded 1.71 g (50% yield) of
product as a wliite solid: Rf = 0.62 (silica; CHCl3:MeOH:H20, 6/4/1); 'H NMR (CD3OD,
500 MHz) 5 3.24-3.27 (m, 2H), 3.44 (t, IH), 3.55 (t, IH), 3.63-3.66 (m, IH), 3.76-3.90 (m,
6H), 3.91 (s, 2H), 4.0 (dd, 2 H), 4.28 (d, IH, J = 4.4), 4.41 (d, IH, J = 3.2), 5.03 (s, IH), 5.10
(m,3H), 7.29-7.3 8(m, lOH).
Preparation of 5-(N~benzyIoxycarboxamido)glycylamldo-3,5-dideoxy-D-^(V'ce/-o-
D-galacto-2-nonulopyranosuronate (1.66 g ,3.6 mmol) was dissolved in 20 mL of 50%
water/methanol. The flask was repeatedly evacuated and placed under argon and then 10%
Pd/C (0.225 g) was added. After repeated evacuation, hydrogen (about 1 atm) was then
added to the flask and the reaction mixture stirred for 18 hr. The reaction mixture was
filtered through celite, concentrated by rotary evaporation and freeze-dried to yield 1.24 g
(100% yield) of product as a white solid: Rf = 0.25 (silica, IP'A/H20/NH40H 7/2/1); 'H
NMR (D2O, 500 MHz) 5 1.83 (t, IH, J = 9.9), 2.23 (dd, IH, J = 12.9, 4.69), 3.51-3.70 (m,
2H), 3.61(s, 2H), 3.75-3.84 (m, 2H), 3.95-4.06(m, 3H).
Preparation of cytidine-5'-monophosphoryI-[5-(N-nuorenylmethoxy-
carboxamido)glycyIamido-3,5-dideoxy-p-D-^/>'ce/'o-D-galacto-2-nonulopyranosuronate],
A solution containing 5-giycylamido-3,5-dideoxy-D-g/>'ceA-o-D-galacto-2-
nonulopyranosuronate (0.55 g, 1.70 mmol) dissolved in 20 mL H2O was added to a solution
of Tris (1.38 g, 11.4 mmol), 1 M MgCh (1.1 mL) and BSA (55 mg). The pH of the solution
was adjusted to 8.8 with IM NaOH (2 mL) and CTP-2Na'' (2.23 g, 4.2 mmol) was added.
The reaction mixture pH was controlled witli a pH controller which delivered 1 M NaOH as
needed to maintain pH 8.8. The fusion protein (sialyltransferase/CMP-neuraminic acid
synthetase) was added to the solution and the reaction mixture was stirred at room
temperatui-e. After 2 days, an additional amount of fusion protein was added and the reaction
stirred an additional 40 hours. The reaction mixture was precipitated in EtOH and the
precipitate was washed 5 times with cold EtOH to yield 2.3 grains of a white solid. Aboui
1.0 g of tlie crude product was dissolved in 1,4 dioxane (4 mL), H2O (4 mL) and saturated
NaHCOs (3 mL) and a solution of FMOC-Cl (308 mg, 1.2 mmol) dissolved in 2 ml dioxane
was added dropwise. Aiter stirring for 16 hr at room temperature, the reaction mixture was
concentrated to about 6 inL by rotary evaporation and purified using chromatography (CI 8
silica, gradient 100% H2O to 30% MeOH/ H2O). Appropriate fractions were combined and
concentrated. The residue was dissolved in water and freeze-dried to yield 253 mg of a white
solid: Rf = 0.50 (silica, IPA/H2O/NH4OH 7/2/1); 'H NMR (D2O, 500 MHz) 6 1.64 (dt, 1H, J
= 12.0, 6.0), 2.50 (dd, IH, J = 13.2, 4.9), 3.38 (d, J = 9.67, IH), 3.60 (dd, J=l 1.65, 6.64, IH),
3.79 (d, J=4.11, IH), 3.87 (dd, J= 12.24, 1.0, IH), 3.97 (m, 2H), 4.07 (td, J = 10.75, 4.84,
IH), 4.17 (dd, J = 10.68, 1.0, 1 H), 4.25 (s, 2H), 4.32 (t, J =4.4, IH), 4.37 (t, J=5.8 IH), 4.6-
4.7 (m, obscured by solvent peak), 5.95 (d, J = 4, 1 H), 6.03 (d, J = 7.4, IH), 7.43-7.53 (m,
3H:), 7.74 (m, 2H), 7.94 (q, J = 7, 3H) . MS (ES); calc. for C35H42N5O18P ([M-H]'), 851.7;
found 850.0.
Preparation of cytidine-5'-monophosphoryl-(5-glycylamido-3,5-dideoxy-p-D-
g^/vcero-D-gaIacto-2-nonulopyranosuronate). Diisopropylamine (83 uL, 0.587 p.mol) was
added to a solution of cytidine-5'-monophosphoryl-[5-(N-fluorenyl-
methoxycarboxaInido)glycylamido-3,5-dideoxy-P-D-g/>'cero-D-galacto-2-
nonulopyranosuronate] (100 mg, 0.117 mmol) dissolved in water (3 mL) and methanol (1
mL). The reaction mixture was stirred 16 hr at room temperature and the reaction methanol
removed from the reaction mixture by rotary evaporation. The crude reaction mixture was
filtered tlirough a CI8 silica gel column using water and the efluant was collected and freeze-
dried to yield (87 mg, 100%) of product as a white solid: Rf = 0.21 (silica, IPA/H2O/NH4OH
7/2/1); 'H NMR (D2O, 500 MHz) 5 1.66 (td, IH, J =5.3), 2.50 (dd, IH, J = 13.2, 4.6), 3.43 (d,
J = 9.58, IH), 3.63 (dd,J= 11.9, 6.44, IH), 3.88 (dd,J= 11.8, 1.0, IH), 3.95 (td, J-9.0, 2.3,
HO, 4.10 (t, J = 10.42, IH), 4.12 (td, J = 10.34, 4.66, 1 H), 4.18 (d, J = 10.36, IH), 4.24 (m,
2H), 4.31 (t, J=4.64, IH), 4.35 (t, IH), 6.00 (d, J = 4.37, 1 H), 6.13 (d, J = 7.71, IH), 7.98,(d,
J=7.64, IH). MS (ES); calc. for C21H32N5O11P ([M-H]"), 629.47; found 627.9.
Preparation of cytidine-5'-monophosphoryl-[5-(;TV-methoxy-poIyoxyethylcne-(l
kDa)-3-oxypropionamido)-gIycyIaraido-3,5-dideoxy-p-D-g^(>'ce/'o-D-gaiacto-2-
nonulopyranosuronate]. Benzyltriazol-l-yloxy-tris(dimethylamino)-phosphoniiim
hexafluorophosphate (BOP, 21 mg, 48 |j.moI) was added to a solution of
metho.\ypolyoxyethyk;!ie-(l kDa average molecular weight)-3-oxypropionic acid (48 mg, 48
lamol) dissolved in anhydrous DMF (700 nL) and triethylamine (13 ^L, 95 nmol). After 30
min, a solution containing cytidine-5'-monophosphoryl-(5-glycylamido-3,5-dideoxy-P-D-
g^ycero-D-galacto-l-nonulopyranosuronate) (30 mg, 48 [imol), water (400 p,L) and
triethylamine (13 \iL, 95 ^mol) was added. This solution was stirred 20 min at room
temperature and then chromatographed (CI8 silica, gradient of methanol/water). Appropriate
fractions were collected, concentrated, the residue dissolved in water and fireeze-dried to
afford 40 mg (50% yield) of a white solid: Rf = 0.36 (silica, IPA/H2O/NH4OH 7/2/1); 'H
NMR (D2O, 500 MHz) 5 1.66 (td, IH, J =5.3), 2.50 (dd, IH, J = 13.2, 4.6), 2.64 (t, J=5.99,
3H) 3.43 (d, J = 9.58, IH), 3.63 (m, IH), 3.71 (s, 70H), 3.79 (m, obscured by 3.71 peak), 3.82
(t, J=6.19, IH) 3.88 (dd, ,J = 11.8, 1.0, IH), 3.95 (td, J= 9.0, 2.3, IH), 3.98 (t, J= 5.06, IH),
4.12 (td, J = 10.34, 4.66, 1 H), 4.18 (d, J = 10.36, IH), 4.23 (d, J=4.85, 2H), 4.31 (t, J=4.64,
IH), 4.35 (t, IH), 6.00 (d, J = 4.55, 1 H), 6.13 (d, J = 7.56, IH), 7.98 (d, J=7.54, IH). MS
(MALDI), observe [M-H]; 1594.5, 1638.5, 1682.4, 1726.4, 1770.3, 1814.4, 1858.2, 1881.5,
1903.5, 1947.3.
^Preparation of cytidine-5'-monophosphoryl-[5-(N-raethoxy-polyoxyethylene-(l()
kDa)-oxycarboxamido)-glycylamido-3,5-dideoxy-p-D-^/Vcero-D-galacto-2-
nonulopyranosuronate]. Cytidine-5'-monophosphoryl-(5-glycylatnido-3,5-dideoxy-P-D-
g/>'c'cero-D-galacto-2-nonulopyranosuronate) (30 rag, 48 jimol) was added as a
solid. Water, pH 8 (330 |-iL) was added and after 30 min, an additional 28 mg of NHS-
acti\'ated PEG was added. After an additional 5 min, the reaction mixture was
chromatographed (C-18 silica, gradient of methanol/water), and appropriate fractions were
concentrated to afford 32 mg (40% yield) of a white solid, Rf = 0.31 (silica, IPA/HzO/NHtOH
7/2/0; '11 NMR (D2O, 500 MHz) 5 1.66 (td, IH, J =5.3), 2.50 (dd, IH, J = 13.2. 4.6), 2.64 (t,
J=5.99, 3H) 3.43 (d, J = 9.58, IH), 3.63 (m, IH), 3.71 (s, 70H), 3.79 (m, obscured by 3.71
peak), 3.82 (t, J=6.19, IH) 3.88 (dd, J = 11.8, 1.0, IH), 3.95 (td, J= 9.0, 2.3, IH), 3.98 (t, J=
5.06, IH), 4.12 (td, J= 10.34, 4.66, 1 H), 4.18 (d, J= 10.36, IH), 4.23 (d, J=4.85, 2H), 4.31
(t, J=4.64, IH), 4.35 (t, IH), 6.00 (d, J = 4.55, 1 H), 6.13 (d, J = 7.56, IH), 7.98 (d, J=7.54,
IH). MS (MALDI), observe [(M-CMP)-H]; 1506.4, 1550.4, 1594.5, 1638.5, 1682.4, 1726.4,
1770.3, 1814.4, 1858.2.
Preparation of Cytidine-5'-monophosphoryl-{5-[N-(2,6-
dimethoxypolyoxyethylene-(20 IiDa)-3oxypropionamidyl-IysyIamido]-gIycylamido-3,5-
dideoxy-p-D-5'/vce/-<7-D-galacto-2-nonuIopyranosuronate}. The 2,6-Di-
[methoxypolyoxyethyleiie-(20 kDa average molecular weight)-3-oxypropionamidyl]-
lysylatnido-N-succinimidyl ester (367 mg, 9 \imol) was dissolved in anhydrous THF (7 ml.)
and triethylamine (5 nL, 36 p,mol). Cytidine-5'-monophosphoryl-(5-glycylamido-3,5-
dideoxy-P-D-g-/yce/"c»-D-galacto-2-nonulopyranosuronate) (30 mg, 48 )LimoI) was dissolved in
1.0 mL of water, and added to the reaction mixture. The reaction was stirred for 4 hours at
room temperature and was then chromotographed (HPLC, Waters Xterra RP8, gradient from
water/NH40H, 100% to 20% methanol/water/NH40H at 1 niL/min) to afford a white solid
with a Rt = 22.8 min. MS (MALDI), observe [(M-CMP)-H]; 43027.01 (40,000 - 45,500).
3. Preparation of UDP-Gal-PEG.
This example sets forth the general procedure for making UDP-Gal-PEG.
Methoxypolyoxyethylenepropionate N-hydroxysuccinimide ester (mPEG-SPA, MW
[,000) 348 mg in THF (0.5 mL) was added to a solution of 25 mg of galactosamine-l-
)hosphate in 1 ml of water, followed by the addition of 67 jiL triethylamine. The resulting
nixture was stirred at room temperature for 17 hr. Concentration at reduce pressures
>rovided a cmde reaction mixture which was purified by cliromatography (C-18 silica, using
. step gradient of 10%, 20%, 30%, 40% aqueous MeOH) to afford 90 mg (74%) of product
fter the appropriate fractions were combined and concentrated to dryness. Rf = 0.5 (silica,
'ropanol/H20/NH,,OH 30,'20/2); MS(MALDI), observed 1356, HOO, 1444, 1488, 1532,
576, 1620.
[a-l-(Uridine-5'-diphosphoryl)]-2-deoxy-2-(methoxypolyoxyethylene-
propionoylamido-1 kDa)-a-D-galactosamine. The 2-deoxy-2-(methoxy-
polyoxyethylenepropionoylamido-1 kDa)-a-l-monophosphate-D-gaIactosamine (58 mg) was
dissolved in 6 mL of DMF and 1.2 mL of pyridine. UMP-rnorpholidate (60 mg) was then
added and the resulting mixture was stirred at 70°C for 48 hi. After concentration, the
residue was chromatographed (C18-silica, using a step gradient of 10%, 20%, 30%, 40%,
50% , 80% MeOH) to yield 50 mg of product after concentration of the appropriate fractions.
Rf= 0.54 (silica, propanol/H20/NH40H 30/20/2). MS(MA]:.DI); Observed 1485, 1529, 1618,
1706.
[a-l-(Uridine-5'-diphosphoryI)]-6-deoxy-6-(methoxypolyoxyethylene-aniino-2
kDa)-a-D-gaIactose. [a-l-(Uridine-5'-diphosphoryl)]-6-carboxaldehyde-a-D-galactose (10
mg) was disssolved in 2 mL of 25 mM sodium phosphate buffer (pH 6.0) and treated with
methoxypolyethyleneglycol amine (MW 2, 000, 70 mg) and then 25 ^L of IM NaBHaCN
solution at 0°C. The resulting mixture was frozen at -20°C for three days. The reaction
mixture was chromatographed (HPLC, Water Xterra P8) using 0.015 M NH4OH as mobile
phase A and MeOH as mobile phase B as eluent at the speed of 1.0 mL/min. The product
was collected, an concentrated to yield a solid; Rt = 9.4 minutes. Rf = 0.27(silica, EtOH/H^iO
7/3).
[a-l-(Uridine-5'-diphosphoryl)]-6-amino-6-deoxy-a-D-galactose. Ammonium
acetate 15 mg was added to a solution of [a-l-(Uridine-5'-diphosphoryl)]-6-carboxaldehyde-
a-D-galactopyranoside (10 mg) in sodium phosphate buffer (pH 6.0). A solution of (25 ^L)
IM NaBHaCN was then added and the mixture was stirred for 24 hr. The solution was
concentrated and the residue was cliromotographed (sephadex Gjo) to afford 10 mg of a white
solid, Rf = 0.62 (silica, EtOH/0.1 MNH4AC).
(a-l-(Uridiue-5'-diphosphor>i)]-6-deoxy-6-(methoxypolyoxyethylenepropionoyl
aniido, -2 kDa)-a-D-gaIactopyranoside. [a-l-(Uridinc-5'-diphosphoryl)j-6-amino-6-
deoxy-a-D-galactopyranoside (5 mg) was dissolved in 1 mL of H2O. Then
metlioxypolyetheneglycolpropionoyl-NHS ester (MW -2,000, 66 mg) was added, followed
by 4.6 [iL triethylainine. The resulting mixture was stirred at room temperature overnight,
and then purified on HPLC (C-8 silica) to afford the product, Rt = 9.0 min.
5. Preparation of UDP-GalNAc-PEG
This example (scheme 18) sets forth the general procedure for making UDP-GalNAc-
PEG. The reaction set fortli above originates with a sugar diphospho-nucleotide, in wliich R
is either a hydroxyl 1 or a protected amine 2. la step a, the starting sugar is treated with a
mixtm'e of an oxidase and a catalase, converting the 6-postion of the sugar into an aideli}'dc
moiety (3 and 4). In step c, the aldehyde is converted to the corresponding amine (7 and 8)
by formation and reduction of a Schiff base. In step e, the amine is optionally treated with em
activated m-PEG derivative, thereby acylating the amine to produce the corresponding m-
PEG amide (11 and 13). Alternatively, in step f, the amine is contacted wdth an activated m-
PEG species, such as a m-PEG active ester, thereby forming the corresponding m-PEG amide
(12 and 14). In step b, the starting material is also treated with a catalase and oxidase,
completely oxidizing the hydroxymethyl moiety, forming a carboxyl group at the 6-position.
In step d, the carboxyl moiety is activated and subsequently converted to a m-PEG adduct (9
and 10) by reaction with a m-PEG amine intermediate. This is shown in scheme 18.
The amino-sugai- phosphate is contacted with a m-PEG N-hydroxy succinimide active
ester, thereby forming the coiresponding sugar-PEG-amide. The amide is contacted with
UMP-morpholidate to form the corresponding active sugar diphospho-nucleotide.
6. Synthesis of CMP-SA-Levulinate
Tliis example sets forth the procedm-e for the synthesis of CMP-SA-leNoilinate.
Preparation of 2-levuIinaraido-2-deoxy-D-mannopyranose. Isobutyichloroformate
(100 pL, 0.77 mmol) was added dropwise to a solution of levulinic acid (86 ^L, 0.84 mmol),
anhydrous THF (3 niL) and triethylamine (127 |iL, 0.91 mmol). This solution was stirred for
3 hours at room temperature and was then added dropwise to a solution containing D-
mannosamine hydrochloride (151 mg, 0.7 mmol), triethylamine (127 fxL, 0.91 mmol), TIfF'
(2 mL) and water (2 mL). The reaction mixture was stirred 15 hours and then concentrated to
dryness by rotary evaporation. Cliromatography (silica, step gradient of 5-15%
MeOH/CH2Cl2) was used to isolate the product yielding 0.156 g (73% yield) of a wliite solid:
Rf = 0.41 (silica, CHCIj/MeOH/water 6/4/1); 'H NMR (D.O, 500 MHz) 8 2.23 (s, 3H), 2.24
(s, 3H), 2.57(td, J = 6.54, 3.68, 2H) 2.63 (t, J=6.71, 2H), 2.86-2.90 (m, 4H), 3.42 (m, IH),
3.53 (t, J=9.76, IH), 3.64 (t, J=9.43, IH), 3.80-3.91 (m, 4H), 4.04 (dd, J = 9.79, 1.71, 1 H).
4.31 (dd, J = 4.63,1.14, IH), 4.45 (dd, J=4.16,1.13, IH), 5.02 (d, J=1.29, IH), 5.11(s, J-1.30,
IH), MS (ES); calculated for CiHigNOy, 277.27; found [M+1] 277.9.
Preparation of 5-levulmamido-3,5-dideoxy-D-g/)'cgro-D-ga/ac/o-2-
nonulopyranosuronate. Sodium pyruvate (0.616 g, 5.6 nunol) and N-acetylneuraminic acid
aldolase (50 U) was added to a solution of 2-levulinaniido-2-deoxy-D-mannopyranose (0.156
g, 0.56 mmol) in 0.1 M HEPES (pH 7.5). The reaction mixture was heated to 37 "C for 20
hours and after freezing. The reaction mixture was then filtered through CI 8 silica, frozen
and freeze-dried. The crude solid was purified using flash chromatography (silica, first using
10-40% MeOH/CHsCb and then CH2Cl2/MeOH/H20 6/4/0.5). Appropriate fractions were
combined and concentrated yielding 45 mg (80% yield) of a white solid: Rf = 0.15 (silica,
CHCla/MeOH/water 6/4/1); 'H NMR (DjO, 500 MHz) 5 1.82 (t, J=l 1.9, IH), 2.21 (dd, J ==
13.76,4.84, IH), 2.23 (s, 3H), 2.57 (app q, J = 6.6, 2H), 2.86-2.95 (m, 2H), 3.15-3.18 (m, IH),
3.28-3.61 (complex,lH), 3.60 (dd, J = 11.91, 6.66, IH), 3.75 (td, J = 6.65, 2.62, IH), 3.84
(dd, J = 11.89, 2.65, 1 H), 3.88-4.01 (complex, 2H), 4.04 (td, J = 11.18, 4.67, IH), MS (ES);
calculated for C14H23NO10, 365.33; found ([M-1]-), 363.97.
Preparation of cytidine-5'-moaophosphoryl-(5-levuUnamido-3,5-dideoxy-P-D-
5'/^'ce/'o-D-^a/flcfo-2-nonuIopyranosuronate). 5-Levulinainido-3,5-dideoxy-D-g/7ce/o-D-
^fl/acro-2-nonulopyranosuronate (50 mg, 137 |j.mol) was dissolved in 2 mL of 100 mM
HEPES pH 7.5 buffer and 1 M MnCh (300 |iL, 300 ^mol) was added. CTP-2Na'' (79 mg,
1.5 nmol) was dissolved in 5 mL HEPES buffer and was added to the sugar. The
sialyltiansferase/CMP-neuraminic acid synthetase fiision enzyme (11 U) was added and the
reaction mixture stirred at room temperature for 45 hours. The reaction mixture was filtered
through a 10,000 MWCO filter and the filtrate, which contained the product of the reaction,
was used directly without further purification: Rf = 0.35 (silica, IPAywater/NH40H 7/2/1).
B. Glycoconjugation and GlycoPEGylation of Peptides
g-Protease Iiiliibitor (g-Antitrypsin)
7. Sialylation of Recombinant Glycoproteins.\ntithrombin III, Fetuin and gl -
¦Ajititrypsiq
Tills example scis forth the preparation of sialylated forms of several recombinant
peptides.
Sialylation of Recombinant Glycoproteins Using ST3Gal III, Several
glycoproteins were examined for their ability to be sialylated by recombinant rat ST3Gal III.
For each of these glycoproteins, sialylation will be a valuable process step in the development
of the respective glycoproteins as commercial products.
Reaction Conditions, Reaction conditions were as summarized in Table 11. The
sialyltransferase reactions were carried out for 24 hour at a temperature between room
temperature and 37°. The extent of sialylation was established by determining the amount of
'''C-NeuAc incorporated into glycoprotein-linked oligosaccharides. See Table 11 for the
reaction conditions for each protein.
Table 11. Reaction conditions.
"Cycle" refers to generation of CMP-NeuAc "in situ" enzymatically using standard
conditions as described in specification (20 mM NeuAc and 2 mM CMP). The buffer was
0.1MHEPES,pH7.5.
The results presented in Table 12 demonstrate that a remarkable extent of sialylation
was achieved in every case, despite low levels of enzyme used. Essentially, complete
sialylation was obtained, based on the estimate of available teiminal galactose. Table 12
shows the relults of the sialylation reactions. The amount of eazyme used per mg of protein
(mU/nig) as a basis of comparison for the various studies. In several of the examples shown,
only 7-13 mU ST3Gal III per mg of protein was required to give essentially complete
sialylation after 24 hours.
Terminal (exposed) Gal content on N-linked oligosaccharides determined by supplier, or
from literatures values (fetuin, asialo-AAAT).
^ NeuAc incorporated determined by incorporation of 14C-NeuAc after separation from free
radiolabeled precursors by gel filtration.
^ The % Rxn refers to % completion of the reaction based on the terminal Gal content as a
theoretical maximum.
'^ Antithrombin III.
^ al Antitrypsin.
These results are in marked contrast to those reported in detailed studies with bovine
ST6Gal I where less than 50 mU/mg protein gave less than 50% sialylation, and 1070 mU/mg
protein gave approximately 85-90% sialylation in 24 hours. Paulson et al. (1977) J. Biol.
Chem. 252: 2363-2371; Paulson et al. (1978) J. Biol. Chem. 253: 5617-5624. A study of rat
a2,3 and a2,6 sialyltransferases by another group revealed that complete sialylation of asialo-
AGP required enzyme concentrations of 150-250 mU/mg protein (Weinstein et al. (1982) J.
Biol. Chem. 257: 13845-13853). These earlier studies taken together suggested that the
ST6Gal I sialyltransferase requires greater than 50 mU/mg and up to 150 mU/mg to achieve
complete sialylation.
This Example demonstrates that sialylation of recombinant glycoproteins using the
ST3 Gal in sialyltransferase required much less enzyme than expected. For a one kilogram
scale reaction, approximately 7,000 units of the ST3Gal III sialyltransferase would be
needed, instead of 100,000-150,000 units that earlier studies indicated. Purification of these
enzymes fi'om natural sources is prohibitive, with yields of only 1-10 units for a large scale
prepaj-ation after 1-2 months work. Assmning that botii tlie ST6Gal I and ST3Gal III
sialyltransferases are produced as recombinant sialyltransferases, with equal levels of
expression of the two eiozjmes being acliieved, a fermentation scale 14-21 times greater (or
more) would be required for the ST6GaI I sialyltransferase relative to the ST3Gal III
sialyltransferase. For the ST6Gal I sialyltransferase, expression levels of 0.3 U/1 in yeast has
been reported (Borsig et al. (1995) Biochem. Biophys. Res. Commun. 210: 14-20).
Expression levels of 1000 U/liter of the ST3 Gal III sialyltransferase have been achieved in
Aspergillus niger. At cuixent levels of expression 300-450,000 liters of yeast fermentation
would be required to produce sufficient enzyme for sialylation of 1 kg of glycoprotein using
tlie ST6Gal Isialyl transferase. In contrast, less than 10 liter fermentation oi Aspergillus niger
would be required for sialylation of 1 kg of glycoprotein using the STBGal III
sialyltransferase. Thus, tlie fermentation capacity required to produce the ST3Gal III
sialyltransferase for a large scale sialylation reaction would be 10-100 fold less than that
required for producing the ST6GaI I; the cost of producing titae sialyltransferase would be
reduced proportionately.
Cri-IgG Antibod\-
8. Glyco-Remodelingof Cri-IgGl Antibodies
This example sets forth the procedures for in vitro remodeling of Cri-IgGl antibodies.
N-glycosylation at one conserved site at Asn 297 in the Fc domain of a monoclonal
antibody can modulate its pharmacokinetic behavior and effector functions (Dwek et al.,
1995, J. Anat. 187:279-292; Boyd et al., 1995, Mol. hnmunol. 32:1311-1318; Lund et al.,
1995,FASEB J. 1995, 9:115-119; Lundetal., 1996, J. hnmimol. 157:4963-4969; Wright &
Morrison, 1998, J. hnmunol. 160:3393-3402; Flynn & Byrd, 2000, Curr. Opin. Oncol.
12:574-581). During cell culture fermentation or in certain pathological conditions,
significant heterogeneity arises in the glycosylation pattern at this site. The resulting
different patterns of glycosylation on the Fc domain are characterized by complex
biantennary structures with zero, one, and two terminal galactose residues (GO, Gl, and G2,
respectively, see Table 13). The observed glycoform variations, such as the variation in
terminal galactosylation, tnancated N-glycoforms and bisecting modification, have been
shown to influence tlie antibody's therapeutic properties, especially its ability to mediate
targeted cell killing tlirough complement binding and activation (Boyd et al., 1995, supra:
Wright & Morrison, 1998, supra, Munura et al., 2000, Molec. Immunol. 37:697-706; Davics
ct al., 2001, Biotechnol. Bioeng. 74:288-294).
In order to obtain different glycoforms of Cri-IgGl antibodies and test their Fc
effector functions, Cri-JgGl antibodies were trimmed back stepwise using exoglycosidases to
generate glycoforms lacking sialic acid (G2, Gl), glycofonns lacking sialic acid and
galactose (GO), and glycoforms lacking sialic acid, galactose and N-acetyl glucosamine
(M3N2F), as illustrated in Table 13. These molecules were subsequently modified using
different glycosyltransferases and appropriate sugars. Modification conditions were
developed that resulted :in the conversion of the original antibody glycan structures into
different glycoforms: M3N2, GnT-I-M3N2 (the M3M2 glycoform with a GlcNAc moiety
added using GnT-I), GO, Bisecting-GO (tlie GO moiety witfi a bisecting GlcNAc added with
GnT-III), galactosylated bisecting-GO (the bisecting-GO glycoform with terminal galactose
moieties added), G2, mono-sialylated Sl(a2,6)'G2 (the G2 glycoform with one terminal
sialic acid moiety added using a2,6-sialyltransferase), Sl(a2,3)-G2 (the G2 glycoform with
one tenninal sialic acid raoiety added using a2,3-sialyltransferase) and disialylated S2(a2,3)-
G2 (the G2 glycoform). After every glycoremodeling step, the glycan structures were
enz)'matically released fiom the antibody protein and were Einalyzed by various methods,
including separation by capillary electrophoresis, 2-AA HPLC profiling and MALDI-TOF
mass spectrometry.
The materials ane methods used in these experiments are now described.
The Cri-IgGl Monoclonal Antibody. The Cri-IgGl .antibody was obtained from R.
Jefferies, MRC Center for knmune Regulation, The Medical School, University of
Birmingham, UK. The antibody is a non-recombinant antibody, and is isolated from a human
myeloma. The antibody was prepared using three methods. In the first method, referred to as
"DEAE," the antibody was isolated under relatively mild conditions using a DEAE ion
exchange column. In the second method, referred to as "SPA," the antibody was purified on
a protein A column {Staphylococcus aureus protein A) with a low pH elution step. In the
third method, referred to as "Fc," the antibody was treated with a protease so that only the Fc
portion of the antibody remained and the antigen binding domains were removed. These
methods for antibody purification are well known to those of skill in the art and are not
repeated in detail here. i
Affinitj' purification of remodeled antibodies. Antibody, modified either by
exoglycosidase or glycosyltransferase, was affinity purified on a ProA-sepharose 4-fast flow
colunm (Amersham Bioscience, Arlington Heights, IL ), eluted with 0.1 M glycine-HCl
buffer, pH 2.7, and immediately neutralized with 1 M Tris, pH 9.5. The eluates were buffer-
exchanged using a NAP-10 column (Amersham Bioscience, Arlington Heights, IL) to an
appropriate buffer for the next step of glycosylation, such as 100 niM MES, pH 6.5 or 50 niM
Tris-HCl, pH 7.2. The remodeled final products were dialyzed extensively against PBS at
4°C in Tubc-O-DialyzersTM (Chemicon International, Temecula, CA) with a MWCO of 8
kDa.
In vitro glycosidase treatment of Cri-antibodies. Antibody was buffer-exchanged into
50 niM Na phosphate/Ciirate, pH 6.0 using NAP-10 column (Amersham Bioscience.
Arlington Heights, IL). In vitro trimming back of sugar moieties was caiTied out stepwise, by
contacting the tuitibody (5 mg/mL) with 20 mU/mg protein neuraminidase at 37 ""C uveniight
(to remove terniinal sialic acid moieties ), 20 mU/mg protein (3-galactosidase at 37°C,
overnight (to remove terminal galactose moieties to result in the GO glycoform), and/or 2
U/mg p-N-acetylhexosaminidase (from Jack Bean, Seikagiaku, Tokyo, Japan) at 37°C,
overnight (to remove terminal N-acetyl glucosamine to result in the M3N2 glycoform). Hie
samples were affinity purified as described above.
In vitro glycosvlation of Cri-antibodies. In vitro GnTl modification was performed
using 1 mg/ml of the M3N2 glycoform antibody as the substrate, and 25 mU/mg of
recombinant human pi,2-mannosyl-UDP-N-acetylglucosaminosyltransferase in a buffer of
100 mM MES, pH 6.5, 5 mM MnCla, 5 mM UDP-GlcNAc, and 0.02% NaNa at 32°C for 24
hr. An aliquot was removed for glycan analysis, and the resulting products were affinity
purified as described above.
In vitro modification of the bisecting-glycoform was carried out using 1 mg/ml of ttie
M3N2 glycoform antibody as the substrate and 25 mU/mg of pi,2-recombinant human
mannosyl-UDP-N-acetylglucosaminosyltransferase I, 25 mU/mg of pi,2-recombinant hiunan
mannosyl-UDP-N-acetylglucosaminosyltransferase II and 3.5 mU/mg of p 1,4-recombinant
mouse mannosyl-UDP-N-acetylglucosaminosyltransferase III in a buffer of 100 mM MES
pH 6.5, 10 mM MnCb, 5 mM UDP-GlcNAc, and 0.02% NaJMj at 32°C for 24 hrs. An aliquot
was removed for glycan tuialysis, and the remaining product was affinity purified as
described above.
In vitro galactosyiation was performed using GO glycoform antibody or bisecting
glycoform antibody by contacting the antibody with 0.6 U/mg recombinant bovine milk p 1,4
galactosyltransferase in a buffer of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM UDP-
galaclose, 5 mM MnCh, at 32°C for 24 hrs. An aliquot was removed for glycan analysis, and
the remaining products were affinity purified as described above.
In vitro sialylation was carried out using the G2 glycoform antibody (1 mg/mL) by
contacting it with 0.1 U/mg ST3Gal3 or 0.1 U/mg ST6Gall, 5 mM CMP-sialic acid, at 32°C
for 24 lir in a buffer of 50 inM Tris pH 7.4, 150 mMNaCl, and 3 mM CMI^-SA. An aliquot
was removed for glycan analj'sis, and the remaining products were affinit}' purified as
descril^ed above.
Give an Analysis:
Capillary Electrophoresis with Laser Induced Fluorescence Dectection. Buffer
components and nucleotide sugars were removed from an aliquot of the glycoremodeled
antibody by dUution and concentration in a Microcon''"** YM-30 microconcentrator
(Millipore, Bedford, MJV). N-linked oligosaccharides were released from the protein by
contacting it with PNGase F (Prozyme, San Leandro, CA) using the methodology provided
by the manufacturer. In brief, the sample was denatured in the buffer of 50 mM sodium
phosphate pH 7.5, 0.1% SDS, and 50 mM p-mercaptoethanol for 10 min at 100°C. TXlOO
was then added to 0.75°/o (v/v) as well as lOU PNGaseF/200 |ig protein. After 3 hours
incubation at 37°C, the {)rotein was ethanol precipitated and the supernatant was dried down.
The released free oligosaccharides were then labeled with 8-aminopyrene-l,3,6-trisulfonic
acid and analyzed by capillary electrophoresis with a carbohydrate labeling and analysis kit
from Beckman-Coulter, Inc. (FuUerton, CA), as indicated by the manufacturer (see also. Ma
andNashabeh, 1999, Anal. Chem. 71:5185-5192).
Capillary electrophoresis (CE) was carried out in an eCAP'"*' N-CHO coated
Capillary (50 |im I.D., length to detector 40 cm; Beckman-Coulter, Inc., Fullerton, CA),
using a P/ACE^^^ MDQ Glycoprotein System (Beckman-Coulter, Inc. Fullerton, CA) with
Laser Induced Fluorescence Detector (Beckman-Coulter, Inc. Fullerton, CA). Samples were
introduced into the cartridge by 20 psi pressure for 10 sec. and separated under 25 kV with
reverse polarity for 20 min. Cartridge temperature was kept at 20°C. The electropherograni
was generated by laser-hiduced fluorescence detection at an excitation wavelength of 488 nm
and an emission wavelength of 520 mn.
Carbohydrate standards (Calbiochem®, EMD Biosciences, Inc., San Diego, CA),
including M3N2 (N-linked trimannosyl core without core fucose), GO (N-linked
oligosaccharide, asialo, agalacto, biantermary with core fucose), G2 (N-linked
oligosaccharide, asialo, bianteimary with core fucose), and G2 without fucose, S1-G2 (mono-
sialylated, galactosylated biantennary oligosaccharide without core fucose) and S2-G2 (di-
sialylated, galactosylated biantennary oligosaccharide without core fucose), (from Glyko, see,
ProZyme, San Leandro, CA), M3N2F (N-linked trimannosyl core with core fucose) and
NGA2F (N-linked oligosaccharide asialo, agalacto, biantcnnaiy with core fucose tmd witii
bisecting GlcNAc) were 'abelcd with l-aminop>Tene-3,6,8-trisuIfonate (APTS. Beckman-
Coulter, Inc. Fullerton, CA) and used to identify the distribution of glycans released from the
antibody.
2-AA HPLC. PNGaseF released glycans were labeled with 2-AA (2-anthranilic acid)
according to the method described by Anumula and Dhume with slight modifications (1998,
Glycobiology 8:685- 694). Reductively-aminated N-glycans were analyzed using a Shodex
Asahipak NH2P-50 4D amino column (4.6 mm x 150 mm) (Showa Denko K.K., Tokyo,
Japan), The two solvents used for the separation are A) 2% acetic acid and 1%
tetrahydrofuran in acetonitrile and B) 5% acetic acid, 3% triethylamine and 1%
tetrahydrofuran in water.
To separate neutral 2AA-labeled glycans, the columji was eluted isocratically with
70% A for 5 minutes, followed by a linear gradient over a period of 60 minutes going from
70% to 50% B, followed by a steep gradient over a period of 5 minutes going from 50% to
5% B and a final isocratic elution with 5% B for 10 minutes. Eluted peaks were detected
using fluorescence detection with an excitation at 230 nm and detection wavelength at 420
nm. In this gradient condition, the GO glycoform will elute at about 30.5 minutes, the Gl
glycofonn at about 34.0 minutes and tlae G2 glycoform at about 37.0 minutes. Under these
conditions, the presence of flicose does not change the elution time.
To separate anionic 2AA-labeled glycans, the coluirm was eluted isocratically with
70% A for 2.5 mmutes, followed by a linear gradient over a period of 97.5 min going from
70% to 5% A and a final isocratic elution with 5% A for 15 minutes. Eluted peaks were
detected using fluorescence detection with excitation at 230 nm and detection at 420 mn. In
this gradient, neutral glycans are expected to elute between 18.00 - 29.00 minutes, glycans
with one charge elute between 30.00 — 40.00 minutes, glycans with two charges elute
between 43.00 - 52.00 minutes, glycans with three charges elute between 54.00 - 63.00
minutes, and glycans with four charges elute between 65.00 - 74.00 minutes.
MALDI analysis of reductively-aminated N-glycans. A small aliquot of the PNGasc-
released N-glycans that were labeled with 2-antliranilic acid (2AA) were then dialyzed for 45
minutes on a MF-Millipore membrane filter (0.025 \xm pore, 47 mm dia.), which was floating
on water. I'he dialyzed aliquot was dried in a Speedvac'''" (ThermoSavant, Holbrook, NY),
redissolved in a small amount of water, and mixed witli a solution of 2,5-dihydroxy benzoic
acid (10 g/L) dissolved in waier/acetonitrile (50:50).
The mixture was dried onto a MALDI target and analyzed using an Applied
Biosystems DE-Pro mass spectrometer (Applied Biosystems, Inc., Foster City, CA) operated
in the linear/negative-ion mode. Oligosaccharide structures were assigned based on the
observed mass-to-charge ratio and literature precedence. No attempt was made to fully
characterize isobaric stnictures.
SDS-PAGE. To determine the stability of the glycoremodeled antibody, all the
samples were analyzed by SDS-PAGE. The final products of the samples were run under
non-reducing conditions using 8-16% Tris-glycine gel (Invitrogen, Carlsbad, CA). Bovine
serum albumin was run under reducing condition as quantitative standards. The gel was
stained with GelCode Blue Stain Reagent (Pierce Chemical Co., Rockford, IL) for
visualization.
The results of the experiments are now described.
Native glycoforms of Cri expressed in human myeloma cells. Cri-IgGl antibody
puritled from the serum of a patient having multiple myeloma contains variable glycoforms.
Figure 97A-97C shows the HPLC profiles of glycans enzymatically released from Cri-IgGl
antibody. Figure 98A-98C shows the MALDI profiles of glycans enzymatically released
from Cri-IgGl antibody expressed in human myeloma cells. The major forms are under-
galactosylated GO, Gl, while G2 and sialylated structures are relatively minor (Table 14 and
Figure 97C). To test the impact of modified glycans on the therapeutic properties of the
monoclonal antibody, Cri-IgGl antibody'was modified by performing in vitro
exoglycosidases trimming and in vitro glycosylation remodeling to generate different
glycoforms of this antibody.
Initially, optimization of each step in exoglycosidases trimming and glycosylation
was performed at small scale (100 ^g of each).
Trimannosvl core glvcoform of Cri-IeGl Antibody (M3N2). M3N2 was created by
stepwise treatment of glycosidases, including neuraminidase, pi,4-galactosidase and pi-2, 3,
4, 6 N-acetyihexosaminidase. To assess the removal of terminal galactose and GlcNAc on
the glycoremodeled Cri-IgGl antibody samples, a quantitative capillary electrophoresis (CE)
method was used. The glycans were enzymatically released from the glycoremodeled
antibody with PNGase F and were derivatized with 8-amLnopyrene-l,3,6-trisulfonic acid
(APTS) at the reducing terminus. The resulting products were analyzed by CE with on-
column laser-induced fluorescence detection (LIF) (Ma & Nashabeh, 1999, supra). Since the
separation of the glycans is based on the differences in hydrodynamic size, the APTS labeled
glycans migrate in order of increasing size (M3N2< M3N2F< GO < Gl < G2).
Figures 99A-99D show the electropherograms indicating the glycans released from
glycoremodeled Cri-IgGl antibody as well as glycan standards derivatized with APTS
(Figure 99A). The glycoforms were identified by comparing their electrophoretic mobilities
to the standards. The relative amount of each glycan species was calculated from the relative
area percentage of each indicated peak, and the results are presented in Table 15. The
M3N2F glycofomi represents 91% of the glycans of DEAE-Cri, 80% of the glycans of SPA-
Cri, and 100% of the glycans of Fc-Cri. Incomplete removal of GlcNAc moiety resulting in
the GnT-I-M3N2F glycofbrm (see. Table 15) was observed in the glycan structures from
DEAE-Cri (8.6%) and SPA-Cri (-20%). Glycoform GnT-I-M3N2F is the M3N2F glycofonn
with one additional GlcNAc, such as would be added by GnT-I.
Table 15. The areas of individual peaks from CE profile in Fig. 99 were calculated,
and relative amounts of tlie M3N2F and GnT-I-M3N2F glycoforms were determined.
Degalactosvlated glvcoform TGO). Cri-IgGl antibody with GO glycoforms was
obtained by stepwise treatment the native Cri-IgGl antibody with neuraminidase and pi,4-
galactosidase in for 24 hours for each reaction. The glycans released from the
glycoremodeled antibody were analyzed by CE, HPLC and MALDI. Figure lOOA shows the
CE profile of the released glycans. In all three samples, only one peak was observed which
was designated as the GO glycoform based on comparison with the standards (Fig. lOOA and
Table 16).
Table 16. The relative amount of the GO glycoform of Cri-IgGl determined by CE
and HPLC.
In addition to the glycan analysis provided by CE, a quantitative HPLC method wa;j
also used to detennine tbie percent of the GO glycoform represented by remodeled glycans of
the Cri-IgGl antibody. The glycan distribution on the glycoremodeled antibody was
monitored by enzymatically releasing the glycans with PNGase F and derivatizing the
released products with 2-anthranilic acid (2-AA) at the reducing terminus. The derivatized
mixture was separated by HPLC on a Shodex Asahipak NHZP-SO 4D column with
fluorescence detection. Figures 101A-IOIC show the chromatograms obtained from the
released glycans. HPLC results confirmed CE analysis, as only one major peak was found in
all tlu-ee samples. In agreement with CE and HPLC data, MALDI analysis also showed
almost complete glycoremodeiing to the GO glycoform (Fig. 102A-102C).
Fully galactosylated G2 glycoform rG2). Cri-IgG antibodies were treated with
neunmiinidase to yield asialo-glycoforms which were also under galactosylated. These
asialoglycofomis were tlien treated with 0.6 U/ml of bovine pi,4 galactosyltransferase and a
galactose donor molecule to glycoremodel the antibody to have the G2 giycoforni.
The extent of tenninal galactosylation was determined by glycan analysis. Only one
major peak was observed in both CE and HPLC profiles (Figure 103A-103C and Fig. 104A-
104C). This peak corresponds to the G2 glycoform in each case. Calculation of the percent
total peak area showed almost complete (-90%) conversion to the G2 from the under
galactosylated glycoforms of the original samples (see. Table 14). These results are
summarized in Table 17. MALDI analysis of the glycans fiorther supported the almost to
complete glycoremodelmg to the G2 glycofonn in all of the samples (Fig. 105A-105C).
Table 17. Relative amount of G2 glycoform of remodeled Cri-Igll antibody
determined by percent total peak area in CE and HPLC analysis.
CE HPLC
RT(min.) % RT (min.) %
glycoremodeled to the GnT-I-M3N2 glycoform by adding one GlcNAc moiety to the
molecule. The molecule was contacted with 25 mU GnT-I/mg antibody and an appropriate
GlcNAc donor molecule. CE, HPLC and MALDI analysis of released glycans (Figures
106A-106D, Figures 107A-107C and Fig. 108A-108C, respectively) indicated that the
original M3N2F glycoform was completely remodeled. Hovrever, only 40-60% of the
modified structures were the GnT-I-M3N2 glycoform, and about 30% were the GO
glycoform. The presence of the GO glycoform may be the result of incomplete GlcNAc
trimming when making the original M3N2 form.
Bisecting glycoform (NGA2F). The M3N2 glycofonn Cri-IgG antibody was
glycoremodeled to the NGA2F glycoform by contacting it with a combination the three
transferases, GnT-1, GnT-lI and GnT-III, and an appropriate N-acetylglucosamine donor
molecule. The reaction was completed in 24 hours. To determine the extent to which thf
biseciing-GlcNAc moiety was added to the glycans, CE analysis was used to detennine Jie
glycoforms preseiit on the glycoremodeled antibody.
Figure 109A-109D shows the electropherograms obtained from CE analysis of the
glycans released from glycoremodeled Cri-IgGl antibody. Four peaks appeared after
remodeling. A major peak migrated at the same retention time as the NGA2F standard
glycoform. The three other minor peaks are likely to be the incompletely remodeled glycans.
For comparison, a quantitative HPLC method was also used, where the 2-AA labeled glycans
eluted in order of increasing size (Gnl < GO < NGA2F). As shown in Figure 1 lOA-i IOC,
similar results were obtained from the CE analysis of the glycans. No M3N2F was found
using either the CE or HPLC analysis. NGA2F glycans were the major peaks I both CE and
HPLC analysis. The Gnl and GO glycans still remaining in the sample likely are the result of
incomplete modification. Most of the original M3N2F glycoforms were remodeled by three
GlcNAc moieties to the NGA2F glycoform (60-70%), about 15-18% were remodeled by the
addition of two GlcNAc moieties to the GO glycoform, and only small amount (~ 7%) were
remodeled by the addition of only one GlcNAc moiety. IvLALDI-MS analysis of the released
glycans (Figure 11 lA-1 IIC) shows peaks of glycoforms with one, two or three terminal
GlcNAc moieties, in agreement with CE and HPLC analysis (Figures 109 and 110). The
relative amount of each glycan species was calculated from the relative area percentage of
each indicated peak, and is summarized in Table 18.
Table 18. Relative amounts of different glycoforms from GnT-I, II, and HI
remodeled Cri-IgGl, as determined by CE and HPLC.
Bisec. 32.078 34.93 68.63 45.64
Galactosvlated Bisecting ('Gal-NGA2F) glvcoforms. NGA2F glycofonns of Cri-IgGl
antibodies were giycoremodeled with bovine pi,4-galactosyrtransferase and an appropriate
galactose donor. Tlie terminal galactose moieties were added using 0.6 U/ml of pi,4
galactosyl transferase. Figure 112A-112D shows the electropherograms obtained using the 2-
AA HPLC method. In brief, the glycoforms terminating in GalNAc were almost 100%
galactosylated. Comparing Figure 112A to Figure 112B for DEAE Cri-IgGl, and Figure
112C to Figure 112D for Fc Cri-IgGl, the 2-AA HPLC profile of GnT-I, II and III modified
glycans (Figures 112A and 112C) is modified by GalTl so that all of the glycan peakes were
shifted to elute later due to the size increase &om added galactose moieties (Fig. 112B and
112D). These results were further confirmed by MALDI-MS analysis.
Sialylated ("S2G2) glycoforms of Cri-IgGl. The giycoremodeled G2 glycoforms of
Cri-IgGl antibody were fiirtlier remodeled using both ST3Gal3 and ST6Gall. Figure 113A-
113C shows the HPLC profile of the G2 glycoforms remodeled with ST3Gal3. Most of the
G2 glycoforms were converted into S2G2 glycoforms (the G2 glycoform with 2 additional
terminal sialic acid moieties; ~70%, see, Table 19), and only small amounts were the S1G2
glycoform (the G2 glycoform with 1 additional terminal sialic acid moiety; <25%, see Table
19). These results were further confirmed in the MALDI analysis shown in Figures 114A-
114C. MALDI data also shows that all the G2 glycofonns were sialylated to either S2G2 or
S1G2 glycoforms.
Table 19. Relative amounts of different glycoforms firom ST3Gal3 remodeled Cri-
IgGl as determined by HPLC.
RTTrainO DEAE ^5PA F^
By comparison, ST6Gall remodeling of the GO glycoform did not reach the level of
completion found with ST3GaI3 remodeling. Figure 115A-115D and Figure 116A-116C
show the results obtained from CE and HPLC analysis, respectively. No S2G2 glycoforms
were seen in any of the glycoremodeled samples. However, all of the G2 glycoforms were
converted into S1-G2. Analysis from MALDI-MS also supports these data (Figures 117A-
117C).
Stability of remodeled glycaas of Cri-IgGl. Lastly, the stability of the Cri-IgGl
glycans remodeled by exoglycosidase treatment and glycosyiation was investigated. Each
glycoremodeled Cri-IgGl antibody was stored at 4°C, and was checked by SDS-PAGE for
degradation at two weeks after remodeling. As shown in Figure 118A-118E, the remodeled
DEAE and SPA antibodies both retained a molecular weight of about 150 kDa, indicating
little to no degradation, regardless of the kind of glycoremodeling performed. The Fc Cri-
IgGl antibody retained a molecular weight of about 38 kDa, also indicating little to no
degradation, regardless oi'the kind of remodeling performed.
Effector Function Bioassay of Remodeled Cri-IgGl antibodies. The effector function
bioassay was derived from the procedure of Mimura et al. (2000, Molecular Immunology
37:697-706). The IC50 of the glycoforms of Cri-IgGl antibody was determined by inhibition
of the superoxide response of U937 cells elicited by red blood cells sensitized with native
anti-NIP antibody.
Monocytic U937 cells were cultured in the presence of 1000 units/mL interferon
gamma for 2 days to induce the differentiation of the cells and their capacity to generate
superoxide. The cells were then washed and resuspended at 2 x 10 cells/mL in Hanks
balanced salt solution without phenol red and containing 20 mM HEPES pH 7.4 and 0.15
mM BSA. The red blood cells were sensitized witli anti-NIP (5-iodo-4-hydroxy-3-
nitrophenacetyl) antibody. Ln the absence or presence of the various glycoforms of Cri-lgG 1
antibody, with incubation at 37°C for 30 minutes. The cells were then washed tlu-ee times
with PBS and resuspended at 2.5 x 10^ cells/mL in HBSS-BSA. The U937 cells (100 y.1, 2 x
10*^ceIls/mL) were added to plastic tubes and lucigenin (20 |il, 2.5 mM) was added to the
tubes. The tubes were waaned in a 37°C water batli for 5 minutes. The sensitized red blood
cells (80 nl, 2.5 X 10 /aiL) were then added to the tubes. Superoxide anion production was
measured by lucigenin-enhanced chemiluminescence at 37°C over a 30 minute period using a
Berthold LV953 luminometer (Berthold Australia Pty Ltd, Bundoora, Australia).
The GO and M3N2 glycoforms Cri-IgGl antibody had relative inhibitory values of
92% and 85%, respectively, as compared with the native antibody. However, the native CRI-
IgGl antibody lacked core fUcose. Shields et al. (2002, J. Biol. Chem. 277:26733-26740)
suggests that the lack of core fucose will improve inhibitory values 10 fold. Based on these;
results, it is anticipated tliat inhibitory values of the galactosylated-bisecting-GO glycoform
will be greater than the bisecting-GO glycoform, which in turn will be much greater than tlie
G2 glycoform, which in inm will be approximately equal to the disialylated-G2 glycoform
and the monosialylated-Cr2 glycoform, which in turn will be greater than the native antibody
glycoform, which in turn will be greater than the GK) glycoform, which in turn will be greater
than the M3N2 glycoform.
Complement Receptor-1
9. Sialylation and Fucosvlation of TPIO
This example sets forth the preparation of TPIO with sialyl Lewis X moieties and
analysis of enhanced biological activity.
Interrupting blood flow to the brain, even for a short time, can trigger inflammatory
events within the cerebral microvasculature that can exacerbrate cerebral tissue damage. The
tissue damage that accrues is amplified by activation of both inflammation and coagulation
cascades. In a murine model of stroke, increased expression of P-selectin and ICAM-1
promotes leukocyte recruitment. sCRl is recombinant form of the extracellular domain of
Complement Receptor-1 (CR-1). sCR-1 is a potent inhibitor of complement activation.
sCRlsLe'^ (CD20) is an alternately glycosylated form of sCFll that is alternately
glycosylated to display sialylated Lewis^ antigen. Previously, sCR-lsLeX that was
expressed and glycosylated in vivo in engineered Led 1 CHO cells was foimd to correctly
localize to ischemic cerebral micro vessels and Clq-expressing neurons, thus uiliibiting
neutrophil and platelet accumulation and reducing cerebral infarct volumes (Huang et al.,
1999, Science 285:595-599). In the present example, sCRlsLe'^ which was prepared in vifru
by remodeling of glycans, exhibited enhanced biological activity similar to that of sCRsLe''^
glycosylated in vivo.
Tlie TPl 0 peptide was expressed in DUK Bl 1 CHO cells. This CHO cell line
produces the TP10 peptide with the typical CHO cell glycosylation, with many but not all
glycans capped with sialic acid.
Sialylation of 66 mg of TPIO. TPIO (2.5 mg/mL), CMPSA (5 mM), and ST3Gal3
(0.1 U/mL) were incubated at 32°C in 50 mM Tris, 0.15M NaCl, 0.05% sodium azide, pH 7.2
for 48 hours. Radiolabelled CMP sialic acid was added to a small aliquot to monitor
incorporation. TPIO was separated from nucleotide sugar by SEC HPLC. Samples analyzed
at 24 hours and 48 hours demonstrated that the reaction was completed after 24 hours. The
reaction mixture was then frozen. The reaction products were subjected to Fluorophore
Assisted Carbohydrate Electrophoresis (FACE®; Glyko, Inc, Novate CA) analysis (Figure
119).
Pharmacokinetic studies. Rats were purchased with a jugular vein cannula. 10
mg/kg of either the pre-sialylation or post-sialylation TP 10 peptide was given by tail vein
injection to three rats for each treatment (n=3).. Fourteen blood samples were taken from 0 to
50 hours. The concentration in the blood of post-sialylation TPIO peptide was higher than
that of pre-sialylation TPIO at every time point past 0 hour (Figure 120). Sialic acid addition
doubled the area under the plasma concentration-time curve (.AUC) of the pharmacokinetic
curve as compared to the starting material (Figure 121).
Fucosylation of sialylated TPIO. 10 mL (25 mg TPIO) of tlie above sialylation mix
was thawed, and GDP-fiicose was added to 5 mM, MnCb to 5 mM, and FTVI
(fiicosyltransferase VI) to 0.05 U/mL. The reaction was incubated at 32°C for 48 hours. The
reaction products were subjected to Fluorophore Assisted Carbohydrate Electrophoresis
(FACE®; Glyko, Inc, Novato CA) analysis (Figure 122). To a small aliquot, radiolabelled
GDP-fucose was added to monitor incorporation. TPIO was separated from nucleotide sugai-
by SEC HPLC. Samples analyzed at 24 hours and 48 hours demonstrated that tlie reaction
was completed at 24 hours. An in vitro assay measuring binding to E-selcctin indicate that
fucose addition can produce a biologically-active E-selectin ligand (Figure 123).
Enbrel™
10. GlvcoPEGvlation of an antibody Enbrer;^
This example sets forth the procedures to PEGylate the O-linked glycans of an
antibody molecule. Here, EnbreF" is used as an example, however one of skill in the art v/ill
appreciate that this procedure can be used with many antibody molecules.
Preparation of EnbreFW-SA-PEG (10 kDa). Enbrel™ cmF-receptor-IgG,-
chimera), either with the O-linked glycans sialylated prior to PEGylation or not, is dissolved
at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCb, 0.05% NaNs, pH 7.2. The
solution is incubated with 5 mM UDP-galactose and 0.1 U/mL of galactosyltransferase at
32°C for 2 days to cap the undergalactosylated glycans with galactose. To monitor the
incorporation of galactose, a small aliquot of the reaction has '""C-galactose-UDP ligand
added; tlie label incorporated into the peptide is separated from the free label by gel filtration
on a Toso Haas G2000SW analytical column in methanol and water. The radioactive label
incorporation into the peptide is quantitated using an in-line radiation detector.
When the reaction is complete, the solution is incubated with 1 mM CMP-sialic acicl-
linker-PEG (10 kDa) and 0.1 U/mL of ST3Gal3 at 32''C for 2 days. To monitor the
incorporation of sialic acid-linker-PEG, the peptide is separated by gel filtration on a Toso
Haas G3000SW analytical column using PBS buffer (pH 7.1). When the reaction is
complete, the reaction mixture is purified using a Toso Haas TSK-Gel-3000 preparative
colunm using PBS buffer (pH 7.1) and collecting fractions based on UV absorption. The
fractions containing product are combined, concentrated, buffer exchanged and then freeze-
dried. The product of the reaction is analyzed using SDS-PAGE and lEF analysis according
to the procedures and reagents supplied by Invitrogen. Samples are dialyzed against water
and analyzed by MALDI-TOF MS.
Erythropoietin (EPO)
11. Addition of GlcNAc to EPO
This example sets forth the addition of a GlcNAc residue on to a tri-mamiosyl core.
Addition of GlcNAc to EPO. EPO was expressed in SF-9 insect cells and purified
(Protein Sciences, Meriden, CT). A 100% conversion from the tri-mannosyl glycofonn of
Epo to the "tri-mannosyl core + 2 GlcNAc" (Peak 1, PI in Figure 124) was achieved in 24
hours of incubation at 32°C with lOOmU/ml of GIcNAcT-I and lOOmU/ml of GIcNAcT-H in
the following reaction final concentrations:
lOOmM MES pH 6.5, or lOOmM Tris pH 7.5
5mM UDP-GlcNAc
20mM MnCl2
lOOmU/ml GlcNAcT-I
lOOmU/mlGIcNAcT-II
1 mg/ml EPO (purified, expressed in Sf9 cells,
purchased from Protein Sciences).
Analysis of glycoforms. This assay is a slight modification on K-R Anumula and ST
Dhume, Glycobiology 8 (1998)685-69. N-glycanase (PNGase) released N-glycans were
reductively labeled with iintivraniiic acid. The reductively-aininated N-glycans were injected
onto a Shodex Asahipak NH2P-50 4D amino column (4.6 mm x 150 mm). Two solvents
were used for the separation: A) 5% (v/v) acetic acid, 1% telxahydrofuran, and 3%
triethylamine in water, and B) 2% acetic acid and 1% tetrahydrofiiran in acetonitrile. The
colunui was then eluted isocratically with 70% B for 2.5 minutes, followed by a linear
gradient over a period of 97.5 minutes going from 70 to 5% B and a final isocratic elution
with 5% B for 15 minutes. Eluted peaks were detected using fluorescence detection with art
excitation of 230 nm and emission wavelength of 420 mn.
Under these conditions, the trimannosyl core had a retention time of 22.3 minutes, aad
the product of the GnT reaction has a retention time of 30 minutes. The starting material was
exclusively trimannosyl core with core GlcNAc (Figure 124).
12. Preparation of EPO with multi-antennary complex elvcans
This example sets fortli the preparation of PEGylated, biantermary EPO, and
triantennary, sialylated EPO from insect cell expressed EPO.
Recombinant huraan erytluopoietin (rliEPO) from the baculovirus/Sf9 expression
system (Protein Sciences Corp., Meriden, CT) was subjected to glycan analysis and the
resulting glycans were shown to be primarily trimaimosyl core with core fucose, with a sm:..ll
percentage of giycans also having a single GlcNAc.
Addition of N-acetylglucosamine with GnT-I and GnT-II. Two lots of rhEPO (1
mg/mL) were incubated with GnT-I and GnT-II, 5 mM UDP-glcNAc, 20 mM MnCb, and
0.02% sodium azide in 100 mM MES pH 6.5 at 32°C for 24hr. Lot A contained 20 mg of
EPO, and 100 mU/mL GnT-I and 60 mU/mL GnT-H. Lot B contained 41 mg of EPO, and 41
mU/mL GnT-I + 50 mU/mL GnT-II. After the reaction, the sample was desalted by gel
filtration (PDIO columns, Pharmacia LKB Biotechnology Inc., Piscataway, NJ).
EPO glycans analyzed by 2-AA HPLC profiling. This assay is a slight
modification on Anumula iind Dhume, Glycobiology 8 (1998) 685-69. Reductively-aminated
N-glycans were injected onto a Shodex Asahipak NH2P-50 4D amino column (4.6 mm x 150
mm). Two solvents were used for the separation, A) 5% (v/v) acetic acid, 1%
tetrahydrofuran, and 3% triethylamine in water and B) 2% acetic acid and 1 % tetrahydrofuran
in acetonitrile. The column was then eluted isocratically with 70% B for 2.5 min, followed
by a linear gradient over a j)eriod of 100 min going from 70 to 5% B, and a final isocratic
elution with 5% B for 20 min. Eluted peaks were detected using fluorescence detection with
an excitation of 230 nm and emission wavelength of 420 nm. Non-sialylated N-linked
glycans fall in the LC range of 23-34 min, monosialylated from. 34-42 min, disialylated from
42-52 min, frisialylated from 55-65 min and tetrasialylated from 68 - 78 min.
Glycan profiling by 2AA HPLC revealed that lot A was 92% converted to a
biantennary structure witli two GlcNAcs (the balance having a single GlcNAc. Lot B showed
97% conversion to the desired product (Figure I25A and 125B ).
Introducing a third antennary branch with GnT-V. EPO (1 mg/mL of lot B) from
the product of the GnT-I and GnT-II reactions, after desalting on PD-10 columns and
subsequent concentration, \\'as incubated with 10 mU/mL GnT-V and 5 mM UDP-GlcNAc in
100 mM MES pH 6.5 containing 5 mM MnCla and 0.02% sodium azide at 32°C for 24 hrs.
2AA HPLC analysis demonstrated that the conversion occurred with 92% efficiency (Figure
126).
,\fter desalting (PD-10) and concentration, galactose was added witli rGalTI: EPO (1
mg/mL) was incubated witli 0.1 U/mL GalTl, 5 mM UDP-galactose, 5 mM MnCl2 at 32"C
for 24 hrs.
MALDI analysis of reductively-aminated N-glycans from EPO. A small aliquot
of the PNGase released N-glycans from EPO that had been reductively labeled witii
anthranilic acid was dialyzed for 45 min on an MF-Millipore membrane filter (0.025 \xm
pore, 47 mm dia), which was floating on water. The dialyzed aliquot was dried in a
speedvac, redissolved in a small amount of water, and mixed with a solution of 2,5-
dihydroxybenzoic acid (10 g/L) dissolved in water/acetonitrile (50:50). The mixture was
dried onto the target and analyzed using an Applied Biosystems DE-Pro MALDI-TOF mass
spectrometer operated in the linear/negative-ion mode. Oligosaccharides were assigned
based on the observed mass-to-charge ratio and literature precedence.
Analysis of released glyeans by MALDI showed that galactose was added
quantitatively to all available sites (Figure 127). Galactosylated EPO from above was then
purified by gel filtration on a Superdex 1.6/60 column in 50 mM Tris, 0.15M NaCl, pH 6.
Sialylation. After concentration and desalting (PD-10), 10 mg galactosylated EPO (1
mg/mL) was incubated with ST3Gal3 (0.05 U/mL), and CMP-SA (3 mM) m 50 mM Tris,
150 mM NaCl, pH 7.2 containing 0.02% sodium azide. A separate aliquot contained
radiolabelled CMP-SA. The resulting incorporated label and free label was separated by
isocratic size exclusion chromatography/HPLC at 0.5mL/min in 45% MeOH, 0.1%TFA
(7.8mm x 30 cm column, particle size 5 |xtn, TSK G2000SWxl, Toso Haas, Ansys
Technologies, Lake Fore,st, CA). Using this procedure, 12% of the coimts were incorporated
(360 micromolar, at 33 micromolar EPO, or about 10.9 moles/mole). Theoretical (3 N-lLnked
sites, tri-antennary) is about 9 moles/mole incorporation. These correspond within the limits
of the method. In an identical reaction with ST6Gall instead of ST3Gal3, 5.7% of the
radiolabel was incorporated into the galactosylated EPO, or about 48% compared with
ST3Gal3.
13. GlycoPEGylation of EPO produced in insect cells
This example sets forth the prepartion of PEGylated biantennary EPO from insect cell
expressed EPO.
Recombinant human erytlaropoietin (rhEPO) from the baculovirus/Sf9 expression
system (Protein Sciences Corp., Meriden, CT) was subjected to glycan analysis and the
resulting glycans were shown to be primarily trimannosyl core with core fucose, with a small
percentage of glycans also having a single GIcNAc (Figure 128).
Addition of N-acetylglucosamine with GnT-I and GnT-II. Two lots of rhEPO (1
mg/mL) were incubated with GnT-I and GnT-II, 5 mM UDP-glcNAc, 20 mM MnCl2, and
0.02% sodium azide in 100 mM MES pH 6.5 at 32°C for 24hr. Lot A contained 20 mg of
EPO, and 100 mU/mL GnT-I and 60 mU/mL GnT-II. Lot B contained 41 mg of EPO, and 41
mU/mL GnT-I + 50 mU/mL GnT-II. After the reaction, the sample was desalted by gel
filtration (PDIO columns, Pharmacia LKB Biotechnology Inc., Piscataway, NJ).
Glycan profiling b)' 2AA HPLC revealed that lot A was 92% converted to a
biantermary structure with two GlcNAcs (the balance having a single glcNAc. Lot B showed
97% conversion to the desired product (Figure 125A and 125B ).
Galactosylation of EPO lot A. EPO (-16 mgs of lot A) was treated with GnT-II to
complete the addition of GlcNAc. The reaction was carried out in 50 mM Tris pH 7.2
contaniing 150 mM NaCl, EPO mg/ml, 1 mM UDP-GlcNAc, 5 mM MnCh, 0.02% sodium
azide and 0.02 U/ml GnT-II at 32 C for 4 hrs. Then galactosylation of EPO was done by
adding UDP-galactose to 3 mM and GalTl to 0.5 U/ml and the incubation continued at 32° C
for 48 hrs.
Galactosylated EPO was then purified by gel filtration on a Superdex75 1.6/60
column in 50 mM Tris, 0.15M NaCl, pH 6. The EPO containing peak was then analyzed by
2AA HPLC. Based on the HPLC data -85% of the glycans contains two galactose and -15%
of the glycans did not have any galactose after galactosylation reaction.
Sialylation of galactosylated EPO. Sialylation of galactosylated EPO was carried
out in 100 mM Tris pH containing 150 mM NaCl, 0.5 mg/ml EPO, 200 mU/ml of ST3Gal3
and either 0.5 mM CMP-SA or CMP-SA-PEG (1 kDa) or CMP-SA-PEG (10 kDa) for 48 hrs
at 32 °C. Almost all of the glycans that have two galactose residues were fully sialylated (2
sialic acids / glycan) after sialylation reaction with CMP-SA. NL4.LDI-T0F analysis
confirmed tlie ITPLC data.
PEGylation of galactosylated EPO. For PEGylation reactions using CMP-SA-PEG
(1 kDa) and CMP-SA-PEG (10 kDa), an aliquot of the reaction mixture was analyzed by
SDS-PAGE (Figuie 129). Ihe molecular weight of the EPO peptide increased with the
addition of each sugai", mid increased more dramatically in molecular weight after the
PEGylation reactions.
In vitro bioassay of EPO. In vitro EPO bioassay (adapted from Hammerling et al,
1996, J. Pharm. Biomed. Anal. 14: 1455-1469) is based on the responsiveness of the TF-1
cell line to multiple levels of EPO. TF-1 cells provide a good system for investigating the
proliferation and differentiation of myeloid progenitor cells. This cell line vi^as established by
T. Kitamura et al. in October 1987 from a heparinized bone marrow aspiration sample from a
35 year old Japanese male with severe pancytopenia. These cells are completely dependent
on Interleukin 3 or Granulocyte-macrophage colony-stimulating factor (GM-CSF).
The TF-1 cell line (ATCC, Cat. No. CRL-2003) was grown in RPMI + FBS 10% +
GM-CSF (12 ng/ml) and iiicubated at 37°C 5% CO2. The cells were in suspension at a
concentration of 5000 cells/ml of media, and 200 jal were dispensed in a 96 well plate. The
cells were incubated with various concentrations of EPO (0.1 |J,g/ml to 10 (xg/ml) for 48 hours.
A MTT Viability Assay was then done by adduig 25 yl of MTT at 5 mg/ml (SIGMA
M5655), incubating the plate at 37°C for 20 min to 4 hours, adding 100 \i\ of
isopropanol/HCl solution (100 ml isopropanol + 333 p.1 HCl 6N), reading the OD at 570 nm,
and 630nm or 690nm, and subtracting the readings at 630 nm or 690 nm from the readings at
570 nm.
Figure 130 contains tlie results when sialylated EPO, find EPO glycoPEGylated with 1
kDa or 10 kDa PEG was subjected to an in vitro EPO bioactivity test. The EPO
glycoPEGylated with IkDa PEG had almost the same activity as the unglycoPEGylated EPC)
when both were at a concentration of approximately 5 fxg/ml. The EPO glycoPEGylated with
10 kl)a PEG had approximately half the activity of the unglycoPEGylated EPO when both
were at a concentration of approximately 5 |j.g/ml.
14. GlycoPEGvlation of 0-Linked Glycans of EPO produced in CHQ Cells
Preparation of O-Iinked EPO-SA-PEG (10 kDa). Asialo-EPO, originally produced
in CHO cells, is dissolved at 2.5 mg/'mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaNa, pH
7.2. The solution is incubated with 5 mM CMP-SA and 0.1 U/mL of ST3Gal3 at 32"C for 2
days. To monitor the mcorporation of sialic acid onto tlie N-liiiked glycans, a small aliquot
of the reaction had CMP-SA-'''C added; the peptide is separated by gel filtration on a To.so
Haas G200CSW anal}^;!^!! column using methanol, water and the product detected using a
radiation detector. When the reaction is complete, the solution is concentrated using a
Centricon-20 filter. The remaming solution is buffer exchanged with 0.05 M Tris (pH 7.2),
0.15 M NaCl, 0.05% NaNa to a final volume of 7.2 mL until the CMP-SA could no longer be
detected. The retentate is then resuspended m 0.05 M Tris (pH 7.2), 0.15 M NaCl, 0.05%
NaNs at 2.5 mg/mL protein. The solution is incubated with 1 mM CMP-SA-PEG (10 kDa)
and STSGall, to glycosylate the 0-linked site, at 32°C for 2 days. To monitor the
incorporation of sialic acid-PEG, a small aliquot of the reaction is separated by gel filtration
suing a Toso Haas TSK-gcl-3000 analytical column eluting with PBS pH 7.0 and analyzing
by U\' detection. When the reaction is complete, the reaction mixture is purified using a
Toso Haas TSK-gel-3000 preparative column using PBS buffer (pH 7.0) collecting fi-actions
based on UV absorption. The product of the reaction is analyzed using SDS-PAGE and lEF
analysis according to the procedures and reagents supplied by Invitrogen. Samples are
dialyzed against water and analyzed by MALDI-TOF MS.
15. EPO-Transferrin
This example sets Ibrth the procedures for the glycoconjugation of proteins to O-
linked glycans, and in particular, transferrin is glycoconjugated to EPO. The sialic acid
residue is removed from O-linked glycan of EPO, and EPO-SA-hnker-SA-CMP is prepared.
EPO-SA-linker-SA-CMP is glycoconjugated to asialotransferrin with ST3Gal3.
Preparation of O-iinked asialo-EPO. EPO (erythropoietin) produced in CHO cells
is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, and is
incubated with 300 mU/niL sialidase {Vibrio cholera)-agarose conjugate for 16 hours at 32
°C. To monitor the reaction a small aliquot of the reaction is diluted with the appropriate
buffer and a lEF gel perfonxied according to Invitrogen procedtores. The mixture is
centrifugcd at 10,000 rpm and the supernatant is collected. The supernatant is concentrated
to a EPO concentration of about 2.5 mg/mL in 50 mM Tris-HCI, 0.15 M NaCl, 0.05% NaN3,
pH 7.2. The solution is incubated with 5 mM CMP-sialic acid and 0.1 U/mL of ST3Gal3 at
32"C for 2 days. To monitor the incoiporation of sialic acid, a small aliquot of the reaction
had CMF-SA-fluorescent ligand added; the label incorporated into the peptide is separated
from the free label by gel filtration on a Toso Haas G3000SW cinalytical column using PBS
buffer (pH 7.1). When tiie reaction is complete, the reaction mixture is purified using a Toso
Haas G3000SW preparative column using PBS buffer (pH 7.1) and collecting fractions bas;ed
on IJV absorption. The product of the reaction is analyzed using SDS-PAGE and lEF
analysis according to the procedures and reagents supplied by Invitrogen. Samples are
dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of EPO-SA-linker-SA-CMP. The 0-linked asialo-EPO 2.5 mg/mL Li
50 mM Tris-HCI, 0.15 M NaCI, 0.05% NaNa, pH 7.2. The solution is incubated with 1 mM
CMI'-sialic acid-linker-SA-CMP and 0.1 U/mL of ST3GaIl at 32°C for 2 days. To monitor
the incorporation of sialic acid-linker-SA-CMP, the peptide is separated by gel filtration on a
Toso Haas G3000SW analytical column using PBS buffer (piH 7.1).
After 2 days, the reaction mixture is purified using a Toso Haas G3000SW
preparative column using PBS buffer (pH 7.1) and collecting fractions based on UV
absorption. The product of the reaction is analyzed usuig SE)S-PAGE and EEF analysis
according to the procedures and reagents supplied by Invitrogen. Samples are dialyzed
against water and analyzed by MALDI-TOF MS.
Preparation of Transferrin-SA-Liaker-SA-EPO. EPO-SA-Linker-SA-CMP from
above is dissolved at 2.5 mg/mL in 50 mM Tris-HCI, 0.15 M NaCI, 0.05% NaNs, pH 7.2.
The solution is incubated with 2.5 mg/mL asialo-transferrin and 0.1 U/mL of ST3Gal3 at
32°C for 2 days. To monitor the incorporation of transferrin, the peptide is separated by gel
filtration on a Toso Haas G3000SW analytical colimin using PBS buffer (pH 7.1) and the
product detected by UV absorption. When the reaction is complete, the solution is incubated
with 5 niM CMP-SA and 0.1 U/mL of ST3Gal3 (to cap any unreacted transferrin glycans) at
32°C for 2 days. The reaction mixture is purified using a Toso Haas G3000SW preparative
column using PBS buffer (pH 7.1) collecting fractions based on UV absorption. The product
of the reaction is analyzed using SDS-PAGE and lEF analysis according to the procedures
and reagents supplied by Invitrogen. Samples are dialyzed against water and analyzed by
MALDI-TOF MS.
16. EPO-GDNF
This example sets forth tlie procedures for the glycoconjugation of proteins, and in
paiticular, the preparation cf EPO-SA-Linker-SA-GDNF.
Preparation of EPO-SA-Linker-SA-GDNF. EPO-SA-Linker-SA-CMP &om above
is dissolved at 2.5 mg/mL in 50 mM Tris-HCI, 0.15 M NaCl, 0.05% NaNs, pH 7.2. The
solution is incubated with 2.5 mg/mL GDNF (produced in NSO) and 0.1 U/mL of ST3Gal3
at 32°C for 2 days. To monitor the incorporation of GDNF, the peptide is separated by gel
filtration on a Toso Haas G3000SW analytical column using PBS buffer (pH 7.1) and the
product detected by UV absorption. When the reaction is complete, the solution is incubated
with 5 mM CMP-SA and 0.1 U/mL of ST3Gal3 (to cap any unreacted GDNF glycans) at
32°C for 2 days. The reaction mixture is purified using a Toso Haas G3000SW preparative
column using PBS buffer (pH 7.1) collecting fractions based on UV absorption. The product
of the reaction is analyzed using SDS-PAGE and lEF analysis according to the procedures
and reagents supplied by Invitrogen. Samples are dialyzed against water and analyzed by
MALDI-TOF MS.
17. Mono -antennary GlycoPEGvIation of EPO
This example sets forth the procedure for the preparation of glycoPEGylated mono-
antennary erythropoietin (TEPO), and its bioactivity in vitro and in vivo.
When EPO (GenBank Accession No. P01588) is expressed in CHO cells, N-linked
glycans are formed at amino acid residues 24, 38 and 83, and an O-linked glycan is formed at
amino acid residue 126 (Fig. 131; Lai et al., 1986, J. Biol. Chem. 261:3116-3121). The
bioactivity of this glycoprotein is directly correlated with the level of NeuAc content.
Increased sialic acid decreases the binding of EPO to its recejjtor in vitro; however increased
sialic acid increases tlie bioactivity of EPO in vivo. The O-linked glycan has no impact on
die in vitro or in vivo activity of EPO, or the pharmacokinetics of the molecule (Wasley et al,
1991, Blood 77:2624-2632).
When EPO is expressed in insect cells, such as is accomplished using a
baculovirus/Sf9 expression system (see also, Wojchowshi et al., 1987, Biochem. Biophys.
Acta 910:224-232; Quelle et al., 1989, Blood 74:652-657), N~Iinked glycans are formed at
amino acid residues 24, 38 and 83, but an O-linked glycan is not formed at amino acid
residue 126 (Fig. 132). This is because the insect cell does not have a glycosyl transferase
that recognizes the amino acid sequence around amino acid residue 126 of EPO. The
majority oi~the N-liiiked glycans are composed of GIcNAc2Man3Fuc. In the present exaiiiplc.
EPO expressed in insect cells was remodeled with high efficiency to achieve the complex
glycan SA2Gal2GIcNAc2Man3FucGlcNAc2 by contacting the protein with, in series, GnTl,2,
GalT-I, and ST in the presence of the appropriate donor molecules. These enzymatic
reactions were performed on insect cell expressed EPO using reaction conditions disclosed
herein, to yield the complex glycans herein with 92% total efficiency (Table 21). Optionally,
0-linked glycans can also be added (O'Connell and Tabak, 1993, J. Dent. Res. 72:1554-
1558; Wang et al., 1993, J. Biol. Chem. 268:22979-22983).
/Vlso in tlie present example, EPO expressed in insect cells was remodeled to form
mono-antennaiy, bi-anntenaiy and tri-antennary glycans, which were subsequently
gfycoPEQylated with 1 kDa, 10 kDaand 20 kOa PEG molecules suing procedures describe./
elsewhere herein. The molecular weights of these EPO forms were detennined, and were
compared to Epoetini'^' having 3 N-linked glycans, and NESP (/uranesp™) having 5 N-linis'' '
glycans (Fig. 133). Examples of the preparation of bi- and tri-antemiary gl}'can strucmres :¦.
given in Example 7, herein.
EPO having monomiteimary PEGylated glycan structures is prepared by expressing
EPO peptide in insect cells, then contacting the EPO peptide with GnTI only (or alternatively
GnTII only) in the presence of a GlcNAc donor. The EPO peptide is then contacted with
GalT-I in the presence of a galactose donor. The EPO peptide is then contacted with ST in
the presence of SA-PEG donor molecules (Fig. 134A) to generate an EPO peptide having
tiaree N-Iinked mono-antennary PEGylated glycan structures (Fig. 134B).
The in vitro bioactivity of EPO-SA and EPO-SA-PEG generated from insect cell
expressed EPO was accessed by measuring the ability of the molecule to stimulate the
proliferation of TF-1 erythroleukemia cells. Tri-antennary EPO-SA-PEG 1 kDa exliibited
almost all of the bioactivity of tri-antennary EPO-SA, and di-antennary EPO-SA-PEG 10
kDa exhibited almost all of the bioactivity of di-antennary EPO-SA over a range of EPO
concentrations (Fig. 135). Remodeled and glycoPEGylated EPO generated in insect cells
exhibited up to 94% of the in vitro bioactivity of Epogen'"''^, which is EPO expressed in CHO
cells without further glycan remodeling or PEGylation (Table 22).
The in vivo pharmacokinetics of glycoPEGylated and non-glycoPEGylated EPO was
detenrined. GlycoPEGylated and non-g lycoPEGylated [I'^^j-labeled EPO was bolus injected
into rats and the phannacol; inetics of the molecules were determined. As compared with bi-
antermary EPO, the AUG of bi-antennary EPO-PEG 1 kDa was 1.8 times greater, and the
AUG of bi-antennary EPO-PEG 10 kDa was 11 times greater (Fig. 136). As compared with
bi-aiitennary EPO, the AUG of bi-antennary EPO-PEG 1 kDa was 1.6 times greater, and the
AUG of bi-antennary EPO-PEG 10 kDa was 46 times greater (Fig. 136). Therefore, the
pharmacokinetics of EPO was greatly improved by glycoPEGylation.
The in vivo bioactivity of glycoPEGylated and non-giycoPEGylated EPO was also
determined by measuring the degree to which the EPO construct could stimulate
reticulocytosis. Reticulocytosis is a measure of the rate of tlie maturation of red blood cell
precursor cells into mature red blood cells (erythrocyte). Eight mice per treatment group
were given a single subcutaneous injection of 10 jig protein/Kg, and the percent reticulocytes
was measured at 96 hours (Fig. 137). Tri- and bi-antennary PEGylated EPO exhibited greater
in vivo bioactivity than non-PEGylated EP(^ forms, including Epogen''"'^.
Further determination of m vivo bioactivity of the EPO constructs was assessed by
measuring tlie hematocrit (the percent of whole blood that is comprised of red blood cells) of
CD-1 female mice 15 days after intraperitoneal injection tliree times per week with 2.5 \ig
peptide/kg body weight of the EPO construct. The hematocrit increment increased with the:
size of the EPO form, with the 82.7 kDa mono-anteimary EPO-PEG 20 kDa having a slightly
greater activity than the 35.6 kDa NESP (Aranesp'^'*^) and about two times the bioactivity of
28.5 kDa Epogen™ (Fig. 138).
This example illustrates that the generation of a longer-acting glycoPEGylated EPO is
feasible. The pharmacokinetic profile of glycoPEGylated EPO can be customized by altering
the number of glycoPEGylation sites and the size of the PEG molecule added to alter the
half-life of the peptide in the bloodstream. Finally, glycoPEGylated EPO retains both in vitro
and in vivo bioactivity.
18. Preparation and Bioactivity of Sialylated and PEGylated Mono-, Bi- and
Tri-Antertnary EPO
This example illustrates the production of glycoPEGylated EPO, in particular
PECiylated EPO having mono-antennary and bi-antennary glycans with PEG Imked thereto.
The following EPO variants were produced: mono-antennary PEG (I kDa) and PEG (20
kDa); bi-antennary 2,3-siaUc acid (SA), bi-antennary SA-PE;G (1 kDa), bi-antennary SA-PEG
(10 kDa); tri-antennary 2.3-SA and tri-antennary 2,6-SA capped with 2,3-SA.
Recombinant erythropoietin (rEPO) expressed in insect cells was obtained from
Protein Sciences (Lot # 060302, Meridan CT). The glycan composition of this batch of EPO
had approximately 98% trimannosyl core structure. Figure 139A depicts the PIPLC analysis
of tile released gl yeans from this EPO, with peak "P2" representing the trimannosyl core
glycan. Figure 139B shows the MALDI analysis of the released glycans with the structures
of tlie released glycans beside the peak they represent.
Mono-antennary branching
Several steps were performed to produce the mono-antennary branched structure. In
briel^ the first step was a GnT-I/GaIT-1 reaction followed by purification using Superdex-75
chromatography. This reaction adds a GlcNAc moiety to one branch of the tri-mannosyl
core, and a galactose moiety onto the GIcNAc moiety. Branching was extended with the
S'OGalS reaction to add the SA-PEG (10 kDa) moiety or the SA-PEG (20 kDa) moiety onto
the terminal galactose moiety. The final purification was accomplished using Superdex-20C
chromatography (Amersham Biosciences, Arluigton Heights, IL).
GnT-I/GalT-1 Reaction. The GnT-I and GaIT-1 reactions were combined and
incubated at 32°C for 36 hours. The reaction contained 1 mg/mL EPO, 100 mM Tris-Cl pH
7.2, 150 mM NaCl, 5 mM MnCb, 0.02% NaNs, 3 mM UDP-GlcNAc, 50 mU/mg GnT-I, 3
mM UDP-Gal, and 200 mU/mg GalT-1. Figure 140 depicts the MALDI analysis of glycans
released fiom EPO after the GnT-I/GalT-I reaction. Glycan analysis showed approximately
90% of the glycans had thgrnphy CAnicrsham Biosciences. Arlington Heights, IL) in PBS containing 0.02%
Tween 20 after the CjuT- !/GalTl reaction. Figure 145 depicts tiie clu-omatogram of the
Superdex 75 gel filtration, where peak 2 is EPO with bi-antennary glycans with terminal
galactose moieties. Figure 146 shows SDS-PAGE analysis of the products of each
remodelmg step indicating the increase in the molecular weight of EPO with each remodeluig
step.
ST3GaI3 Reaction. The ST3Gal3 reaction was incubated at 32°C for 24 hours. The
reaction contained 0.5 mg/mL EPO, 100 mM Tris-Cl pH 12, 150 mM NaCl, 0.02% NaNs,
100 mU/mg STSGalS, and 0.5 mM CMP-SA, 0.5 mM CMP-SA-PEG (1 kDa), or 0.5 mM
CMP-SA-PEG (10 kDa). Figure 147 shows the results of SDS-PAGE analysis of EPO before
and after the ST3Gal3 reaction. Based on this SDS-PAGE analysis, bi-antennary EPO
containing terminal Gal can no longer be visually detected after each ST3Gal3 reaction. All
sialyl ated EPO variants show an increase in size compared to non-sialylated EPO at the start
of the reaction.
Superdex 200 Purification. EPO was purified from the contaminants of the
ST3Gal3 reactions by a 1.6 cm x (^Q cm Superdex-200 gel filtration chromatography
(Amersham Biosciences, .\rlington Heights, IL) in PBS containing 0.02% Tween-20. Table
23 summaries the distribution of glycan structures at each remodeling step.
Diamonds represent fiicose, and squares represent GlcNAc, circles represent mannose, open
circles represent galactose.
Tri-antennary Branching
Several reaction;; were performed to accomplish the tri-antennary branching of EPO.
Briefly, the first reaction combined the GnT-I and GnT-II reactions to add a GlcNAc moiety
to the two outer tri-mannosyl core branches of the glycan. The second reaction, GnT-V
reaction, adds a second GlcNAc moiety to one of the two outer trimannosyl core branches so
that there are now three GlcNAc moieties. The third reaction, GalT-1 reaction, adds a
galactose moiety to each terminal GlcNAc moiety. The EPO products were then separated
by Superdex 75 chromatography. The tri-antennary branching was further extended with the
ST.3Gal3 reaction to add either a 2,3-SA moiety or a 2,6-SA moiety, and capped with a 2,3-
SA moiety. Final purification was accompUshed using Superdex 75 chromatography.
GnT-I/GnT-II Reaction. The GnT-I and GnT-II reactions were combined and
incubated at 32°C for 24 hours. The reaction contained 1 mg/mL EPO, 100 mM MES pH
6.5, 150 mMNaCl, 20 mM MnCb, 0.02% NaNs, 5 mM UDP GlcNAc, 50 mU/mg GnT-1 and
41 mU/mg GnT-II. The reaction achieved 97% completion of the addition of the bi-
antennary GlcNAc moiety, with 3% tri-mannosyl core remaining. Figure 148 depicts the
HPLC analysis of the glycans released from EPO after the GnT-I/GnT-II reaction.
GnT-V Reaction. The GnT-V reaction contaming 100 mM MES pH 6.5, 5 mM
UDP-GlcNAc, 5 mM MnCb, 0.02% NaNs, 10 mU/mg GnT-V and 1 mg/mL EPO, was
incubated at 32°C for 24 hours. This reaction adds a GlcNAc moiety to ein outer mannose
moiety already containing a GlcNAc moiety. Figure 149 depicts the HPLC analysis of t!ie
glycans released from EPO after the GnT-V reaction. Approximately 92% the glycans
released from EPO were the desired product, tri-antennary branched EPO with terminal
GlcNAc moieties, based on glycan and MALDI analysis. The remaining 8% of the glycans
were bi-antennary branched structures containing terminal GlcNAc moieties.
GaIT-1 Reaction. The GalT-1 reaction containing 100 niM Tris pH 7.2, 150 niM
NaCl, 5 mM UDP Gal, 100 mU/mg GaIT-1, 5 mM MnCb, 0.02% NaNi and 1 mg/mL EPO
was incubated at 32°C :or 24 hours. Figure 150 depicts the HPLC analysis of tlie glycans
released from EPO after this reaction. Glycan and MALDI analysis indicates that 97% of tlie
released glycans had terminal galactose moieties on the tri-antennary branched structures.
The remaining 3% was a bi-antennary structure containing a terminal galactose.
Superdex 75 Purification. After the GnT-I/GalTl reaction, EPO was purified from
the enzyme protein contimiinants and nucleotide sugars using a 1.6 cm x 60 cm Superdex-75
gel filtration chromatography (Amersham Biosciences, Arlington Heights, IL) in PBS
containing 0.02% Tween 20. The purified material was divided into two batches to produce:
the tri-antennary glycan with tenninal 2,6-SA moieties and the tri-antennary glycan with
terminal 2,6-SA moieties capped with 2,6-SA moieties.
ST3Gal3 Reaction. The ST3Gal3 reaction was incubated at 32°C for 24 hours. The
reaction contained 1 mg/rnL galactosylated EPO, 100 mM Tris-Cl pH 7.2, 150 mM NaCI,
0.02% NaNs, 50 mU/mg ST3Gal3, and 3 mM CMP-SA. Figure 151 depicts the HPLC
analysis of glycans released fi-om EPO after tliis step. Based on glycan and MALDI analysis,
approximately 80% of the released glycans were tri-antennar)' branched structures with
terminal 2,3-SA moieties. The remaining 20% of the released glycans were bi-antennary
structures with terminal 2,3-SA moieties.
ST6Gall sialylation Reactloa following the ST3GaB Reaction. The ST6GaIl
reaction was incubated at 32°C for 24 hours. The reaction contained 1 mg/mL siaiylated
galactosylated EPO, 100 mM Tris-Cl pH 7.2, 150 mM NaCl, 0.02% NaNj, 50 mU/mg
ST6Gall, and 3 mM CMP-SA. Figure 152 depicts the results of HPLC analysis of the
glycans released from EPO after the ST6Gall reaction. Based on glycan and MALDI
analysis, approximately 80% of the tri-antennary branched glycans contained terminal 2,3-';/.
moieties. The remaining 20% of the glycans were bi-antennary with terminal 2,3-SA
moieties.
Superdex 75 Purification. EPO was purified from the contaminants of the ST3Gal3
reactions by a 1.6 cm x 60 cm Superdex-75 gel filtration chromatography (Amersham
Biosciences, Arlington Heights, IL) in PBS containing 0.02% Tween-20.
Bioassay of Tri-antenaary and Bi-antennaiy Siaiylated or PEGylated EPO. The
activity of the tri-antennary and bi-antennary siaiylated EPO gl)^co forms, and the PEG 10
kDa and I kDa bi-antennary glyco forms were assayed using the TF-1 cell line and the MTi
viability test, as described above. Figure 153 depicts die results of the MTI' cell proliferatio.
assay. At 2 jag/ml EDP, the bi-antennary sialylated EPO had nearly the activity of the control
Epogen, while the tri-antennary sialylated EPO had significanly less activity.
Factor IX
19. GlvcoPEGvlation of Factor IX produced m CHQ cells
This example sets forth the preparation of asialoFactor IX and its sialylation with
CMl^-sialic acid-PEG.
Desialylation of rFactor IX. A recombinant form of Coagulation Factor IX (rFactor
IX ) was made in CHO cells. 6000 lU of rFactor IX were dissolved in a total of 12 mL USP
H2O. This solution was transferred to a Centricon Plus 20, PL-10 centrifugal filter with
another 6 mL USP H2O. The solution was concentrated to 2 mL and then diluted with 15 mJL
50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.05% NaNa and then reconcentrated.
The dilution/concentration was repeated 4 times to effectively change the buffer to a final
volume of 3.0 mL. Of this solution, 2.9 mL (about 29 mg of rFactor IX) was transferred tc
small plastic tube and to ii: was added 530 mU a2-3,6,8-Neuraniinidase- agarose conjugate
(Vibrio cholerae, Calbiochem, 450 \iL). Tlie reaction mixture was rotated gently for 26.5
hours at 32 °C. The mixture was centrifuged 2 minutes at 10,000 ipm and the supernatant
was collected. The agarose beads (containing neuraminidase) were washed 6 times with 0.5
mL 50 mM Tris-HCl pH 7.12, 1 M NaCl, 0.05% NaNs- The pooled washings and
supernatants were centrifuged again for 2 minutes at 10,000 rpm to remove any residual
agarose resin. The pooled, desialylated protein solution was diluted to 19 mL with the same
buffer and concentrated down to ~ 2 mL in a Centricon Plus 20 PL-10 centrifugal filter. Thf"
solution was twice diluted wth 15 mL of 50 mlv4 Tris-HCl pH 7.4, 0.15 M NaCl, 0.05%
NaNs and reconcentrated to 2 mL. The final desialyated rFactor IX solution was diluted I' ¦ 5
niL final volume (-10 mg/noL) with the Tris Buffer. Native and desialylated rFactor IX
samples were analyzed by lEF-Electrophoresis. Isoelectric Focusing Gels (pH 3-7) were .
using 1.5 pL (15 pg) samples first diluted with 10 ^L Tris buffer and mixed with 12 pL
sample loading buffer. Gels were loaded, run and fixed using standard procedures. Gels
were stained with Colloidal Blue Stain (Figure 154), showing a band for desialylated Fat
IX.
Preparation of PEG (1 kDa and 10 kDa)-SA-Factor IX. Desialylated rFactor-EX.
(29 mg, 3 mL) was divided into two 1.5 mL (14.5 mg) samples in two 15 mL centrifuge
tubes. Each solution was diluted with 12.67 mL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl,
0.05% NaNs and either CMP-SA-PEG-lk or 10k (7.25 ^mioi) was added. The tubes were
inverted gently to mix and 2.9 U ST3GaI3 (326 nL) was added (total volume 14.5 mL). The
tubes were inverted again and rotated gently for 65 hours at 32 "C. The reactions were
stopped by freezing at -20 °C. 10 [ig samples of the reactions were analyzed by SDS-PAGE.
The PEGylated proteins were pimfied on a Toso Haas Biosep G3000SW (21.5 x 30 cm, 13
um) ILPLC column with Dulbecco's Phosphate Buffered Saline, pH 7.1 (Gibco), 6 mL/min.
The reaction and purification were monitored using SDS Page and lEF gels. Novex Tris-
Glycine 4-20% 1 mm gels were loaded with 10 p.L (10 ^g) of samples after dilution with 2
^iL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaNs buffer and mixing with 12 pL
sample loading buffer and 1 pL 0.5 M DTT and heated for 6 rainutes at 85 °C. Gels were
stained with Colloidal Blue Stain (Figure 155) showing a band for PEG (1 kDa and 10 kDa)-
SA-Factor DC.
20. Direct Sialyl-GlycoPEGylation of Factor IX
This example sets forth the preparation of sialyl-PEGylation of Factor IX without
prior sialidase treatment.
Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(10 KDa). Factor IX (1100
lU), wtiich was expressed in CHO cells and was fully sialylated, was dissolved in 5 mL of 20
mM histidine, 520 niM glycine, 2% sucrose, 0.05% NaNa and 0.01% polysorbate 80, pH 5.0.
The CMP-SA-PEG-(10 kDa) (27 mg, 2.5 pmol) was then dissolved in the solution and 1 U of
ST3Ga[3 was added. The reaction was complete after gently mixing for 28 hours at 32°C.
The reaction was analyzed by SDS-PAGE as described by Invitrogen. The product protein
was purified on an Amershaxn Superdex 200 (10 x 300 mm, 13 pm) HPLC colunm with
phosphate buffered saline, pH 7.0 (PBS), 1 mL/min. Rt = 9.5 min.
Sialyl-PEG) lation of Factor-LX with CMP-SA-PEG-(20 kDa). Factor IX (1100
lU), which was expressed in L'HO cells and was fully sialylated, was dissoh'ed in 5 niL oi'.
mM hisudine, 520 mM glycine, 2% sucrose, 0.05% NaN3 and 0.01% polysorbate 80. pi 1 5.0
The CMP-SA-PEG-(20 kDa) (50 mg, 2.3 pmol) was then dissolved in the solution and CST-
II was added. The reaction mixture was complete after gently mixing for 42 hours at 32°C.
The reaction was analyzed by SDS-PAGE as described by Invitrogen.
The product protein was purified on an Amersham Superdex 200 (10 x 300 mm, 13
|im) HPLC column witli phosphate buffered saline, pH 7.0 (Fisher), 1 mL/min. Rt = 8.6 min.
21. Sialic Acid Capping of GlvcoPEGvlated Factor EK
This examples sets forth the procedure for sialic acid capping of sialyl-
glycoPEGylated peptides. Here, Factor-IX is the exemplary peptide.
Sialic acid capping of N-linked and O-Iinked Glycans of Factor-IX-SA-PEG (10'
kDa). Purified r-Factor-IX-PEG (10 kDa) (2.4 mg) was concentrated in a Centricon® Plus 20
PL-10 (Millipore Corp., Bedford, MA) centrifugal filter and the buffer was changed to 50
mM Tris-HCl pH 7.2, 0.] 5 M NaCl, 0.05% NaNs to a final volume of 1.85 mL. The protein
solution was diluted with 372 p.L of the same Tris buffer and 7.4 mg CMP-SA (12 lamol) was
added as a solid. The solution was inverted gently to mix and 0.1 U STSGall and 0.1 U
ST3Gal3 were added. The reaction mixture was rotated gently for 42 hours at 32 °C.
A 10 )ig sample of the reaction was analyzed by SDS-PAGE. No vex Tris-Glycine 4~
12% 1 mm gels were performed and stained using Colloidal Blue as described by Invitrogen.
Briefly, samples, 10 pL (10 ]xg), were mixed with 12 pL sample loading buffer and 1 pL 0.5
M DTT and heated for 6 minutes at 85 °C (Figure 156, lane 4).
Factor Vila
22. GlycoPEGylation of Recombinant Factor Vila produced in BHK cells
This example sets forth the PEGylation of recombiucuit Factor Vila made in BHK
cells.
Preparation of Asiaio-Factor Vila. Recombinant Factor Vila was produced in
BHK cells (baby hanister kidney ceils). Factor Vila (14.2 rag) was dissolved at 1 mg/ml in
buffer solution (pH 7.4, 0.05 M Tris, 0.15 M NaCI, 0.001 M CaCb, 0.05% NaNs) and was
incubated with 300 mU/mL sialidase (Vibrio cholera)-agmos- is based on the dose-response relationship that exists between tlie
anjount of estiadiol produced when FSH, but not lutenizing hormone (LH), is added to
cultured Sertoli cells obtained from immature old rats. Exogenous testosterone is converted
to 1 yp-estradiol in the presence of FSH.
Seven to 10 days old Sprague-Dawley rats were used to obtain Sertoli cells. After
sacrifice, testes were decapsulated and tissue was dispersed by incubation in collagenase (1
mg/ml), trypsin (Img/ml), hyaluronidase (1 mg/ml) and DNases (5 ^ig/ml) for 5 to 10 min.
The tubule fragments settled to the bottom of the flask and were washed in PBS (Ix). Tlie
tubule fragments were reincubated for 20 min with a media containing the same enzymes:
collagenase (1 mg/ml), trypsin (Img/ml), hyaluronidase (1 mg/ml) and DNases (5 \ig/ml).
The tubule fragments were homogenized and plated into a 24 well plate in a serum
free media. 5x10^ cells were dispersed per well. After 48h incubation at 37° C and 5%
CO2. fresh media was added to the cells. Composition of the serum free media: DMEM (1
vol), Ham's FIO nutrient mixture (1 vol), insulin 1 }ig/ml. Transferrin 5 ^g/ml, EGF 10
ng/ml, T4 20 pg/ml. Hydrocortisone 10" M, Retinoic acid 10" M.
The stimulation experiment consists of a 24 hour incubation with standard FSH or
samples at 37°C and 5% CO2. The mean intra-assay coefficient of variation is 9% and the
mean inter-assay coefficient of variation is 11%.
The 17B-estradiol Elisa Kit DE2000 (R&D Systems, Minneapolis, MN) was used to
quantify the level of estradiol after incubation with FSH, FSH-SA-PEG (1 kDa) and FSH-
SA-PEG(lOkDa).
The procedure wa;; as follows: 100 p.1 of Estradiol Standard (provided with kit and
prepared as per instructions with kit) or sample was pipetted into wells of 17B-estradiol Elisa
plate(s); 50 fj,l of 17B-estradiol Conjugate (provided with kit, prepared as per instructions
with kit) was added to each well; 50 (il of 17B-estradiol antibody solution (provided with kit
and prepared as per instructions with kit) was added to each well; plates were incubated for 2
hour at room temperature ..U 200 rpm; the liquid was aspirated from each well; the wells were
washed 4 times using the washing solution; all the liquid was removed from the wells: 200 |,ii
of pNPP Substrate (provided with kit and prepai"ed as per instructions with kit) was added to
all wells and incubated for 45 min; 50 (J.1 of Stop solution (pro\'ided with kit and prepared as
per instmctions with kit) was added and the plates were read it at 405 nm (Figure 170).
While FSH-PEG(10 kDa) cxiiibited a modest stimulation of Sertoli cells, at 1 |J,g/ml, I'Sli-
PEG(] kDa) stimulated S'-loli cells up to 50% more than unPEGylated FSH.
27. Steelman-Pohlev Bioassav of In Vivo Activity of GlvcoPEGvlated FSH
In this example, the Steelman-Pohley bioassay (Steehnan and Pohley, 1953,
Endocrinology 53:604-615) was used to determine the in vivo activity of glycoPEGylated
FSH. The Steelman-Pohley assay uses the change in ovary weight of a rat to measure the in
vivo activity of FSH that is coinjected with human chorionic gonadotropin.
Tlie Steelman-Poliley bioassay was performed according to the protocol described in
CIiristin-Maitre et al. (2000, Methods 21:51-57). Seventy female Sprague-Dawley Rats
(Charles River Laboratories, Wilmington, MA), aged 21 to 22 days, were housed in the
testing facility for at least 5 days before the beginning the assay procedure. Throughout the
procedure, the animal room was cUmate controlled at 18 to 26°C, 30 to 70% relative
humidity, and 12 lir. artificial light/12 hr. dark. All animals were fed Certified Rodent Chow
(Harlan Teklad, Madison WI) or the equivalent, and water, both ad libitum. Animal
procedures were performed at Calvert Preclinical Services, Inc. (Olyphant, PA).
Recombinant FSH. was expressed in CHO cells, purified by standard techniques and
glycoPEGylated with PE(j (1 kDa). The rats were divided into seven test groups, with ten
animals per group. On days -1 and 0, animals of all groups were subcutaneously injected
with 20 I.U. of human chorionic gonadotropin (HCG) in 0.5 inl of 0.9 % NaCl. On days 1, 2
and 3, the control animals were subcutaneously injected with a dose of 0.5 ml containing 20
I.U. HCG in 0.9% NaCl, while in the other groups, the HCG dose was augmented with either
rFSH or rFSH-SA-PEG (1 kDa) at either 0.14 ^g, 0.4 ^g or 1.2 pg per dose. On day 4, the
animals were euthanized biy CO2 inlialation. The ovaries were removed, trimmed and
weighted. The average o\'ary weight was determined for each group.
Figure 171 presents the average ovary weight of the test groups on day 4. The group •
receiving HCG alone (control) or the low dose (0.14 pg) of either rFSH or rFSH-SA-PEG (I
kDa) had ovary weights that were roughly equivalent. The groups receiving the medium (0
(ig) or high (1.2 pg) doses of rFSH or rFSH-SA-PEG (1 kDa) had ovary weights roughly
twice that of the control group. At the medium dose (0.4 pg), the glycoPEGylated rFSH had
roughly the same in vivo activity (as determined by ovary weight) as the unPEGyiated rFSi
At the high dose (1.2 [ag), the glycoPEGylated rFSH had somewhat higher in vivo activity
than the unPEGylated rFSH.
G-CSF
28. GlvcoPEGvIation of G-CSF produced m CHQ cells
Preparation of Asialo-Granulocyte-Colony Stimulation Factor (G-CSF). G-CSF
produced in CHO cells is dissolved at 2.5 mg/noL in 50 mM Tris 50 mM Tris-HCI pH 7.4,
0.15 M NaCl, 5 mM CaCb and concentrated to 500 \xL in a Centricon Plus 20 centrifugal
filter. The solution is incubated with 300 mU/mL Neuraminidase II (Vibrio cholerae) for 16
hours at 32 °C. To monitor the reaction a small aliquot of the reaction is diluted with the
appropriate buffer and a lEF gel performed. The reaction mixtxire is then added to prewashsd
N-(p-aminophenyl)oxamic acid-agarose conjugate (800 [aL/rnL reaction volume) and the
washed beads gently rotated for 24 hours at 4 °C. The mixtire is centrifuged at 10,000 rpm
and the supernatant was collected. The beads are washed 3 times with Tris-EDTA buffer,
once witli 0.4 mL Tris-EDTA buffer and once with 0.2 mL of the Tris-EDTA buffer and all.
supematants are pooled. The supernatant is dialyzed at 4 °C against 50 mM Tris —HCl pH
7.4, 1 M NaCl, 0.05% NiiNs and then twice more against 50 mM Tris -HCl pH 7.4, 1 M
NaCl, 0.05% NaNa- The dialyzed solution is then concentrated using a Centricon Plus 20
centrifugal filter and stored at -20 °C. The conditions for the lEF gel were run according to
the procedures and reagents provided by Invitrogen. Samples of native and desialylated G--
CSF are dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of G-CSF-(aIpha2,3)-SiaIyI-PEG. Desialylated G-CSF was dissolved
at 2.5 mg/mL in 50 mM Tris-HCI, 0.15 M NaCl, 0.05% NaJvfs, pH 7.2. The solution is
incubated with 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST3Gall at 32°C for 2 days.
To monitor the incorporation of sialic acid-PEG, a small aliquot of the reaction had CMP-
SA-PEG-fluoresceni ligmid added; tiie label incorporated into the peptide is separated from
the free label by gel filtration on a Toso Haas G3000SW analytical column using PBS bufler
(pH 7.1). The fluorescent label incorporation into the peptide is quantitated using an in-line
fluorescent detector. .After 2 days, the reaction mixture is purified using a Toso Haas
G3000SW prepai-ative column usmg PBS buffer (pH 7.1) and collecting fractions based ou
UV absorption. Tlie product of the reaction is analyzed using SDS-PAGE and lEF analysis
according to the procedures and reagents supplied by Invitrogen. Samples of native and
PEGylated G-CSF are dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of G-CSF-(alpha2,8)-Sialyl-PEG. G-CSF produced in CHO cells,
which contains an alpha2,3-sialylated 0-linked glycan, is dissolved at 2.5 mg/mL in 50 mlvl
Tris-HCl, 0.15 M NaCI, 0.05% NaNy, pH 7.2. The solution is incubated with 1 mM CMP-
sialic acid-PEG and 0.1 U/mL of CST-II at 32°C for 2 days. To monitor the incorporation of
sialic acid-PEG, a small aliquot of the reaction has CMP-SA-PEG-fluorescent ligand added;
the label incorporated into the peptide is separated from the free label by gel filtration on a
Toso Haas G3000SW analytical column usmg PBS buffer (pH 7.1). The fluorescent label
incorporation into the peptide is quantitated using an in-line fluorescent detector. After 2
days, the reaction mixture is purified using a Toso Haas G3000SW preparative column using,
PBS buffer (pH 7.1) and collecting fractions based on UV absorption. The product of the
reaction is analyzed using SDS-PAGE and lEF analysis according to the procedures and
reagents supplied by Invitrogen. Samples of native and PEGylated G-CSF are dialyzed
against water and analyzed by MALDI-TOF MS.
Preparation of G-CSF-(alpha2,6)-Sialyl-PEG. G-CSF, containing only 0-lmked
GalNAc, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaNj, pH 7.2.
The solution is incubated with 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST6GalNAcI or
II at 32°C for 2 days. To monitor the incorporation of sialic acid-PEG, a small aliquot of the
reaction has CMP-SA-PEG-fluorescent ligand added; the label incorporated into the peptide
is sepai-ated from the free label by gel filtration on a Toso Haas G3000SW anal3^ical column
using PBS buffer (pH 7.1). The fluorescent label incorporation into the peptide is quantitated
using an in-line fluorescent detector. After 2 days, the reaction mixture is purified uskig a
Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and collecting fractions
based on UV absorption. The product of the reaction is analyzed using SDS-PAGE and lEF
analysis according to the procedures and reagents supplied by Invitrogen. Samples of native-
and PEGylated G-CSF are d.ialyzed against water and analyzed by MALDI-TOF MS.
G-CSF produced in CHO cells was treated with Artlu'obacter sialidjise and was then
purified by size exclusion on Superdex75 and w^as treated with ST3GaIl or ST3 Gal2 and
then with CMI'-SA-PEG 20Kda. The resulting molecule was purified by ion exchange and
gel filtration and analysis by SDS PAGE demonstrated that the PEGylation was complete.
This is tlie first demonstration of glycoPEGylation of an 0-linked glycan.
Glucocerebrosidase
29. Glucocerebrosidase-mamiose-6-phosphate produced in CHO cells
This example sets forth the procedure to glycoconjugate mannose-6-phosphate to a
peptide produced in CHO cells such as glucocerebrosidase.
Preparation of asialo-glucoceramidase. Glucocerebrosidase produced in CHO cells
is dissolved at 2.5 rag/mL. in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, and is
incubated with 300 mU/mL sialidase-agarose conjugate for 16 hours at 32 °C. To monitor
the reaction a small aliquot of the reaction is diluted with the appropriate buffer and a lEF gel
and SDS-PAGE performed according to Invitrogen procedures. The mixture is centrifuged at
10,000 rpm and the supernatant is collected. The beads are washed 3 times with Tris-EDTA
buffer, once with 0.4 mL Tris-EDTA buffer, and once with 0.2 mL of the Tris-EDTA buffer.
All supematants are pooled. The supernatant is dialyzed at 4 "C against 50 mM Tris-HCl pH
7.4, 1 M NaCl, 0.05% NaNa and then twice more against 50 niM Tris-HCl pH 7.4, 1 M
NaCl, 0.05% NaN3. The dialyzed solution is then concentrated using a Centricon Plus 20
centrifiigal filter. The product of the reaction is analyzed using SDS-PAGE and lEF analysis
according to the procedures and reagents supphed by Invitrogen. Samples are dialyzed
against water and analyzed by MALDI-TOF MS.
Preparation of GIucocerebrosidase-SA-Iinker-Mannose-6-phosphate (proccdui e
1). Asialo-glucocerebrosidasefrom above is dissolved at 2.5 mg/mL in 50 mM Tris-HCl,
0.15 M NaCl, 0.05% NaN:i, pH 7.2. The solution is incubated with 1 mM CMP-sialic acid-
linker-Man-6-phosphate and 0.1 U/mJ. of ST3Gal3 at 32°C for 2 days. To monitor the
incorporation of sialic acid-linker-Man-6-phosphate, a small aliquot of the reaction had CM.P-
SA-PEG-fluorescent ligand added; the label incorporated into the peptide is separated trom
the free label by gel filtration on a Toso Haas TSK-Gel-3000 analytical column using PBS
buffer (pH 7.1). The fluorescent label incorporation into the peptide is quantitated using an
in-line fluorescent detector. When the reaction is complete, the reaction rnixtui-e is purifieJ
using a Toso Haas TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) and
collecting fractions based on UV absorption. The product of tlie reaction is analyzed using
SDS-PAGE and lEF analysis according to the procedures and reagents supplied by
Invitrogen. Samples are dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of Glucocerebrosidase-SA-linker-Mannose-6-phosphate (procedure
2). Glucocerebrosidase, produced in CHO but incompletely sialylated, is dissolved at 2.5
mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaNs, pH 7.2. The solution is incubated
with 1 mM CMP-sialic acid-linker-Man-6-phosphate and 0.1 U/mL of ST3Gal3 at 32''C for 2
days. To monitor the incorporation of siaUc acid-linker-Man-6-phosphate, a small aliquot of
the reaction had CMP-SA-PEG-fluorescent ligand added; the label incorporated into the
peptide is separated from the free label by gel filtration on a Toso Haas TSK-Gel-3000
analytical column using PBS buffer (pH 7.1). The fluorescent label incorporation into the
peptide is quantitated using an in-line fluorescent detector. When the reaction is complete,
tlie reaction mixture is purified using a Toso Haas TSK-Gel-3000 preparative column using
PBS buffer (pH 7.1) and collecting fractions based on UV absorption. The product of the
reaction is analyzed using SDS-PAGE and lEF analysis according to the procedures and
reagents supplied by hivitrogen. Samples are dialyzed agJiinst water and analyzed by
MALDI-TOF MS.
30. Glucocerebrosidase-transferrin
This example sets forth the procedures for the giycoconjugation of proteins, and in
particular, transferrin is glycoconjugated to glucocerebrosidase. The GIcNAc-ASN structures
are created on glucoceraminidase, and Transferrin-SA-Linker-Gal-UDP is conjugated to
GNDF GlcNAc-ASN structures using galactosyltransfera;5e.
Preparation of GlcNAc-glucocerebrosidase (Cerczyme''^'^. Cerezyme^'*'^
(glucocerebrosidase) produced in CHO cells is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM
Tris-HCl pH 7.4, 0.15 M NaCl, and is incubated with 300 mU/mL Endo-H-agarose conjugate
for 16 hours at 32 °C. To monitor the reaction a small aliquot of the reaction is diluted with
tlie appropriate buffei and a lEF gel and SDS-PAGE performed according to Invitrogen
procedures. The mixture is centrifuged at 10,000 rpm and the supernatant is collected. I'he
beads are washed 3 times with Tris-EDTA buffer, once witli 0.4 mL Tris-EDTA buffer Jmd
cnce with 0.2 niL of the Tris-EDTA buffer and all supematants are pooled. The supernatant
is dialyzed at 4 °C against 50 niM Tris -FICI pH 7.4, 1 M NaCI, 0.05% NaNs and tlien twice
more against 50 mM Tris -HCl pH 7.4, 1 MNaCl, 0.05% NaNs- The dialyzed solution is
then concentrated using a Centricon Plus 20 centrifugal filter. The product of the reaction is
analyzed using SDS-PAGE and lEF analysis according to the procedures and reagents
supplied by Invitrogen. Samples are dialyzed against water and analyzed by MALDI-TOF
MS.
Preparation of Transferrin-SA-Linker-Gal-glucocerebrosidase. Transferrin-SA-
Linker-Gal-UDP from above is dissolved at 2.5 mg/niL in 50 mM Tris-HCl, 0.15 M NaCl, 5
mM MnCb, 0.05% NaNs, pH 7.2. The solution is incubated with 2.5 mg/mL GlcNAc-
glucocerebrosidaseand 0.1 U/mL of galactosyltransferase at 32°C for 2 days. To monitor tlie
incorporation of glucocerebrosidase, the peptide is separated by gel filtration on a Toso Haas
G3000SW analytical column using PBS buffer (pH 7.1) and the product detected by UV
absorption. The reaction mixture is then purified using a Toso Haas G3000SW preparative
column using PBS buffer (pH 7.1) collecting Inactions based on UV absorption. The produci;
of the reaction is analyzed using SDS-PAGE and lEF analysis according to the procedures
and reagents supplied by Invitrogen. Samples are dialyzed against water and analyzed by
MALDI-TOF MS.
GM-CSF
31. Generation and PEGvlation of GlcNAc-ASN Structures: GM-CSF
produced in Saccharomyces
This example sets forth the preparation of Tissue-type Activator with PEGylated
GlcNAc-Asn structures.
Recombinant GM-CSF expressed in yeast is expected to contain 2 N-linked and 2 O-
linked glycans. The N-lirtked glycans should be of the branched mannan type. Tlus
recombinant glycoprotein is treated with an endoglycosidase from the group consisting of
endoglycosidase H, endoglycosidase-Fl, endoglycosidase-F2, endoglycosidase-F3,
endoglycosidase-M either alone or in combination with mannosidases I, II and III to generate
GlcNAc nubs on the asparagine (Asn) residues on the peptide/protein backbone.
The GIcNAc-Asn stmctures on the peptide/protein backbone is then be modified will;
galactose or galactose-PEG using UDP-galactose or UDP-galactose-6-PEG, respectiveh., anci
a galactosyltransferase such as GalTl. In one case the galactose-PEG is tlie tenninal residue
In the second case the galactose is further modified with SA-PEG using a CMP-SA-PEG
donor and a sialyltransferase such as ST3GalIII. In another embodiment the GIcNAc-Asn
structures on the peptide/protem backbone can be galactosylated and sialylated as described
above, and then further sialylated using CMP-SA-PEG and an a2,8-sialyltranferase such as
the enzyme encoded by the Campylobacter jejuni cst-II gene.
Herceptin'^^
32. Glycoconjugation of mitliramycin to Herceptin'^'^
This example sets Ibrth the procedures to glycoconjugate a small molecule, such as
mithramycin to Fc region glycans of an antibody molecule produced in mammalian cells.
Here, the antibody HerceptinTw is used, but one of skill in the art will appreciate that the
method can be used with many other antibodies.
Preparation of Herceptin''''^-Gal-linker-mithraraycin. Herceptin''"'^ is dissolved at
2.5 mg/mL in 50 mM Tris-HCI, 0.15 M NaCI, 5 mM MnClz, 0.05% NaNa, pH 7.2. The
solution is incubated with 1 mM UDP-galactose-linker-mithramycin and 0.1 U/mL of
galactosyltransferase at 32°C for 2 days to introduce the mithramycin in the Fc region
glycans. To monitor the incorporation of galactose, a small aliquot of the reaction has C-
galactose-UDP ligand added; the label incorporated into the peptide is separated from the free
label by gel filtration on a Toso Haas G3000SW analytical column using PBS buffer (pH
7.1). The radioactive label incorporation into the peptide is quantitated using an in-line
radiation detector.
When the reaction is complete, the reaction mixture is purified using a Toso Haas
TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) aiid collecting fractions based
on UV absorption. The fractions containing product are combined, concentrated, buffer
exchanged and then freeze-dried. The product of the reaction is analyzed using SDS-PAGE
and lEF analysis according to the procedures and reagents supplied by Invitrogen. Samples
are dialyzed against water and analyzed by MALDI-TOF MS.
Interferon a and Interferon p
33. GlycoPEGvlation of Proteins expressed in Mammalian or Insect Systems:
EPO, Interferon a and Interferon 6
This example sets forth the preparation of PEGylated peptides that are expressed in
mammalian and insect systems.
Preparation of acceptor from mammalian expression systems. The peptides to be
glycoPEGylated using CMP-siaUc acid PEG need to have giycans terminating in galactose.
Most peptides from mammalian expression systems will have terminal sialic acid that first
needs to be removed.
Sialidase digestion. The peptide is desialylated using a sialidase. A typical
procedure involves incubating a 1 mg/mL solution of the peptide in Tris-buffered saline, pH
7.2, with 5 mM CaC^ added, with 0.2 U/mL immobilized sialidase from Vibrio cholera
(Calbiochem) at 32°C for 2.4 hours. Microbial growth can be halted either by sterile filtration
or the inclusion of 0.02% sodium azide. The resin is then removed by centrifiigation or
filtration, and then washed to recover entrapped peptide. At this point, EDTA may be added
to the solution to inhibit any sialidase that has leached from the resin.
Preparation from insect expression systems. EPO, interferon-alpha, and
interferon-beta may also be expressed in non-mammalian systems such as yeast, plants, or
insect cells. The peptides to be glycoPEGylated using CMP-sialic acid PEG need to have
giycans terminating in galactose. The majority of the N-glycans on peptides expressed in
insect cells, for example, aie the trimannosyl core. These giycans are first built out to
giycans terminating in galactose before they are acceptors for sialyltransferase.
Building acceptor giycans from trimannosyl core. Peptide (1 mg/mL) in Tris-
buffered saline, pH 7.2, containing 5 mM MnClz, 5 mM UDP-glcNAc, 0.05 U/mL
GLCNACT I, 0.05 U/mL GLCNACT II, is incubated at 32°C for 24 hours or until the
reaction is substantially complete. Microbial growth can be halted either by sterile filtration
or the inclusion of 0.02% sodium azide. After buffer exchange to remove UDP and other
small molecules, UDP-galactose and MnCl2 are each added to 5 mM, galactosyltransferase h
added lo 0.05 U/ml., and is incubated at 32°C for 24H or until the reaction is substantially
complete. Microbial growth can be halted either by sterile filtration or the inclusion of 0.02/:
sodium azide. The peptides are tlien ready for glycoPEGylation.
Building O-linked glycans. A similar strategy may be employed for interferon alpha
to produce enzymatically the desired 0-glycan Gal-GalNAc. If necessary, GalNAc linked to
serine or threonine can be added to the peptide using appropriate peptide GalNAc
transferases (e.g. GalNAc Tl, GalNAc T2, T3, T4, etc. ) and UDP-GalNAc. Also, if needed,
galactose can be added using galactosyltransferase and UDP-galactose.
GlycoPEGylation using sialyltransferase. The glycopeptides (1 mg/mL) bearing
terminal galactose in Tris buffered saline + 0.02% sodium azide are incubated with CMP-Si\-
PEG (0.75 nM) and 0.4 U/mL sialyltransferase (ST3Gal3 or ST3Gal4 for N-glycans on EPO
and interferon beta; ST3Gal4, or ST3Gall for 0-glycans on interferon alpha) at 32''C for 24
hours. Other transferases that may work include the 2,6 sialyltransferase from
Photobacterium damsella. The acceptor peptide concentration is most preferably in the range
of 0.1 mg/mL up to the solubility limit of the peptide. The concentration of CMP-SA-PEG
should be sufficient for there to be excess over the available sites, but not so high as to cause;
peptide solubility problems due to the PEG, and may range fiom 50 fxM up to 5 mM, and the
temperature may range from 2°C up to 40°C. The time required for complete reaction will
depend on the temperature, the relative amounts of enzyme to acceptor substrate, the donor
substrate concentration, aiid the pH.
34. GlycoPEGylation of Interferon a produced in CHO cells
Preparation of Asialo-Interferon a. Interferon alpha produced from CHO cells is
dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl.
and concentrated to 500 \iL in a Centricon Plus 20 centrifugal filter. The solution is
incubated with 300 mU/mL Neuraminidase II {Vibrio cholerae) for 16 hours at 32 °C. To
monitor the reaction a small aliquot of the reacfion is diluted with the appropriate buffer arnl i
lEF gel performed. The reaction mixture is then added to prewashed N-(p-
aminophenyl)oxamic acid-agarose conjugate (800 jiL/mL reaction volume) and the washed
beads gently rotated for 24 hours at 4 °C. The mixture is centrifuged at 10,000 rpm and the
supernatant was collected. The beads are washed 3 times with Tris-EDTA buffer, once wiih
0.4 mL Tris-EDTA buffer \md once with 0.2 niL of the Tris-EDTA buffer :uid all
supematants were pooled. The supernatant is dialyzed at 4 °C against 50 mM Tris -HCl pi 1
7.4, 1 M NaCl, 0.05% NaN, and then twice more against 50 mM Tris -HCl pH 7.4, 1 M
NaCl, 0.05% NaNs. The dialyzed solution is then concentrated using a Centricon Plus 20
centrifugal filter and stored at -20 °C. The conditions for the lEF gel are run according to the
procedures and reagents provided by Invitrogen. Samples of native and desialylated G-CSF
are dialyzed against water and analyzed by MALDI-TOFIVIS.
Preparation of Interferon-alpha-(alpha2,3)-SiaIyl-PEG. Desialylated mterferon-
alpha is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaNa, pH 7.2.
Tlie solution is incubated with 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST3GaIl at
32°C for 2 days. To monitor the mcorporation of sialic acid-PEG, a small aliquot of the
reaction had CMP-SA-PE(j-fluorescent ligand added; the label incorporated into the peptide
is separated from the free label by gel filtration on a Toso Haas G3000SW analytical column
using PBS buffer (pH 7.1). The fluorescent label incorporation mto the peptide is quantitated
using an in-line fluorescent detector. After 2 days, the reaction mixture is purified using a
Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and collecting fractions
based on UV absorption. The product of tlie reaction is analyzed using SDS-PAGE and lEF
analysis according to the procedures and reagents supplied by Invitrogen. Samples of native
and desialylated Interferon-alpha are dialyzed against water and analyzed by MALDI-TOF
MS.
Preparation of Interferon-alpha-(aIpha2,8)-SiaIyl-PEG. Interferon-alpha
produced in CHO, which contains an alpha2,3-sialylated O-linked glycan, is dissolved at 2.5
mg/mL in 50 mM Tris-HCl, 0.15 M NaCi, 0.05% NaNs, pH 7.2. The solution is incubated
with I loM CiVlP-siaJic acid-PEG and 0.1 U/mL of CST-H at 32''C for 2 days. To monitor the
incorporation of sialic acid-1'EG, a small aliquot of the reaction has CMP-SA-PEG-
fluorescent ligand added; the label incorporated into the peptide is separated from the free
label by gel filtration on a Toso Haas G3000SW analytical column using PBS buffer (pH
7.1). The fluorescent label incorporation into the peptide is quantitated using an m-hne
fluorescent detector. After 2 days, tlie reaction mixture is purified using a Toso Haas
G3000SW preparative colunm using PBS buffer (pH 7.1) and collecting fractions based on
UV absorption. 71ie product of the reaction is analyzed using SDS-PAGE aiid lEF analysis
according to die procedures cind reagents supplied by Invitrogen. Samples of native and
FEGylated interferon-alpha are dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of Interferon-alpha-(alpha2,6)-Sialyl-PEG. Interferon-alpha,
containing only O-linked GalNAc, was dissolved at 2.5 mg/mL in 50 mM Tris-HCI, 0.15 M
NaCl, 0.05% NaNs, pH 7.2. The solution is incubated with 1 raM CMP-sialic acid-PEG and
0.1 U/mL of ST6GaINAcI or II at 32°C for 2 days. To monitor the incorporation of sialic
acid-PEG, a small aliquot of the reaction had CMP-SA-PEG-fluorescent ligand added; the
label incorporated into the peptide is separated from the free label by gel filtration on a Tosc'
Haas G3000SW analytical column using PBS buffer (pH 7.1). The fluorescent label
incorporation into the peptide is quantitated using an in-line fluorescent detector. After 2
days, the reaction mixture is purified using a Toso Haas G3000SW preparative column using
PBS buffer (pH 7.1) and collecting fractions based on UV absorption. The product of the
reaction is analyzed using SDS-PAGE and lEF analysis according to the procedures and
reagents supplied by Invitrogen. Samples of native and PEGylated interferon-alpha are
dialyzed against water and analyzed by MALDI-TOF MS.
35. GlvcoPEGylation of Interferon-B-la with PEG (10 kPa) and PEG r20
kPa)
This example illustrates a procedure PEGylate Interferon-P with either PEG (10 kDa)
or PEG (20 kDa).
Briefly, Interferon-p-1 a (INF-p) was obtained from Biogen (Avonex'r'^). The IFN-P
was first purified by Supeidex-75 chromatography. The IFN-p was then desialylated with
Vibrio cholerae sialidase. The INF-p was then PEGylated with SA-PEG (10 kDa) or SA-
PEG (20 kDa) and purified with Superdex-200 chromatography.
Superdex-75 chromatography purification. ESTF-P (150 pg) was applied to a
Superdex-75 column (Amersham Biosciences, Arlington Heights, IL) and eluted with PBS
witli 0.5 M NaCl, 0.02 Tween-20, 20 mM histidine and 10% glycerol. The eluant was
monitored for absorbance at 280 nm (Figure 172A and 172B) ;and fractions were collected.
Peaks 4 and 5 were pooled, concentrated in an Amicon Ultra 15 spin filter (Millipore,
Billerica, MA), and the buffer was exchanged to TBS with 5 mM CaCls, 0.02% T\vcen-20,
20 mM histidine and 10% glycerol.
Sialidase Reaction. The INF-P was then desialydated with Vibrio cholera salidase
(70 mU/niL CALBIOCHEMcg), EMD Biosciences. Inc., San Diego, CA) on agaiose in TBS
with 5 mM CaCh, 0.02% Tween-20, 20 mM histidine and 10% glycerol. The reaction was
carried out at 32°C for 18 hours. The INF-p was removed from the agarose with a 0.22 |am
Spin-X'T'^ filter (Coming Technology, Inc., Norcross, GA). Figure 173A depicts the MALDI
analysis of glycans released from native INF-p. The native INF-p has many glycoforms
containing terminal sialic acid moieties. Figure 173B depicts the MALDI analysis of glycans
released from desialylated INF-p. The desialylated INF-P has primarily one glycoform
which is bi-antennary with terminal galactose moieties.
Lectin Dot-Blot Analysis of Sialylation. Samples of the DSfF-P from the desialidase
reaction were dot-blotted onto nitrocellulose and then blocked with Tris buffered saline
(TBS.: 0.05M Tris, 0.15M NaCl, pH 7.5) and DIG kit (glycan differentiation kit available
from Roche #1 210 238) blocking buffer. Some of the blots were incubated with Maackia
amurensis agglutinin (MAA) labeled with digoxogenin (DIG) (Roche Applied Science,
Indianapolis, IL) to detect a2,3-sialylation of INF-p. Tliese blots were washed with TBS
then incubated with anti-digitonin antibody labeled with alkaline phosphatase, then washed
again with TBS and developed withNBT/X-phosphate solution, wherein NBT is 4-nitro blue
tetrazolium chloride and X-phosphate is 5-bromo-4-chloro—3indoyl phosphate. The left side
of Figure 174 depicts the results of the MAA blot of INF-P after the desialylation reaction.
The ENF-P is partially disialylated, as indicated by the decrease in dot development as
compared to native INF-p in the desialylated samples.
Other blots were incubated with Erthrina cristagalli lectin (ECL) labeled with biotin
(Vector Laboratories, Burlingame, CA) to detect exposed galactose residues orl INF-p. After
incubation with 2.5 |J.g/ml ECL, the blots were washed in TBS and incubated with
streptavidin labeled with alkaline phosphatase. The blots were then washed again and
developed. The right side of Figure 174 depicts the ECL blot after development. The
increased intensity of the dot of desialylated INF-P as compai-ed to the native INF-P indicate
more exposed galactose mioieties and therefore extensive desialylation.
PEGylation of Desialylated INF-p with SA-PEG (10 kDa). Desialylated INF-P
(0.05 mg/ml) was PEGylated with ST3Gal3 (50 mU/ml) and CMP-SA-PEG (10 kDa) (25u
\xM) in an appropriate buffer of TBS + 5 mM CaCb, 0.02% Tween 20, 20 niM histidine, 10%
glycerol for 50 hours at 32 °C. Figure 175 depicts the SDS-PAGE analysis of the reaction
products showing PEGylated INF-p at approximately 98 kDa.
PEGylation of Desialylated INF-P with SA-PEG (20 kBa). Desialylated INF-p
(0.5 mg/ml) was PEGylated with ST3Gal3 (170 mU/ml) and CMP-SA-PEG (20 kDa) in an
appropriate buffer of TBS + 5 mM CaCl2, 0.02% Tween 20, 20 mM histidine, 10% glycerol
for 50 hours at 32°C. Figure 176 depicts the SDS-PAGE analysis the products of the
PEGylation reaction. Tlie PEGylated INF-P has many higher molecular weight bands not
found in the unmodified INF-P indicating extensive PEGylation.
Superdex-200 Purification of INF-p PEGylated with PEG (10 kDa). The products
of the PEGylation reaction were separated on a Superdex-200 column (Amersham
Biosciences, Arlington Heights, IL) in PBS v/ith 0.5 NaCI, 0.02 Tween-20, 20 mM histidme
and 10% glycerol at Iml/min and 30 cm/hr flow. The eluant was monitored for absorbance at
280 nm (Figure 177) and fractions were collected. Peaks 3 and 4 were pooled and
concentrated in an Amicon Ultra 15 spin filter.
Bioassay of INF-P PEGylated with PEG (10 kDa).
The test is inhibition of the proliferation of the lung carcinoma cell line, A549. The
A549 cell line are lung carcinoma adherent cells growing in RPMI + 10% FBS at 37°C 5%
CO2. They can be obtained from ATCC # CCL-185. Wash the cells with 10 ml of PBS and
remove the PBS. Add 5 ml of trypsin, incubate for 5 minutes at room temperature or 2
minutes at 37°C. When the cells are detached resuspend into 25 ml of media and count the
cells. Dilute the cells at a concentration of 10000 cells/ml and add 200 ul / well (96 wells
plate). Incubate for 4 hours at 37°C 5% CO2. Prepare 1 ml of IFN B at a concentration of
0.1 ug/'ml. Filter it under the hood with a 0.2 um filter. Add 100 ul per well (8 replicates = 1
lane). Incubate for 3 days (do not let the cells go to confluence). Remove 200 ul of media
(only lOOuI per well left). Add 25 |al of MTT (Sigma) (5 mg/ml filtered 0.22^im). hicubate
for 4 hours at 37°C and 5% CO2 . Aspirate the media gently and add 100 ^1 of a mixture of
isopropanol (100 ml and 6N HCl. Aspirate up and down to homogenize tlie crystal violet.
Read CD 570nm (remove die background at 630 or 690 nm).
Figure 178 depicts the results of the bioassay of the pcciks containing INF-P
PEGylated with PEG (10 l'
molecule by introducing PEG molecules to the Fc region glycans. Here Remicade'^'^, a TNV-
R;lgG Fc region fusion protein, is the exemplary peptide.
Preparation of RemicadeTW-Gal-PEG (10 kDa). Ren:ricade™ is dissolved at 2.5
mg/ml. in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCb, 0.05% NaNs. pH 7.2. The solution
is incubated witla 1 mM UDP-galactose-PEG (10 kDa) and 0.1 U/raL of galactosyltransferase
at 32°C for 2 days to introduce the PEG m tlie Fc region glycans. To monitor the
incorporation of galactose, a small aliquot of the reaction has "C-galactose-UDP ligand
added; the label incorporated into the peptide is separated from the free label by gel liltratioii
on a Toso Haas G3000SW ajialytical column using PBS buffer (^H 7.1). The radioactive
label incorporation into the peptide is quantitated using an in-line radiation detector.
Mien the reaction is complete, the reaction mixture is purified usmg a Toso Haas
TSK-Gel-3000 preparative colunm using PBS buffer (pH 7.1) and collecting fractions based
on UV absorption. The fractions containing product are combined, concentrated, buffer
exchanged and then fireeze-dried. The product of the reaction is analyzed using SDS-PAGE
and lEF analysis according to the procedures and reagents supplied by Invitrogen. Samples
are dialyzed against water and analyzed by MALDI-TOF MS.
Rituxan"^^
37. Glycoconjugation of geldanamvcin to Rituxan'^'^
This example sets forth the glycoconjugation of a small molecule, such as
geldanamycin, to tlie Fc region glyeans of an antibody produced in CHO cells, such as
Rituxan'T'*^. Here, the antibody Rituxan^M is used, but one of skill in the art will appreciate
that the method can be used with many other antibodies.
Preparation of Rituxan"*-Gal-linker-geldanamycin. Rituxan''^'^ is dissolved at 2.5
mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl2, 0.05% NaN3, pH 7.2. The solution
is incubated with 1 mM UDP-galactose-linker-geldanamycin and 0.1 U/mL of
galactosyltransferase at 32"C for 2 days to introduce the geldanamycin in the Fc region
glycans. To monitor the incorporation of galactose, a small aliquot of the reaction has '''C-
galactose-UDP ligand added; the label incorporated into the peptide is separated from the free
label by gel filtration on a 'J^oso Haas G3000SW anal5^ical column using PBS buffer (pH
7.1). The radioactive label incorporation into the peptide is quantitated using an in-line
radiation detector.
When the reaction is complete, the reaction mixture is purified using a Toso Haas
TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) and collecting fractions based
on UV absorption. The fractions containing product are combined, concentrated, buffer
exchanged and then freeze-dried. The product of the reaction is analyzed using SDS-PAGE
and lEF analysis according to the procedures and reagents supplied by Invitrogen. Samples
are dialyzed against water and analyzed by MALDI-TOF MS.
Rnase
38. Remodeling high mannose N-glycans to hybrid and complex N-glycans:
Bovine pancreatic RNase
This example sets forth the preparation of bovine pancreas RNase with hybrid or
complex N-glycans. The high mtimiose N-linked glycans of the RNase are enzymatically
digested and elaborated to create hybrid N-linked glycans. Additionally, the high mannose
N-linked glycans of the RNase are enzymatically digested and elaborated to create complex
N-linked glycans.
High mannose structures of A'-Iinked oligosaccharides in glycopeptides can be
modified to hybrid or complex forms using the combination of a-mannosidases and
glycosyltransferases. This example summarizes the results in such efforts using a simple N-
Glycan as a model substrate.
Ribonuclease B (RNaseB) purified from bovine pancreas (Sigma) is a glycopeptide
consisting of 124 amino acid residues. It has a single potential TV-glycosylation site modified
with high marmose structures. Due to its simplicity and low molecular weight (13.7 kDa to
15.5 kDa), ribonuclease B is a good candidate to demonstrate the feasibility of the A^-Glycan
remodeling from high mannose structures to hybrid or complex A'-linked oligosaccharides.
The MALDI-TOF spectrum of RNaseB (Figure 180 A) and HPLC profile for the
oligosaccharides cleaved from RNaseB by N-Glycanase (Figure 180B) indicated that, other
than a small portion of the non-modified peptide, the majority of//-glycosylation sites of the
peptide are modified with high mannose oligosaccharides consisting of 5 to 9 mannose
residues.
Conversion of high mannose N-GIycans to hybrid N-Glycans. High mannose A'^-
Glycans were converted to hybrid 7^-Glycans using the combination of al,2-mannosidase,
GlcNAcT-1 (P-l,2-/-acety.l glucosamuiyl transferase), GalT-1 (pi,4-galactosyltransfease) and
a2,3-sialyltransf6rase /or a2,6-sialyltransferase as shown in Figure 181.
As an example, high mannose structures in RNaseB were successfiilly converted to
hybrid structures.
Man5GlcNAc2-R was obtained from Man5.9GlcNAc2-R catalyzed by a single al,2-
mannosidase cloned from Trichoderma reesei (Figure 182). RNase B (1 g, about 67 }.imol)
was incubated at 30°C for 45 iir with 15 mU of the recombinant T. reesei al,2-mannosidase
in MES buffer (50 mM, pH 6.5) in a total volume of 10 mL. Man6-9GlcNAc2-protein
structures have been succes;^fully converted to Man5GlcNAc2-protein with iaigh efficiency l:'y
the recombinant mannosidase.
Alternately, Man5GlcNAc2-R was obtained from Man5.9GlcNAc2-R catalyzed by a
single al,2-mannosidase purified from Aspergillus saitoi (Figure 183). RNase B (40 ng,
about 2.7 nmol) was incubated at 37°C for 42.5 hr with 25 M.U of the commercial A. saitoi
al,2-mannosidase (Glyko or CalBioChem) in NaOAC buffer (100 mM, pH 5.0) in a total
volume of 20 \il. Man6-9CjlcNAc2-protein structures were successfully converted to
Man5GlcNAc2-protein by the commercially available maimosidase. However, a new peak
corresponding to the GlcNAc-protein appears in the spectrum, indicating the possible
contamination of endoglycosidase H in the preparation. Although several mammalian alpha-
mannosidases were required to achieve this step, the fungal al,2-mannosidase was very
efficient to remove all al,2-linked mannose residues.
GlcNAcT-I then added a GlcNAc residue to the Man5GlcNAc2-R (Figure 184). The
reaction mixture after the T. reesei al,2-maimosidase reaction containing RNase B (600 (j.g,
about 40 nmol) was incubated with non-purified recombinant GlcNAcT-I (34 mU) in MES
buffer (50 mM, pH 6.5) containing MnCb (20 mJM) and UDP-GlcNAc (5 mM) in a total
volume of 400 \i\. at 37°C' for 42 hr. A GlcNAc residue was quantitatively added to
Man5GIcNAc2-protein by the recombinant GlcNAcT-I.
A Gal residue was then added using GalT 1 (Figiore 185). The reaction mixture after
the GnT-I reaction containing RNase B (120 p.g, about 8 nmol) was incubated at 37°C for 20
hr with 3.3 mU of the recombinant GalT-1 in Tris-HCl buffer (100 mM, pH 7.3) containing
UDP-Gal (7.5 mM) and MnCb (20 mM) in a total volume of 100 \i\. A Gal residue was
added to about 98% of the GlcNAc-Man5GlcNAc2-protein by the recombinant GalT 1.
The next step was the addition of a siaUc acid using Ein a2,3-sialyltransferase or an
a2,6-sialyltransferase (Figure 186). As an example, ST3Gal III, an a2,3-sialyltransferase was
used. The reaction mixture after the GalT-1 reaction containing RNase B (13 (ig, about 0.87
nmol) was incubated at 37°C for 16 hr with 8.9 mU of recombinant ST3Gal 111 in Tris-HCl
buffer (100 mM, pH 7.3) containing CMP-Sialic acid (5 mM) and MnClz (20 mM) in a total
volume of 20 |j,l. A sialic acid residue was added to about 90% of the Gal-GlcNAc-
Man 5 GlcNAcz-protein by recombinant ST3Gal III using CMP-SA as the donor. The yield
can be ftarther improved by adjusting the reaction conditions.
For convenience, no purification or dialysis step was required after each reaction
described above. More interesting, GalT 1 and ST3Gal IH can be combined in a one-pot
reaction. Similar yields were obtained as compared with the separate reactions. The reaction
mixture after tlie GlcNAcT-I reaction containing RNase B (60 jag, about 4 imiol) was
incubated at 37°C for 20 hr with 1.7 mU of recombinant GalT 1, 9.8 mU of recombinant
ST3Gal III in Tris-HCl buffer (100 mM, pH 7.3) containing IJDP-Gal (7.5 mM), CMP-sialic
acid (5 mM) and MnCb (20 mM) in a total volume of 60 fil.
As shown in Figuie 187, SA-PEG (10 kDa) was successftiUy added to the RNaseB.
The reaction mixture after the GalT-1 reaction containing KN'ase B (6.7 (jg, about 0.45 nmol)
was dialyzed against H2O for 1 hour at room temperature and incubated at 37°C for 15.5
hours with 55 mil of the recombinant ST3Gal III in Tris-HCl buffer (50 mM, pH 7.3)
containing CMP-SA-PEG (10 kDa) (0.25 mM) and MnCh (20 mM) in a total volume of 20
[il. PEG-modified sialic acid residues were successfully added to the Gal-GlcNAc-
Man5GlcNAc2-peptide by the recombinant ST3Gal III. The yield can be further improved by
adjusting the reaction conditions.
Conversion of high mannose N-Glycans to complex N-GIycans. To achieve tliis
conversion, a GlcNAcp 1,2Man3GlcNAc2-peptide intermediate is obtained. As shown in
Figure 188, there are at legist four feasible routes to carry out the reaction from
Man5GlcNAc2-peptide to this intermediate:
Route I: The Man5GlcNAc2-peptide produced by the fungal al,2 mannosidase is a
substrate of GlcNAc transferase I (GlcNAcT-I, enzyme 2) which adds one GlcNAc. The
terminal a 1,3- and al,6-lirLk:ed mannose residues of GlcNAcMan5GlcNAc2-peptide is
removed by Golgi a-manriosidase II (Manll, enzyme 5). Tliis route is a part of the natural
pathway for the processing of A''-linked oligosaccharides carried out in higher organisms.
Route II: Two majinose residues are first removed by an a-mannosidase (enzyme 6),
then a GlcNAc is added b}' GlcNAcT-I (enzyme 2). Other than its natural acceptor
Man5(jlcNAc2-R, GlcNAcT-I can also recognize Man3GlcNAc2-R as its substrate and add
one GlcNAc to the mannose core structure to form GlcNAcMan3GlcNAc2-peptide.
Route III: The al,6-linked mannose is removed by an al,6-mannosidase, followed
by the addition of GlcNAc by GlcNAcT-I and removal of the terminal al,3-linked mannose
by an al,3-mannosidase. From the experimental data obtained, GlcNAcT-I can recognize
this MantGIcNAca-peptide as acceptor and add one GlcNAc residue to form
GlcNAcMan4GlcNAc2-peptide.
Route IV: Similar to Route III, aI,3-lLnked mannose is removed by an al ,3-
mannosidase, followed by GlcNAcT-I reaction. Then the terminal al,6-linked mannose can
be removed by an al,6-mannosidase.
After the function of GlcNAcT-I (responsible for the addition of the GlcNAc pi,2-
linked to the al,3-mannose on the mannose core) and GlcNAcT-II (responsible for the
addition of a second GlcNAc pi,2-linked to the al,6-mannose on the mannose core), the
GlcNAc2Man3GlcNAc2-peptide can be processed by GalT 1 and sialyltransferase to form bi-
antennary complex N- Glycans. Other GlcNAc transferases such as GlcNAcT-FV, GlcNAcT-
V, and/or GlcNAcT-VI (Figure 188 and Figure 189) can also glycosylate the
GlcNAc2Man3GlcNAc2-peptide. Additional glycosylation by the GalT 1 and
sialyltransferases will form multi-antennary complex N-glycans. The enzyme GlcNAcT-IIl
catalyzes the insertion of a bisecting GlcNAc, thus preventing the actions of Manll and
subsequent action of transferases GlcNAcT-II, GlcNAcT-FV and GlcNAcT-V.
Tissue-Type Plasminogen Activator (TPA)
39. Fucosyiation of TPA to create Sialvl Levels X
This example sets forth the preparation of Tissue Tissue-type Plasminogen Activator
(TPA) with N-linked sialyl Lewis X antigen.
Sialylation. TPA expressed in mammalian cells will often contain a majority of the
glycans terminating in sialic acid, but to ensure complete sialylation, it would be beneficial to
first perform an in vitro sialylation. TPA in a suitable buffer (most preferably between pH
5.5 and 9, for example Tris buffered saline, pH 7.2) is incubated with CMP sialic acid and
sialyltransferase for a time sufficient to convert any glycans lacking sialic acid to sialylated
species. Typical conditions would be 1 mg/mL TPA, 3 mM CMP sialic acid, 0.02 U/mL
ST3Gal3, 32°C for 24 hours. Microbial grow1:h can be halted either by sterile filtration or the
inclusion of 0.02% sodium azide. The TPA concentration is most preferably m the range 0.1
mg/niL up to the solubility limit of the peptide. The concentration of CMP-SA should be
sufficient for there to be excess over the available sites, and might range from 50 pM up to 50
mM, and the temperature from 2°C up to 40°C. The time required for complete reaction will
depend on the temperature, the relative amounts of enzyme to acceptor subsfrate, the donor
substrate concentration, and the pH. Other sialyltransferases that may be capable of adding
sialic acid in 2,3 linkage include ST3Gal4; microbial transferases could also be used.
Fucosylation. Typical conditions for flicosylation would be 1 mg/mL TPA, 3 mM
GDP-fiicose, 0.02 U/mL FTVI, 5 mM MnCh, 32°C for 24H in Tris buffered saline.
Microbial growth can be halted either by sterile filtration or the inclusion of 0.02% sodium
azide. The TPA concentration is most preferably in the range 0.1 mg/mL up to the solubility
limit of the peptide. The concentration of GDP-fiacose should be sufficient for there to be
excess over the available sites, and might range from 50 jxM up to 50 mM, and the
temperature from 2°C up to 40°C. The time required for complete reaction will depend on
the temperature, the relative amounts of enzyme to acceptor substrate, the donor substrate
concentration, and the pti. Other fiicdsylfransferases that may be capable of making sialyl
Lewis X include FTVII, FTV, FTIII, as well as microbial transferases could also be used.
40. Trimming of high mannose to tri-maimose core structure: Tissue-type
Plasminogen Activator produced in CHO
This example sets forth the preparation of Tissue-type Plasminogen Activator with a
trimarmose core by trimming back from a high mannose glycan.
Tissue-type plasminogen activator (TPA) is currently produced in Chinese Hamster
Ovary (CHO) cells and contains a low amount of high mannose N-linked oligosaccharide.
The mannoses can be trinmied down using a variety of the specific mannosidases. The first
step is to generate Man5GlcNAc2(FucO-l) from Man9GlcNAc2(FucO-l). This can be done
using mannosidase I. Then either GlcNAcTl (GlcNAc transferase I) is used to make
GlcNAclMan5GlcNAc2(FucO-I) or Maimosidase III is used to make Man3GlcNAc2(FucO-
1). From Man3GlcNAc2(FucO-l), GIcNAclMan3GlcNAc2(FucO-l) can be produced using
GlcNAcTl or from GIcNAclMan5GlcNAc2(FucO-l), GlcNAclMan3GlcNAc2(FucO-l) can
be produced using Mamiosidase II. GlcNAclMan3GlcNAc2(FucO-l) is then converted into
GlcNAc2Maj-i3GlcNAc2(FucO-l) using GlcNAcTransferase II (GlcNAcTII). The two
terminal GlcNAc residues are then galactosylated using GalTI and then sialylated with SA-
PEG using ST3GalIir.
Conversely, TPA can be produce in yeast or fungal systems. Similar processing
would be required for fungal derived material.
41. Generation and PEGvlation of GlcNAc-ASN structures: TPA produced in
Yeast
This example sets forth the preparation of PEGylated GlcNAc-Asn structures on a
peptide such as TPA expressed in yeast.
Yeast expression is expected to result in a TPA which contains a single N-linked
mannan-type structure. Tliis recombinant glycoprotein is first treated with endoglycosidase
H to generate GlcNAc structures on the asparagine (Asn) residues on the peptide.
The GlcNAc-Asn structures on the peptide/protein backbone are then be modified
with galactose or galactose-PEG using UDP-galactose or UDP-galactose-6-PEG,
respectively, and a galactosyltransferase such as GalTl. In one case, the galactose-PEG is
the termmal residue. In the second case, die galactose is further modified with SA-PEG
using a CMP-SA-PEG donor and a sialyltransferase such as ST3GalIII. In another
embodiment, the GlcNAc-Asn structures on the peptide/protein backbone may be
galactosylated and sialylated as described above, and then further sialylated using CMP-SA-
PEG and an a2,8-sialyltransferase such as the enzyme encoded by the Campylobacter jejuni
cst-II gene.
Transferrin
42. GlycoFEGylation of Transferrin
This example sets forth tlie preparation of asialotransferrin and its sialylation with
PEG-CMP-sialic acid.
Preparation of Asialo-transferrin. Human-derived holo-Transferrin, (10 mg) was
dissolved in 500 ^iL of 50 mM NaOAc, 5 mM CaCl2, pH 5.5. To this solution was added
500 mU Neuraminidase II {Vibrio cholerae) and the reaction mixture was shaken gently for
20.5 hours at 37 °C. The reaction mixture was added to the prewashed N-(p-
aminophenyl)oxamic acid-agarose conjugate (600 \iL) and the washed beads gently rotated
for 24 hours at 4 "C. The mixture was centrifiiged at 10,000 rpm and the supernatant was
collected. Thereactionmixture was adjusted to 5 mMEDTA by addition of 100 jiLofSO
mM EDTA to the washed beads, which were gently rotated for 20 hours at 4 °C. The
suspension was centrifiiged for 2 minutes at 10,000 rpm and the supernatant was collected.
The beads were washed 5 times with 0.35 nxL of 50 mM NaOAc, 5 mM CaCl2, 5 mM
EDTA, pH 5.5 and ail supematants were pooled. The enzyme solution was dialyzed twice at
4 °C into 15 mM Tris-HCl, I M NaCl, pH 7.4. 0.3 mL of the transferrin solution (3.3 mL
total) was removed and dialyzed twice against water. The remainder was dialyzed twice
more at 4 °C against phosj)hate buffered saline. The dialyzed solution was stored at —20 ° C.
Protein samples were analyzed by lEF Electrophoresis. Samples (9 |aL, 25 ^g) were diluted
with 16 |J.L Tris buffer and mixed with 25 jj.L of the sample loading buffer and applied to
Isoelectric Focusing Gels (pH 3-7). Gels were run and fixed using standard procedures. Gels
were stained with Colloidal Blue Stain.
Sialyl-PEGylation of asialo-Transferrin. Desialylated transferrin (250 ^j,g) and
CMP-sialic acid or CMP-SA-PEG (1 kDa or 10 kDa)(0.05 ^imol) were dissolved in 69 fiL 50
mM Tris-HCl, 0.15 M NaCI, 0.05% NaNs, pH 7.2 in 1.5 mL plastic tubes. The tubes were
vortexed briefly and 100 mU ST3Gal3 (90 p. L) were added (total volume 250 ^ L). The
tubes were vortexed again and mixed gently for 24 hours at 32 °C. The reactions were
stopped by freezing at -80 'C. Novex Tris-Glycine 8-16% 1 mm gels were used for SDS
PAGE analysis (Figure 190). Samples (25 pL, 25 pg) were mixed with 25 fiL of sample
loading buffer and 0.4 pL of p-mercaptoethanol and heated for 6 minutes at 85 °C. Gels
were run using standard conditions and stained with Colloidal Blue Stain. lEF gels were also
performed as described above Figure 191). Samples were also dialyzed against water
analyzed by MALDI-TOF.
Results. MALDI was also performed. Native transferrin (78729); asialotransfeirin
(78197); resialylated transferrin (79626/80703); with SA-PEG Ik (79037 (1); 80961 (2);
82535 (3); 84778 (4)); with SA-PEG 5k (90003 (2); 96117 (3); 96117 (4)); with SA-PEG 1 Ok
(100336(2); 111421 (3); 122510(4)).
43. Transfenin-GDNF
This example sets forth the procedures for the glycoconjugation of proteins, and ini
particular, transferrin is glycoconjugated to GDNF. Transferrin-SA-Linker-Gal-UDP is
prepared from transfenin. The galactose residue is removed from GNDF glycans, and
Transferrin-SA-Linker-Gal-UDP is conjugated to GNDF glycans using a
galactosyltransferase.
Preparation of agalacto-GDNF. GDNF produced in NSO cells (NSO murine
myeloma cells) is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M
NaCI, and is incubated with 300 mU/mL beta-galactosidase-agarose conjugate for 16 hours at
32°C. To monitor the reaction a small aliquot of the reaction is diluted with the appropriate
buffer and a lEF gel performed according to Invitrogen procedures. The mixture is
centrifiiged at 10,000 rpm and the supernatant is collected. The supernatant is dialyzed at 4
°C against 50 mM Tris --HC1 pH 7.4, 1 M NaCl, 0.05% NaNj and then twice more against 50
mM Tris -HCl pH 7.4, 1 M NaCl, 0.05% NaNa. The dialyzed solution is then concentrated
using a Centricon Plus 20 centrifrigal filter and stored at -20 °C. The conditions for the LEF
gel are run according to the procedures and reagents provided by Invitrogen. Samples are
dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of Transferrin-SA-Linker-Gal-UDP. Asialo-transferrin is dissolved at
2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaNs, pH 7.2. The solution is
incubated with CMP-sialic acid-linker-Gal-UDP (molar amount to add 1 molar equivalent of
nucleotide sugar to transferrin) and 0.1 U/mL of ST3Gal3 at 32°C for 2 days. To monitor the
incorporation of sialic acid, a small aliquot of the reaction has ''*C-SA-UDP ligand added; the
label incorporated into the peptide is separated from the free label by gel filtration on a Toso
Haas G3000SW analytical column using PBS buffer (pH 7.1). The radioactive label
incorporation into the peptide is quantitated using an in-line radiation detector.
The solution is incubated with 5 mM CMP-sialic acid and 0.1 U/mL of ST3Gal3 (to
cap ;my unreacted transferrin glycans) at 32°C for 2 days. The incorporation into the peptide
is quantitated using an in-line UV detector. After 2 days, the reaction mixture is purified
using a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and collecting
fractions based on UV absorption. The product of the reaction is analyzed using SDS-PAGE
and lEF analysis according to the procedures and reagents supplied by Invitrcgen. Samples
are dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of Transferrin-SA-Linker-Gal-GDNF. The transferrin-SA-Linker-
Gal-UDP prepared as described above is dissolved at 2.5 mg/rtiL in 50 mM Tris-HCl, 0.15 M
NaCl, 5 mM MnCl2, 0.05% NaN3, pH 7.2. The solution is incubated with 2.5 mg/mL
agalacto-GDNF and 0.1 U/mL of galactosyltransferase at 32°C for 2 days. To monitor the
incorporation of galactose, a small aliquot of the reaction has '"^C-galactose-UDP ligand
added; the label incorporated into the peptide is separated from the free label by gel filtration
on a Toso Haas G3000SW analytical column using PBS buffer (pH 7.1). The radioactive
label incorporation into the peptide is quantitated using an in-line radiation detector.
When the reaction is complete, the solution is incubated with 5 raM UDP-Gal and 0.1
U/mL of galactosyltransferase (to cap any unreacted transferrin glyeans) at 32°C for 2 days
followed by addition of 5 mM CMP-SA and 0.1 U/mL of ST3GaI3. After 2 additional days,
the reaction mixtxire is purified using a Toso Haas G3000SW preparative coluinn using PBS
buffer (pH 7.1) collecting fractions based on UV absorption. The product of the reaction is
analyzed using SDS-PAGE and lEF analysis according to the procedures and reagents
supplied b}' Invitrogen. Scunples are dialyzed against water and analyzed by MALDI-TOF
MS.
The disclosures of each and every patent, patent application, and publication cited
herein are hereby incorporated herein by reference in their entirety.
While this invention has been disclosed with reference to specific embodiments, it is
apparent that otlier embodiments and variations of this invention may be devised by others
skilled in ihe art without departing from the true spirit and scope of the invention. The
appended claims are intended to be constmed to include all such embodiments and equivalent
variations.
1. A cell-free, in \'itro method of forming a co\ alent conjugate of a
precursor peptide ha\ ing the formula:
wherein
AA is a terminal or internal amino acid residue of the peptide;
x'-x- is a saccharide covalentlv linked to the AA. wherein
X' is a first ghcosyl residue: and
X" is a second glycosx 1 residue covalentK linked to X . wherein X and X"
are selected from monosaccharyl and oligosacchar\ 1 residues:
the method comprising:
(a) remo\ ing X' or a saccharyl subunit thereof from the peptide.
thereb> formmg a truncated ghcan
(h) contacting said truncated glycan with at least one
glycosN'ltransferase and at least one modified sugar donor under conditions suitable for
said at least one ghcosyltransferase to transfer a modified sugar moiet\ of said at least one
modified sugar donor to said truncated ghcan. wherein said modified sugar moielx
comprises poly(eth\ lene glvcol). thereby forming said covalent conjugate of said peptide.
2. The method as claimed in claim 1 wherein said truncated gl\can i^^
formed b\ remo\ ing a Sia residue.
-v The method as claimed in claim 1 wherein said peptide has the
formula:
wherein
X . X X\ X\ X . and X' .are independently selected monosacchar\ I or
oligosaccharyl residues; and
a. b. c. d. e. and x are independently selected from the integers 0. ) and 2.
4. The method as claimed in claim 3 wherein said oligosacchar\ I
residue is a member selected from GIcNAc-Gal-Sia and
GlcNAc-Gal.
5. ! he method as claimed in claim 3 wherein at least one member
selected from a. b. c. d. e and x is ! or 2.
6. The method as claimed in claim 3. wherein said remo\ ing of step
(a) produces a truncated gl>can in which at lea^-t one ot a. b. c. e and
\ are 0.
'^. The method as claimed in claim 6. wherein X\ X' and X are
members independentU selected from (mannose)^ and
(mannose)/-(X')
wherein
X''^ is a gKcosyl moiety selected from mono- and oligo-saccharides; and
z is an integer between 1 and 20. wherein
when z is 3 or greater, each (mannose)y is independent)} selected froni
linear and branched structures.
8 1 he method as claimed in claim 6 wherein X is selected from the
group consisting of GlcM.Ac and xylose.
9. 1 he method as claimed in claim 6. wherein X'. .X^ and X are
(man nose )ii
wherein
u is selected from the integers between 1 and 20. and when u is 3 or greater,
each (mannose)u is independenth' selected from linear and branched structures.
lo. The method as claimed in claim 3 wherein said peptide has the
t'ormula:
wherein
r. s. and t are integers independently selected from 0 and 1.
1 1. The method as claimed in claim 1, wherein said peptide has the
brmula:
wherein
X' and X are independenth selected monosaccharx 1 or oligosacchar\ 1
residues: and
m. n and fare integers independenth selected from 0 and 1.
12. 1 he method as claimed in claim 11. wherein said peptide has ihe
fornuila:
wherein
X"' is a member selected from:
wherein
425
s and i .ire integers independently selected from 0 and 1.
13. The method as claimed in claim 12. wherein said peptide has tiie
formula:
wherein
X'\X'''. andX'-' are independently selected glycossl residues; and
g. h. i. j. k. and p are independenth' selected from the integers 0 and 1
14. The method as claimed in claim 13 wherein at least one of g. h. i. j.
k and p is 1.
15. The method as claimed in claim 13. wherein
X'"* and X''^ are members independently selected from GlcNAc and Sia.
and
i and k are independently selected from the integers 0 and 1.
16. The method as claimed in claim 15 wherein at least one of i and k i^
I. and if k is 1. g. h. .ind j are 0.
1 7. The method as claimed in claim 1 wherein said gl\cos\ I donor
comprises a modifying group covalently linked thereto.
IS. The method as claimed in claim 1. comprising:
(c) remo\ingX . thereb\ exposing AA.
U). The method ass claimed in claim 18. optionalK comprising:
(d) contcicting AA with at least one glvcosyltransferase and at leas\ one
glycosyl donor under conditions suitable to transfer said at least one glycos\ 1 donor to
AA. thereby remodeling said peptide comprising poly(ethylene ghcol).
20 The method as claimed in claim 19 wherein said at least one
gl\cosyl donor comprises a modifying group covalentl} linked thereto.
21. The method as claimed in claim 20 wherein said modifying group is
poly(ethylene glxcob.
22 The method as claimed in claim 21 wherein said pol>(ethylene
iiKcol) has a molecular weight distribution that is essentially homodisperse.
23. The method as claimed in claim 17. comprising:
(e) prior to step (b). removing a group added to said saccharide during
post-translational modification.
24. The method as claimed in claim 23 wherein said group is a member
selected from phosphate, sulfate. carbo\\ late and esters thereof
25. The method as claimed in claim 1 wherein said peptide has the
formula:
wherein Z is a member selected from O. S. NM and a cross-linker.
^^. The method as claimed in claim 1. wherein said peptide has the
formula:
wherein
II I "*
X and X " are independently selected glycosyl moieties: and
r and .\ are integers independently selected from 0 and 1.
27. The method as claimed in claim 17, wherein X'' and .\'~ are
(mannose)^,. wherein
q is selected from the integers between I and 20. and when q is three or
greater, (mannose},, is selected from linear and branched structures.
427
28 A cell-tree, in vitro method of remodeling a peptide comprising
poly(ethylene glycol), said peptide ha\ing the formula:
wherein
AA is a terminal or internal amino acid residue of said peptide;
X is a glycosyl residue coylently linked to said AA. selected from
monosaccharyl and oligosaccharyl residues: and
u is an integer selected from 0 and 1.
said method comprising:
contacting said peptide with at least one glycosyltransferase and at least one
glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said
truncated glycan. thereby remodeling said peptide.
29. The method as claimed in claim 28 wherein said at least one
glycosyl donor comprises a modifying group covalently linked thereto.
30 I he method as claimed in claim 28 wherein said modifying group is
poly(ethylene glycol).
31. The method as claimed in claim 30 wherein said poly(ethylene
glycol) has a molecular weight distribution that is essentially homodisperse.
The present invention discloses a cell-free, in vitro method ot forming a
covalent conjugate of a precursor peptide having the formula:
wherein
AA is a terminal or internal amino acid residue of the peptide:
X1-X2 is a saccharide covalently linked to the AA. wherein
X' is a first glycosyl residue: and
X2 is a second glycosyl residue covalently linked to X1 . wherein X1 and X2 are
selected from monosaccharyl and oligosaccharyl residues:
the method comprising:
(a) removing X2 or a saccharyl subunit thereof from the peptide. thereby
forming a truncated glycan
(b) contacting said truncated glycan with at least one glycosyltransferase
and at least one modified sugar donor under conditions such as herein before described
suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at
least one modified sugar donor to said truncated glycan, wherein said modified sugar moiety
comprises poly(ethylene glycol). thereby forming said covalent conjugate of said peptide.
