CONJUGATES OF AN IL-15 MOIETY AND A POLYMER
CROSS REFERENCE TO RELATEDAPPLICATION
[0001] This application claims the benefit of priority under 35 U.S.C. § 1 19(e) to U.S.
Provisional Patent Application Serial No. 61/974,914, filed on April 3, 2014, the disclosure of
which is incorporated herein by reference.
FIELD
[0002] Among other things, one or more embodiments of the present invention relate
generally to conjugates comprising an IL-15 moiety (i.e., a moiety having at least some activity
similar to human IL-15) and a water-soluble, non-peptidic polymer. In addition, the invention
relates to (among other things) compositions comprising conjugates, methods for synthesizing
conjugates, and methods of administering a composition.
BACKGROUND
[0003] Interleukin- 15 ("IL-15") is a pleiotropic cytokine that was first reported by
Grabstein et al. [Grabstein et al. (1994) Science 264:965-968]. Secreted as a 162-amino acid
precursor, human IL-15 contains a 29-amino acid leader sequence and an 19-amino acid pro
sequence; the mature protein is therefore 114 amino acids in length. Belonging to the four a-helix
bundle family of cytokines, IL-15 binds to a heterotrimeric receptor, wherein a unique a subunit
(IL-15Ra) confers receptor specificity to IL-15, and the b and g subunits of this receptor share
commonality with one or more other cytokine receptors. Giri et al. (1995) EMBO J . 14:3654-
3663.
[0004] As a cytokine, IL-15 has effects on both the innate immune system and the adaptive
immune system. DiSabitino et al. (201 1) Cytokine Growth Factor Rev. 22:19-33. With respect to
the innate immune system (which defends the host from foreign invaders generically), IL-15
causes the development of and maintains the survival of natural killer cells ("NK cells") and
natural killer-T cells ("NK-T cells"), among other properties. Consistent with its role in the innate
immune system, NK cells do not specifically attack the invading pathogen; rather, these cells
destroy compromised host cells (such as tumor cells or virus-infected cells). NK-T cells generate
immunomodulatory cytokines, particularly interferon- g , which result in a general activation of the
immune response.
[0005] With respect to the adaptive immune system (which defends the host from a
specific foreign invader following an initial encounter with that particular pathogen), IL-15 is
necessary for the maintenance of the immunomodulatory cytokine-generating helper T cells.
Importantly, IL-15 also supports the long-term maintenance of "antigen-experienced" memory T
cells, which have the ability to rapidly reproduce, thereby generating a faster and stronger immune
response upon re-exposure to the particular foreign pathogen invading the host.
[0006] Finally, notwithstanding its specific roles within the innate and adaptive immune
systems, IL-15 has significant and broad effects across both categories of immune systems. In
particular, IL-15 inhibits or reduces apoptosis (or cell death) of a number of cells types (including
dendritic cells, neutrophils, eosinophils, mast cells, CD4+ T cells, and B cells) associated within
both categories of immune systems.
[0007] Because it stimulates the proliferation and maintenance of many cells within the
immune system that can fight against cells that appear to the host as foreign (or "non-self), IL-15
has been proposed for use in the treatments of individuals suffering from cancer. Steel et al.
(2012) Trends Pharmacol. Sci. 33(1):35-41 . For example, an IL-1 5-based agonist has been
proposed to treat myelomas. Wong et al. (2013) Oncolmmunology 2(1 1), e26442:l-3. In
addition, IL-15 pharmacotherapy has been proposed for treating individuals suffering from viral
infections, such as HIV infection.
[0008] Despite its potential for use in the treatment of individuals suffering from a number
of diseases, IL-1 5-based therapies face a number of challenges. For example, IL-15 is rapidly
cleared and is relatively unstable under physiological conditions. Certain approaches attempt to
overcome these limitations by complexing IL- 15 with the IL- 15 receptor alpha subunit. Such an
approach, however, may abrogate the desirable signaling that occurs uniquely through the IL-15
receptor alpha, expressed on multiple cell types. A non-releasable PEGylation with a
succinimidyl carbonate-terminated polymer of relatively small molecular weight (5kDa) has been
reported, but this resulted in significant alteration of IL-15's biological activity. Pettit et al. (1997)
J . Biol. Chem. 272(4):23 12-23 18.
[0009] Notwithstanding these conjugates, however, there remains a need for new
conjugates of IL-15 having improved characteristics and profiles. Among other things, one or
more embodiments of the present invention is therefore directed to such conjugates as well as
compositions comprising the conjugates and related methods as described herein, which are
believed to be new and completely unsuggested by the art.
SUMMARY
[0010] Accordingly, in one or more embodiments of the invention, a conjugate is
provided, the conjugate comprising a residue of an IL-15 moiety covalently attached to a
water-soluble polymer.
[001 1] In one or more embodiments of the invention, a conjugate is provided, the
conjugate comprising a residue of an IL-15 moiety covalently attached to a water-soluble polymer,
wherein the residue of the IL-15 moiety is covalently attached to the water-soluble polymer via a
releasable linkage.
[0012] In one or more embodiments of the invention, a conjugate is provided, the
conjugate comprising a residue of an IL-15 moiety covalently attached to a water-soluble polymer,
wherein the residue of the IL-15 moiety is covalently attached to the water-soluble polymer via a
non-releasable linkage, preferably wherein the water-soluble polymer has a weight-average
molecular weight of greater than 5,000 Daltons.
[0013] In one or more embodiments of the invention, a conjugate is provided, the
conjugate comprising a residue of an IL-15 moiety covalently attached to a water-soluble polymer,
wherein the IL-15 moiety is free of cysteine residues not involved with disulfide bonding.
[0014] In one or more embodiments of the invention, a conjugate is provided, the
conjugate comprising a residue of an IL-15 moiety covalently attached to a water-soluble polymer,
wherein the IL-15 moiety has an additional cysteine residue compared to human IL-15, and the
water-soluble polymer is covalently attached to the additional cysteine residue.
[0015] In one or more embodiments of the invention, a conjugate is provided, the
conjugate comprising a residue of an IL-15 moiety covalently attached to a branched
water-soluble polymer.
[0016] In one or more embodiments of the invention, a conjugate is provided, the
conjugate comprising a residue of an IL-15 moiety covalently attached to a water-soluble polymer,
wherein an amine of the IL-15 moiety is covalently attached to the water-soluble polymer via a
linkage other than an amide linkage.
[0017] In one or more embodiments of the invention, a conjugate is provided, the
conjugate comprising a residue of an IL-15 moiety covalently attached to a water-soluble polymer,
wherein an amine of the IL-15 moiety is covalently attached to the water-soluble polymer via an
amine linkage.
[0018] In one or more embodiments of the invention, a composition is provided, the
composition comprising a conjugate as described herein along with a pharmaceutically acceptable
excipient.
[0019] In one or more embodiments of the invention, a method for delivering a conjugate
is provided, the method comprising the step of subcutaneously administering to the patient a
composition comprised of a conjugate of a residue of an IL-15 and a water-soluble polymer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is an SDS-PAGE gel of conjugation reaction mixtures of IL-15 with 20K
PEG2-ru-NHS.
[0021] FIG. 2 is an SDS-PAGE gel of conjugation reaction mixtures of IL-15 with 40
PEG2-ru-NHS.
[0022] FIG. 3 is a plot showing the percent body weight change following administration
of IL-15 (in unconjugated form), conjugated rIL-15, or vehicle control in established B16F10
subcutaneos tumors in mice.
DETAILED DESCRIPTION
[0023] Before describing one or more embodiments of the present invention in detail, it is
to be understood that this invention is not limited to the particular polymers, synthetic techniques,
IL-15 moieties, and the like, as such may vary.
[0024] It must be noted that, as used in this specification and the intended claims, the
singular forms "a," "an," and "the" include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a polymer" includes a single polymer as well as two
or more of the same or different polymers, reference to "an optional excipient" refers to a single
optional excipient as well as two or more of the same or different optional excipients, and the like.
[0025] In describing and claiming one or more embodiments of the present invention, the
following terminology will be used in accordance with the definitions described below.
[0026] "PEG," "polyethylene glycol" and "poly(ethylene glycol)" as used herein, are
interchangeable and encompass any nonpeptidic, water-soluble poly(ethylene oxide). Typically,
PEGs for use in accordance with the invention comprise the following structure -(OCH2CH2)n-"
where (n) is 2 to 4000. As used herein, PEG also includes "-CH2CH2-0(CH 2CH20 ) -CH2CH2-
and "-(OCH2CH2)„0-," depending upon whether or not the terminal oxygens have been displaced,
e.g., during a synthetic transformation. Throughout the specification and claims, it should be
remembered that the term "PEG" includes structures having various terminal or "end capping"
groups and so forth. The term "PEG" also means a polymer that contains a majority, that is to say,
greater than 50%, of -OCH2CH2- repeating subunits. With respect to specific forms, the PEG can
take any number of a variety of molecular weights, as well as structures or geometries such as
"branched," "linear," "forked," "multifunctional," and the like, to be described in greater detail
below.
[0027] The terms "end-capped" and "terminally capped" are interchangeably used herein
to refer to a terminal or endpoint of a polymer having an end-capping moiety. Typically, although
not necessarily, the end-capping moiety comprises a hydroxy or Ci-2oalkoxy group, more
preferably a C Oalkoxy group, and still more preferably a Ci- alkoxy group. Thus, examples of
end-capping moieties include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as aryl,
heteroaryl, cyclo, heterocyclo, and the like. It must be remembered that the end-capping moiety
may include one or more atoms of the terminal monomer in the polymer [e.g., the end-capping
moiety "methoxy" in CH30(CH 2CH20 )n- and CH3(OCH2CH2) -]. In addition, saturated,
unsaturated, substituted and unsubstituted forms of each of the foregoing are envisioned.
Moreover, the end-capping group can also be a silane. The end-capping group can also
advantageously comprise a detectable label. When the polymer has an end-capping group
comprising a detectable label, the amount or location of the polymer and/or the moiety (e.g., active
agent) to which the polymer is coupled can be determined by using a suitable detector. Such
labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme
labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, and the like. Suitable detectors
include photometers, films, spectrometers, and the like. The end-capping group can also
advantageously comprise a phospholipid. When the polymer has an end-capping group
comprising a phospholipid, unique properties are imparted to the polymer and the resulting
conjugate. Exemplary phospholipids include, without limitation, those selected from the class of
phospholipids called phosphatidylcholines. Specific phospholipids include, without limitation,
those selected from the group consisting of dilauroylphosphatidylcholine,
dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,
behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin. The end-capping
group may also include a targeting moiety, such that the polymer ~ as well as anything, e.g., an
IL-15 moiety, attached thereto - can preferentially localize in an area of interest.
[0028] "Non-naturally occurring" with respect to a polymer as described herein, means a
polymer that in its entirety is not found in nature. A non-naturally occurring polymer may,
however, contain one or more monomers or segments of monomers that are naturally occurring, so
long as the overall polymer structure is not found in nature.
[0029] The term "water soluble" as in a "water-soluble polymer" polymer is any polymer
that is soluble in water at room temperature. Typically, a water-soluble polymer will transmit at
least about 75%, more preferably at least about 95%, of light (e.g., of a wavelength of 600 nm)
transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer will
preferably be at least about 35% (by weight) soluble in water, more preferably at least about 50%
(by weight) soluble in water, still more preferably about 70% (by weight) soluble in water, and
still more preferably about 85% (by weight) soluble in water. It is most preferred, however, that
the water-soluble polymer is about 95% (by weight) soluble in water or completely soluble in
water.
[0030] Molecular weight in the context of a water-soluble polymer, such as PEG, can be
expressed as either a number average molecular weight or a weight average molecular weight.
Unless otherwise indicated, all references to molecular weight herein refer to the weight average
molecular weight. Both molecular weight determinations, number average and weight average,
can be measured using gel permeation chromatography or other liquid chromatography
techniques. Other methods for measuring molecular weight values can also be used, such as the
use of end-group analysis or the measurement of colligative properties (e.g., freezing-point
depression, boiling-point elevation, or osmotic pressure) to determine number average molecular
weight or the use of light scattering techniques, ultracentrifugation, or viscometry to determine
weight average molecular weight. The polymers of the invention are typically polydisperse (i.e.,
number average molecular weight and weight average molecular weight of the polymers are not
equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less
than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about
1.05, and most preferably less than about 1.03.
[0031] The terms "active," "reactive" or "activated" when used in conjunction with a
particular functional group, refer to a reactive functional group that reacts readily with an
electrophile or a nucleophile on another molecule. This is in contrast to those groups that require
strong catalysts or highly impractical reaction conditions in order to react (i.e., a "non-reactive" or
"inert" group).
[0032] As used herein, the term "functional group" or any synonym thereof is meant to
encompass protected forms thereof as well as unprotected forms.
[0033] The terms "spacer moiety," "linkage" and "linker" are used herein to refer to a bond
or an atom or a collection of atoms optionally used to link interconnecting moieties such as a
terminus of a polymeric reagent and an IL-15 moiety or an electrophile or nucleophile of an IL-15
moiety. The spacer moiety may be hydrolytically stable or may include a physiologically
hydrolyzable or enzymatically degradable linkage. Unless the context clearly dictates otherwise, a
spacer moiety optionally exists between any two elements of a compound (e.g., the provided
conjugates comprising a residue of IL-15 moiety and water-soluble polymer can be attached
directly or indirectly through a spacer moiety).
[0034] "Alkyl" refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms
in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be
branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups
include methyl, ethyl, propyl, butyl, pentyl, 3-methylpentyl, and the like.
[0035] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, and
may be straight chain or branched, as exemplified by methyl, ethyl, rc-butyl, z-butyl, and /-butyl.
[0036] "Cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon chain,
including bridged, fused, or spiro cyclic compounds, preferably made up of 3 to about carbon
atoms, more preferably 3 to about 8 carbon atoms. "Cycloalkylene" refers to a cycloalkyl group
that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring
system.
[0037] "Alkoxy" refers to an -OR group, wherein R is alkyl or substituted alkyl, preferably
Ci-6 alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth).
[0038] The term "substituted" as in, for example, "substituted alkyl," refers to a moiety
(e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not
limited to: alkyl, C3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro,
chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like.
"Substituted aryl" is aryl having one or more noninterfering groups as a substituent. For
substitutions on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or
para).
[0039] "Noninterfering substituents" are those groups that, when present in a molecule, are
typically nonreactive with other functional groups contained within the molecule.
[0040] "Aryl" means one or more aromatic rings, each of 5 or 6 core carbon atoms. Aryl
includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl
rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or
heterocyclic rings. As used herein, "aryl" includes heteroaryl.
[0041] "Heteroaryl" is an aryl group containing from one to four heteroatoms, preferably
sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one
or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
[0042] "Heterocycle" or "heterocyclic" means one or more rings of 5-12 atoms, preferably
5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom
that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.
[0043] "Substituted heteroaryl" is heteroaryl having one or more noninterfering groups as
substituents.
[0044] "Substituted heterocycle" is a heterocycle having one or more side chains formed
from noninterfering substituents.
[0045] An "organic radical" as used herein shall include akyl, substituted alkyl, aryl, and
substituted aryl.
[0046] "Electrophile" and "electrophilic group" refer to an ion or atom or collection of
atoms, which may be ionic, having an electrophilic center, i.e., a center that is electron seeking,
capable of reacting with a nucleophile.
[0047] "Nucleophile" and "nucleophilic group" refers to an ion or atom or collection of
atoms that may be ionic having a nucleophilic center, i.e., a center that is seeking an electrophilic
center or with an electrophile.
[0048] An "enzymatically degradable linkage" means a linkage that is subject to
degradation by one or more enzymes.
[0049] A "hydrolyzable" bond is a bond that reacts with water (i.e., is hydrolyzed) under
physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on
the general type of linkage connecting two central atoms but also on the substituents attached to
these central atoms. Appropriate hydrolytically unstable or weak linkages include but are not
limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether,
imines, orthoesters, peptides and oligonucleotides. A "releasable bond" is a covalent linkage that
cleaves under physiological conditions at a rate that is clinically useful and includes, for example
and without limitation, hydrolyzable bonds and enzymatically degradable linkage.
[0050] A "hydrolytically stable" linkage or bond refers to a chemical bond, typically a
covalent bond, which is substantially stable in water, that is to say, does not undergo hydrolysis
under physiological conditions to any appreciable extent over an extended period of time.
Examples of hydrolytically stable linkages include, but are not limited to, the following:
carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like. Generally,
a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per
day under physiological conditions. Hydrolysis rates of representative chemical bonds can be
found in most standard chemistry textbooks.
[0051] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may
optionally be included in the compositions of the invention and that causes no significant adverse
toxicological effects to the patient.
[0052] "Pharmacologically effective amount," "physiologically effective amount," and
"therapeutically effective amount" are used interchangeably herein to mean the amount of a
polymer-(IL-15) moiety conjugate that is needed to provide a desired level of the conjugate (or
corresponding unconjugated IL-1 5 moiety) in the bloodstream or in the target tissue. The precise
amount will depend upon numerous factors, e.g., the particular IL-15 moiety, the components and
physical characteristics of the therapeutic composition, intended patient population, individual patient
considerations, and the like, and can readily be determined by one skilled in the art, based upon the
information provided herein.
[0053] "Multi-functional" means a polymer having three or more functional groups
contained therein, where the functional groups may be the same or different. Multi-functional
polymeric reagents of the invention will typically contain from about 3-100 functional groups, and
can contain, for example, a number satisfying one or more of the following ranges: from 3-50
functional groups; from 3-25 functional groups; from 3-15 functional groups; from 3 to 10
functional groups. For example, the number of functional groups can be selected from the group
consisting of 3, 4, 5, 6, 7, 8, 9 and 10 functional groups within the polymer backbone.
[0054] The term "IL-15 moiety," as used herein, refers to a peptide or protein moiety
having human IL-15 activity. The IL-15 moiety will also have at least one electrophilic group or
nucleophilic group suitable for reaction with a polymeric reagent. In addition, the term "IL-15
moiety" encompasses both the IL-15 moiety prior to conjugation as well as the IL-15 moiety
residue following conjugation. As will be explained in further detail below, one of ordinary skill
in the art can determine whether any given moiety has IL-15 activity. Proteins comprising an
amino acid sequence corresponding to any one of SEQ ID NOs: 1 through 3 is an IL-15 moiety, as
well as any protein or polypeptide substantially homologous thereto. As used herein, the term "IL-
15 moiety" includes such peptides and proteins modified deliberately, as for example, by site
directed mutagenesis or accidentally through mutations. These terms also include analogs having
from 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the
carboxy terminal end of the peptide or protein wherein the additional amino acid(s) includes at
least one glycosylation site, and analogs having an amino acid sequence which includes at least
one glycosylation site. The term includes naturally, recombinantly and synthetically produced
moieties.
[0055] The term "substantially homologous" means that a particular subject sequence, for
example, a mutant sequence, varies from a reference sequence by one or more substitutions,
deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity
between the reference and subject sequences. For puiposes of the present invention, sequences
having greater than 95 percent homology, equivalent biological activity (although not necessarily
equivalent strength of biological activity), and equivalent expression characteristics are considered
substantially homologous. For purposes of determining homology, truncation of the mature
sequence should be disregarded. Exemplary IL-15 moieties for use herein include those sequences
that are substantially homologous SEQ ID NO: 1.
[0056] The term "fragment" means any protein or polypeptide having the amino acid
sequence of a portion or fragment of an IL-15 moiety, and which has the biological activity of
IL-15. Fragments include proteins or polypeptides produced by proteolytic degradation of an
IL-15 moiety as well as proteins or polypeptides produced by chemical synthesis by methods
routine in the art.
[0057] The term "patient," refers to a living organism suffering from or prone to a
condition that can be prevented or treated by administration of an active agent (e.g., conjugate),
and includes both humans and animals.
[0058] "Optional" or "optionally" means that the subsequently described circumstance may
or may not occur, so that the description includes instances where the circumstance occurs and
instances where it does not.
[0059] "Substantially" means nearly totally or completely, for instance, satisfying one or
more of the following: greater than 50%, 51% or greater, 75% or greater, 80% or greater, 90% or
greater, and 95% or greater of the condition.
[0060] Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or
F; Leucine is Leu or L; Isoleucine is e or I; Methionine is Met or M; Valine is Val or V; Serine is
Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y;
Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic
Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Tip or W;
Arginine is Arg or R; and Glycine is Gly or G.
[0061] Turning to one or more embodiments of the invention, a conjugate is provided, the
conjugate comprising a residue of an IL-15 moiety covalently attached (either directly or through a
spacer moiety) to a water-soluble polymer. The conjugates of the invention will have one or more
of the following features.
[0062] The IL-15 Moiety
[0063] As previously stated, the conjugate comprises a residue of an IL-15 moiety
covalently attached, either directly or through a spacer moiety, to a water-soluble polymer. As
used herein, the term "IL-15 moiety" shall refer to the IL-15 moiety prior to conjugation as well as
to the IL-1 5 moiety following attachment to a nonpeptidic, water-soluble polymer. It will be
understood, however, that when the original IL- 5 moiety is attached to a nonpeptidic,
water-soluble polymer, the IL-15 moiety is slightly altered due to the presence of one or more
covalent bonds associated with linkage to the polymer(s). Often, this slightly altered fo m of the
IL-15 moiety attached to another molecule is referred to a "residue" of the IL-15 moiety.
[0064] The IL-1 5 moiety can be derived from non-recombinant methods and from
recombinant methods and the invention is not limited in this regard. In addition, the IL-15 moiety
can be derived from human sources, animal sources (including insects), fungi sources (including
yeasts), and plant sources.
[0065] The IL-15 moiety can be obtained according to the procedures described by
Grabstein et al. See. Grabstein et al. (1994) Science 264:965-968.
[0066] The IL-15 moiety can be derived from recombinant methods. See, for example,
EP 0 772 624 B2.
[0067] The IL-15 moiety can be purchased commercially from, for example, GenScript
USA Inc. (Piscataway NJ) and Peprotech (Rockyhill, NJ).
[0068] The IL-15 moiety can be expressed in bacterial [e.g., E. coli, see, for example,
Fischer et al. (1995) Biotechnol. Appl. Biotechnol. 2J_(3):295-3 11], mammalian [see, for example,
Kronman et al. (1992) Gene 121 :295-304], yeast [e.g., Pichia pastoris, see, for example, Morel et
al. (1997) Biochem. J. 328(1): 121 -129], and plant [see, for example, Mor et al. (2001) Biotechnol.
Bioeng. 75(3):259-266] expression systems. The expression can occur via exogenous expression
(when the host cell naturally contains the desired genetic coding) or via endogenous expression.
[0069] Although recombinant-based methods for preparing proteins can differ,
recombinant methods typically involve constructing the nucleic acid encoding the desired
polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host
cell (e.g., plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as Chinese hamster
ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired
polypeptide or fragment. Methods for producing and expressing recombinant polypeptides in
vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art.
[0070] To facilitate identification and purification of the recombinant polypeptide, nucleic
acid sequences that encode for an epitope tag or other affinity binding sequence can be inserted or
added in-frame with the coding sequence, thereby producing a fusion protein comprised of the
desired polypeptide and a polypeptide suited for binding. Fusion proteins can be identified and
purified by first running a mixture containing the fusion protein through an affinity column
bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding
sequence in the fusion proteins, thereby binding the fusion protein within the column. Thereafter,
the fusion protein can be recovered by washing the column with the appropriate solution (e.g.,
acid) to release the bound fusion protein. The recombinant polypeptide can also be purified by
lysing the host cells, separating the polypeptide, e.g., by ion-exchange chromatography, affinity
binding approaches, hydrophobic interaction approaches, and thereafter identify by MALDI or
western blot, and collecting the polypeptide. These and other methods for identifying and
purifying recombinant polypeptides are known to those of ordinary skill in the art. In one or more
embodiments of the invention, however, the IL-1 5 moiety is not in the form of a fusion protein.
[0071] Depending on the system used to express proteins having IL-1 5 activity, the IL-1 5
moiety can be unglycosylated or glycosylated and either may be used. That is, the IL-1 5 moiety
can be unglycosylated or the IL-1 5 moiety can be glycosylated. In one or more embodiments of
the invention, the IL-1 5 moiety is unglycosylated.
[0072] The IL-1 5 moiety can advantageously be modified to include and/or substitute one
or more amino acid residues such as, for example, lysine, cysteine and/or arginine, in order to
provide facile attachment of the polymer to an atom within the side chain of the amino acid. An
example of substitution of an IL-15 moiety is described in U.S. Patent No. 6,177,079. In addition,
the IL-15 moiety can be modified to include a non-naturally occurring amino acid residue.
Techniques for adding amino acid residues and non-naturally occurring amino acid residues are
well known to those of ordinary skill in the art. Reference is made to J . March, Advanced Organic
Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992).
[0073] In addition, the IL-1 5 moiety can advantageously be modified to include
attachment of a functional group (other than through addition of a functional group-containing
amino acid residue). For example, the IL-1 5 moiety can be modified to include a thiol group. In
addition, the IL-1 5 moiety can be modified to include an N-terminal alpha carbon. In addition, the
IL-1 5 moiety can be modified to include one or more carbohydrate moieties. In addition, the IL-
5 moiety can be modified to include an aldehyde group. In addition, the IL-1 5 moiety can be
modified to include a ketone group. In some embodiments of the invention, it is preferred that the
IL-1 5 moiety is not modified to include one or more of a thiol group, an N-terminal alpha carbon,
carbohydrate, adehyde group and ketone group.
[0074] Exemplary IL-1 5 moieties are described herein, in the literature, and in, for
example, U.S. Patent Application Publication No. US 2006/0104945, Pettit et al. (1997) J . Biol.
Chem. 272(4):23 12-23 8, and Wong et al. (2013) Oncolmmunology 2(1 1), e26442:l-3. Preferred
IL- 15 moieties include those having an amino acid sequence comprising sequences selected from
the group consisting of SEQ ID NOs: 1 through 3, and sequences substantially homologous
thereto (wherein even if SEQ ID NOs 2 and 3, and sequences substantially homologous thereto do
not meet the in vitro activity standard of an IL-1 moiety provided herein, it will be understood for
purposes of the present invention that these sequences are also understood to be "IL-1 5 moieties").
A preferred IL-1 5 moiety has the amino acid sequence corresponding to SEQ ID NO: 1.
[0075] In some instances, the IL-1 moiety will be in a "monomer" form, wherein a single
expression of the corresponding peptide is organized into a discrete unit. In other instances, the
IL-1 5 moiety will be in the form of a "dimer" (e.g., a dimer of recombinant IL-1 5) wherein two
monomer forms of the protein are associated to each other.
[0076] In addition, precursor forms IL-1 5 can be used as the IL-1 5 moiety. An exemplary
precursor form of IL-1 has the sequence of SEQ ID NO: 3.
[0077] Truncated versions, hybrid variants, and peptide mimetics of any of the foregoing
sequences can also serve as the IL-1 moiety. Biologically active fragments, deletion variants,
substitution variants or addition variants of any of the foregoing that maintain at least some degree
of IL-1 5 activity can also serve as an IL-1 5 moiety.
[0078] For any given peptide, protein moiety or conjugate, it is possible to determine
whether that peptide, protein moiety or conjugate has IL-15 activity. Various methods for
determining in vitro IL- 15 activity are described in the art. An exemplary approach is based on a
pSTAT assay. Briefly, if an IL-15-dependent CTLL-2 cell is exposed to a test article having IL-15
activity, initiation of a signaling cascade results that includes the phosphorylation of STAT5 at
tyrosine residue 694 (Tyr694) that can be quantitatively measured. Assay protocols and kits are
known and include, for example, the MSD Phospho(Tyr694)/Total STATa,b Whole Cell Lysate
Kit (Meso Seal Diagnostics, LLC, Gaithersburg, MD), which was used in connection with
Example 1; using this approach, a proposed IL-15 moiety that exhibits a pSTAT5 EC50 value of at
least 300 ng/mL (more preferably at least 150 ng/mL) at least one of 5 minutes or at 10 minutes is
considered an "IL-15 moiety" in connection with the present invention. It is preferred, however,
that the IL-15 moiety used in the present invention is more potent (e.g., having a pSTAT5 EC50
value of less than 150 ng/mL at at one of least 5 minutes or 10 minutes, such as less than 1 ng/mL,
and even more preferably less than 0.5 ng/mL at at least one of 5 minutes or at 10 minutes). It is
preferred that conjugates containing a stable linkage exhibit a pSTAT5 EC50 value of at least 300
ng/mL (more preferably at least 150 ng/mL) at 10 minutes, and it is more preferred that conjugates
containing a stable linkage exhibit a pSTAT5 EC50 value of less than 150 ng/mL at 10 minutes.
[0079] Other methodologies known in the art can also be used to assess IL-1 5 function,
including electrometry, spectrophotometry, chromatography, and radiometric methodologies. See,
for example, Ring et al. (2012) Nat. Immunol. 13(12): 1187-1 195 for one such additional type of
assay.
[0080] Assays for use in connection with measuring the activity of an IL-1 5 moiety can
also be used to measure the activity of conjugates described herein. Due to a given conjugate's
properties (e.g., incorporation of a releasable linkage, ability to withstand metabolism, increased
half-life, selective binding properties, and so forth), however, the conjugate need not necessarily
exhibit the same activity as an IL-15 moiety defined herein.
[0081] The Water-Soluble Polymer
[0082] As previously discussed, each conjugate comprises an IL-1 5 moiety attached to a
water-soluble polymer. With respect to the water-soluble polymer, the water-soluble polymer is
nonpeptidic, nontoxic, non-naturally occurring and biocompatible. With respect to
biocompatibility, a substance is considered biocompatible if the beneficial effects associated with
use of the substance alone or with another substance (e.g., an active agent such as an IL-15
moiety) in connection with living tissues (e.g., administration to a patient) outweighs any
deleterious effects as evaluated by a clinician, e.g., a physician. With respect to
non-immunogenicity, a substance is considered non-immunogenic if the intended use of the
substance in vivo does not produce an undesired immune response (e.g., the formation of
antibodies) or, if an immune response is produced, that such a response is not deemed clinically
significant or important as evaluated by a clinician. It is particularly preferred that the nonpeptidic
water-soluble polymer is biocompatible and non-immunogenic.
[0083] Further, the polymer is typically characterized as having from 2 to about 300
termini. Examples of such polymers include, but are not limited to, poly(alkylene glycols) such as
polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and
propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),
poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines
("POZ") (which are described in WO 2008/106186), poly(N-acryloylmorpholine), and
combinations of any of the foregoing.
[0084] The water-soluble polymer is not limited to a particular structure and can be linear
(e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g.,
forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or
without one or more degradable linkages. Moreover, the internal structure of the water-soluble
polymer can be organized in any number of different repeat patterns and can be selected from the
group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer,
alternating tripolymer, random tripolymer, and block tripolymer.
[0085] Typically, activated PEG and other activated water-soluble polymers (i.e.,
polymeric reagents) are activated with a suitable activating group appropriate for coupling to a
desired site on the IL-15 moiety. Thus, a polymeric reagent will possess a reactive group for
reaction with the IL-15 moiety. Representative polymeric reagents and methods for conjugating
these polymers to an active moiety are known in the art and further described in Zalipsky, S.,
et al., "Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides" in
Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J.M. Harris, Plenus
Press, New York (1992), and in Zalipsky (1995) Advanced Drug Reviews 16:157-182. Exemplary
activating groups suitable for coupling to an IL-15 moiety include hydroxyl, maleimide, ester,
acetal, ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinyl ketone, thione, thiol,
vinyl sulfone, hydrazine, among others.
[0086] Typically, the weight-average molecular weight of the water-soluble polymer in the
conjugate is from about 100 Daltons to about 150,000 Daltons. Exemplary ranges, however,
include weight-average molecular weights in the range of greater than 5,000 Daltons to about
100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range
of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons
to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the
range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000
Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000
Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of
from about 40,000 Daltons to about 120,000 Daltons. For any given water-soluble polymer, PEGs
having a molecular weight in one or more of these ranges are preferred.
[0087] Exemplary weight-average molecular weights for the water-soluble polymer
include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500
Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900
Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons,
about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500
Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons,
about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about
11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000
Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000
Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000
Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000
Daltons, and about 75,000 Daltons. Branched versions of the water-soluble polymer (e.g., a
branched 40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton polymers) having
a total molecular weight of any of the foregoing can also be used. In one or more embodiments,
the conjugate will not have any PEG moieties attached, either directly or indirectly, with a PEG
having a weight average molecular weight of less than about 6,000 Daltons.
[0088] When used as the polymer, PEGs will typically comprise a number of (OCH2CH2)
monomers [or (CH2CH2O) monomers, depending on how the PEG is defined]. As used
throughout the description, the number of repeating units is identified by the subscript "n" in
"(OCH2CH2)„." Thus, the value of (n) typically falls within one or more of the following ranges:
from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about
136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200
to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to
about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to
about 1,900. For any given polymer in which the molecular weight is known, it is possible to
determine the number of repeating units (i.e., "n") by dividing the total weight-average molecular
weight of the polymer by the molecular weight of the repeating monomer.
[0089] One particularly preferred polymer for use in the invention is an end-capped
polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such
as a lower C - alkoxy group, although a hydroxyl group can also be used. When the polymer is
PEG, for example, it is preferred to use a methoxy-PEG (commonly referred to as mPEG), which
is a linear form of PEG wherein one terminus of the polymer is a methoxy (-OCH3) group, while
the other terminus is a hydroxyl or other functional group that can be optionally chemically
modified.
[0090] In one form useful in one or more embodiments of the present invention, free or
unbound PEG is a linear polymer terminated at each end with hydroxyl groups:
HO-CH2CH20-(CH 2CH20)„-CH 2CH2-OH,
wherein (n) typically ranges from zero to about 4,000.
[0091] The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can be
represented in brief form as HO-PEG-OH where it is understood that the -PEG- symbol can
represent the following structural unit:
-CH2CH20-(CH 2CH20)n-CH CH2-,
wherein (n) is as defined as above.
[0092] Another type of PEG useful in one or more embodiments of the present invention is
methoxy-PEG-OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group,
while the other terminus is a hydroxyl group. The structure of mPEG is given below.
CH30-CH 2CH20-(CH 2CH20 )n-CH2CH2-OH
wherein (n) is as described above.
[0093] Multi-armed or branched PEG molecules, such as those described in U.S. Patent
No. 5,932,462, can also be used as the PEG polymer. For example, PEG can have the structure:
p°iya— P
R"— C—
polyb—Q
wherein:
polya and poly ar PEG backbones (either the same or different), such as methoxy
poly(ethylene glycol);
R" is a nonreactive moiety, such as H, methyl or a PEG backbone; and
P and Q are nonreactive linkages. In a preferred embodiment, the branched PEG polymer
is methoxy poly(ethylene glycol) disubstituted lysine. Depending on the specific IL- 15 moiety
used, the reactive ester functional group of the disubstituted lysine may be further modified to
form a functional group suitable for reaction with the target group within the IL-15 moiety.
[0094] In addition, the PEG can comprise a forked PEG. An example of a forked PEG is
represented by the following structure:
Z
/
PEG-X-CH
Z
wherein: X is a spacer moiety of one or more atoms and each Z is an activated terminal group
linked to CH by a chain of atoms of defined length. International Patent Application Publication
WO 99/45964 discloses various forked PEG structures capable of use in one or more
embodiments of the present invention. The chain of atoms linking the Z functional groups to the
branching carbon atom serve as a tethering group and may comprise, for example, alkyl chains,
ether chains, ester chains, amide chains and combinations thereof.
[0095] The PEG polymer may comprise a pendant PEG molecule having reactive groups,
such as carboxyl, covalently attached along the length of the PEG rather than at the end of the
PEG chain. The pendant reactive groups can be attached to the PEG directly or through a spacer
moiety, such as an alkylene group.
[0096] In addition to the above-described forms of PEG, the polymer can also be prepared
with one or more weak or degradable linkages in the polymer, including any of the
above-described polymers. For example, PEG can be prepared with ester linkages in the polymer
that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer
into fragments of lower molecular weight:
-PEG-CO2-PEG- + H20 -PEG-C0 2H + HO-PEG-
[0097] Other hydrolytically degradable linkages, useful as a degradable linkage within a
polymer backbone and/or as a degradable linkage to an IL-15 moiety, include: carbonate linkages;
imine linkages resulting, for example, from reaction of an amine and an aldehyde (see, e.g., Ouchi
et al. (1997) Polymer Preprints 38(l):582-3); phosphate ester linkages formed, for example, by
reacting an alcohol with a phosphate group; hydrazone linkages which are typically formed by
reaction of a hydrazide and an aldehyde; acetal linkages that are typically formed by reaction
between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction
between a formate and an alcohol; amide linkages formed by an amine group, e.g., at an end of a
polymer such as PEG, and a carboxyl group of another PEG chain; urethane linkages formed from
reaction of, e.g., a PEG with a terminal isocyanate group and a PEG alcohol; peptide linkages
formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of a
peptide; and oligonucleotide linkages formed by, for example, a phosphoramidite group, e.g., at
the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.
[0098] Such optional features of the conjugate, i.e., the introduction of one or more
degradable linkages into the polymer chain or to the IL-1 5 moiety, may provide for additional
control over the final desired pharmacological properties of the conjugate upon administration.
For example, a large and relatively inert conjugate (i.e., having one or more high molecular weight
PEG chains attachedthereto, for example, one or more PEG chains having a molecular weight
greater than about 10,000, wherein the conjugate possesses essentially no bioactivity) may be
administered, which is hydrolyzed to generate a bioactive conjugate possessing a portion of the
original PEG chain. In this way, the properties of the conjugate can be more effectively tailored to
balance the bioactivity of the conjugate over time.
[0099] The water-soluble polymer associated with the conjugate can also be "releasable."
That is, the water-soluble polymer releases (either through hydrolysis, enzymatic processes,
catalytic processes or otherwise), thereby resulting in the unconjugated IL-1 5 moiety. In some
instances, releasable polymers detach from the IL-15 moiety in vivo without leaving any fragment
of the water-soluble polymer. In other instances, releasable polymers detach from the IL-15
moiety in vivo leaving a relatively small fragment (e.g., a succinate tag) from the water-soluble
polymer. An exemplary cleavable polymer includes one that attaches to the IL-15 moiety via a
carbonate linkage.
[0100] Those of ordinary skill in the art will recognize that the foregoing discussion
concerning nonpeptidic and water-soluble polymer is by no means exhaustive and is merely
illustrative, and that all polymeric materials having the qualities described above are contemplated.
As used herein, the term "polymeric reagent" generally refers to an entire molecule, which can
comprise a water-soluble polymer segment and a functional group.
[0101] As described above, a conjugate of the invention comprises a water-soluble
polymer covalently attached to an IL-15 moiety. Typically, for any given conjugate, there will be
one to three water-soluble polymers covalently attached to one or more moieties having IL-1 5
activity. In some instances, however, the conjugate may have 1, 2, 3, 4, 5, 6, 7, 8 or more
water-soluble polymers individually attached to an IL-15 moiety. Any given water-soluble
polymer may be covalently attached to either an amino acid of the IL-15 moiety, or, when the IL-
15 moiety is (for example) a glycoprotein, to a carbohydrate of the IL-15 moiety. Attachment to a
carbohydrate may be carried out, e.g., using metabolic functionalization employing sialic
acid-azide chemistry [Luchansky et al. (2004) Biochemistry 43(38): 12358-12366] or other suitable
approaches such as the use of glycidol to facilitate the introduction of aldehyde groups [Heldt et
al. (2007) European Journal of Organic Chemistry 32:5429-5433].
[0102] The particular linkage within the moiety having IL-1 5 activity and the polymer
depends on a number of factors. Such factors include, for example, the particular linkage
chemistry employed, the particular IL-15 moiety, the available functional groups within the IL-15
moiety (either for attachment to a polymer or conversion to a suitable attachment site), the
presence of additional reactive functional groups within the IL-15 moiety, and the like.
[0103] The conjugates of the invention can be, although not necessarily, prodrugs,
meaning that the linkage between the polymer and the IL-15 moiety is hydrolytically releasable to
allow release of the parent moiety. Exemplary releasable linkages include carboxylate ester,
phosphate ester, thiol ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters,
peptides and oligonucleotides. Such linkages can be readily prepared by appropriate modification
of either the IL-15 moiety (e.g., the carboxyl group C terminus of the protein, or a side chain
hydroxyl group of an amino acid such as serine or threonine contained within the protein, or a
similar functionality within the carbohydrate) and/or the polymeric reagent using coupling
methods commonly employed in the art. Most preferred, however, are hydrolyzable linkages that
are readily formed by reaction of a suitably activated polymer with a non-modified functional
group contained within the moiety having IL-15 activity.
[0104] Alternatively, a hydrolytically stable linkage, such as an amide, urethane (also
known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide)
linkage can also be employed as the linkage for coupling the IL- 5 moiety. Again, a preferred
hydrolytically stable linkage is an amide. In one approach, a water-soluble polymer bearing an
activated ester can be reacted with an amine group on the IL-15 moiety to thereby result in an
amide linkage.
[0105] The conjugates (as opposed to an unconjugated IL-1 5 moiety) may or may not
possess a measurable degree of IL-15 activity. That is to say, a polymer-IL-15 moiety conjugate
in accordance with the invention will possesses anywhere from about 0.1% to about 100% of the
bioactivity of the unmodified parent IL-15 moiety. In some instances, the polymer-IL-15 moiety
conjugates may have greater than 100% bioactivity of the unmodified parent IL-15 moiety.
Preferably, conjugates possessing little or no IL-15 activity contain a hydrolyzable linkage
connecting the polymer to the moiety, so that regardless of the lack (or relatively lack) of activity
in the conjugate, the active parent molecule (or a derivative thereof) is released upon
aqueous-induced cleavage of the hydrolyzable linkage. Such activity may be determined using a
suitable in-vivo or in-vitro model, depending upon the known activity of the particular moiety
having IL-15 activity employed.
[0106] For conjugates possessing a hydrolytically stable linkage that couples the moiety
having IL-15 activity to the polymer, the conjugate will typically possess a measurable degree of
bioactivity. For instance, such conjugates are typically characterized as having a bioactivity
satisfying one or more of the following percentages relative to that of the unconjugated IL-15
moiety: at least about 2%, at least about 5%, at least about 10%, at least about 15% , at least about
25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about
100%, and more than 105% (when measured in a suitable model, such as those well known in the
art). Preferably, conjugates having a hydrolytically stable linkage (e.g., an amide linkage) will
possess at least some degree of the bioactivity of the unmodified parent moiety having IL- 15
activity.
[0107] Exemplary conjugates in accordance with the invention will now be described.
Typically, such an IL-15 moiety is expected to share (at least in part) a similar amino acid
sequence as the sequence provided in at least one of SEQ ID NOs: 1 through 3. Thus, while
reference will be made to specific locations or atoms within SEQ ID NOs: 1 through 3, such a
reference is for convenience only and one having ordinary skill in the art will be able to readily
determine the corresponding location or atom in other moieties having IL-15 activity. In
particular, the description provided herein for native human IL-15 is often applicable to fragments,
deletion variants, substitution variants or addition variants of any of the foregoing.
[0108] Amino groups on IL-15 moieties provide a point of attachment between the IL-1 5
moiety and the water-soluble polymer. Using the amino acid sequence provided in SEQ ID NOs:
1 through 3, it is evident that there are several lysine residues in each having an e-amino acid that
may be available for conjugation. Further, the N-terminal amine of any protein can also serve as a
point of attachment.
[0109] There are a number of examples of suitable polymeric reagents useful for forming
covalent linkages with available amines of an IL-15 moiety. Specific examples, along with the
coiTesponding conjugate, are provided in Table 1, below. In the table, the variable (n) represents
the number of repeating monomelic units and "-NH-(IL-15)" represents the residue of the IL-15
moiety following conjugation to the polymeric reagent. While each polymeric portion [e.g.,
(OCH2CH2)n or (CH2CH20 )n] presented in Table 1 terminates in a "CH3" group, other groups
(such as H and benzyl) can be substituted therefor.
Table 1
Amine-Selective Pol meric Rea ents and the IL-15 Moiet Con u ate Formed Therefrom
mPEG-Benzotriazole Carbonate Reagents
mPEG-Succinimidyl Reagents

un er certa n react on con t ons suc as p > Secondary Amine Linkage
O Conjugation of a polymeric reagent to an amino group of an IL-15 moiety can be
accomplished by a variety of techniques. In one approach, an IL-15 moiety can be conjugated to a
polymeric reagent functionalized with a succinimidyl derivative (or other activated ester group,
wherein approaches similar to those described for these alternative activated ester
group-containing polymeric reagents can be used). In this approach, the polymer bearing a
succinimidyl derivative can be attached to the IL-15 moiety in an aqueous media at a pH of 7 to
9.0, although using different reaction conditions (e.g., a lower pH such as 6 to 7, or different
temperatures and/or less than 15 °C) can result in the attachment of the polymer to a different
location on the IL-15 moiety. In addition, an amide linkage can be formed by reacting an
amine-terminated nonpeptidic, water-soluble polymer with an IL-15 moiety bearing an activating
a carboxylic acid group.
[0111] Exemplary conjugates are encompassed within the following structure
OI
I
H3CO-(CH2CH20 )n-X-CH-C-NH-(IL-1 5)
R
wherein:
(n) is an integer having a value of from 2 to 4000;
X is a spacer moiety;
R1 is an organic radical; and
IL-15 is a residue of an IL-15 moiety.
[0112] Exemplary conjugates are encompassed by the following structure:
O
II
H3CO-(CH2CH20) -CH2-CH-C-NH-(IL-1 5)
CH3
wherein (n) an integer having a value of from 2 to 4000 and IL-15 is a residue of an IL-15 moiety.
[01 13] Typical of another approach useful for conjugating the IL-1 5 moiety to a polymeric
reagent is use of reductive amination to conjugate a primary amine of an IL-15 moiety with a
polymeric reagent functionalized with a ketone, aldehyde or a hydrated form thereof (e.g., ketone
hydrate, aldehyde hydrate). In this approach, the primary amine from the IL-15 moiety reacts with
the carbonyl group of the aldehyde or ketone (or the corresponding hydroxyl-containing group of a
hydrated aldehyde or ketone), thereby forming a Schiff base. The Schiff base, in turn, can then be
reductively converted to a stable conjugate through use of a reducing agent such as sodium
borohydride. Selective reactions (e.g., at the N-terminus) are possible, particularly with a polymer
functionalized with a ketone or an alpha-methyl branched aldehyde and/or under specific reaction
conditions (e.g., reduced pH).
[0114] Exemplary conjugates of the invention wherein the water-soluble polymer is in a
branched form include those wherein the water-soluble polymer is encompassed within the
following structure:
wherein each (n) is independently an integer having a value of from 2 to 4000.
[0115] Exemplary conjugates of the invention are encompassed within the following
structure:
O
H3CO-(CH2CH20 ) -CH 2CH2-NH-C-0-| R
O -0-X-(CH 2CH20 ) C- -NH-(IL-1 5)
H3CO-(CH2CH20 ) -CH 2CH2-NH-C-0-
wherein:
each (n) is independently an integer having a value of from 2 to 4000;
X is spacer moiety;
(b) is an integer having a value 2 through 6;
(c) is an integer having a value 2 through 6;
R , in each occurrence, is independently H or lower alkyl; and
IL- 15 is a residue of an IL- 15 moiety.
[0116] Exemplary conjugates of the invention are encompassed within the following
structure:
o
H3CO-(CH CH20 ) -CH 2CH2-NH-C-CH o
II
O -OCH2CH2CH2-C-NH-(CH2CH20 )4-CH 2CH2CH2CH2-NH-(IL-15)
H3CO-(CH2CH20 )n-CH 2CH2-NH-C-0-
wherein:
each (n) is independently an integer having a value of from 2 to 4000; and
IL-15 is a residue of an IL-15 moiety.
Other exemplary conjugates of the invention are encompassed within following
O
H3CO-(CH2CH20 )n-CH 2CH2-NH-C-O R2
-0-(X)a-(CH 2CH20 )b. - C- -NH-(IL-1 5) O
I I I
H3CO-(CH2CH20 ) -CH 2CH2-NH-C -0- 1 R3
wherein:
each (n) is independently an integer having a value of from 2 to 4000;
(a) is either zero or one;
X, when present, is a spacer moiety comprised of one or more atoms;
(b') is zero or an integer having a value of one through ten;
(c) is an integer having a value of one through ten;
2, in each occurrence, is independently H or an organic radical;
R3, in each occurrence, is independently H or an organic radical; and
IL-15 is a residue of an IL-15 moiety.
[0118] Still further exemplary conjugates of the invention are encompassed within the
following structure:
OI
I H3CO-(CH2CH20 ) -CH2CH 2-NH-C- O
O -CH 2CH2CH2C-NH-(IL-1 5)
II
H3CO-(CH2CH20) -CH 2CH2-NH-C-0 -
wherein:
each (n) is independently an integer having a value of from 2 to 4000; and
IL- 15 is a residue of IL- 15 moiety.
[0119] Exemplary conjugates that include a releasable linkage include those in which an
IL-1 5 moiety are conjugated to a polymeric reagent encompassed within the following formula:
wherein:
POLY is a first water-soluble polymer;
POLY2 is a second water-soluble polymer;
X1 is a first spacer moiety;
X is a second spacer moiety;
H < is an ionizable hydrogen atom;
R1 is H or an organic radical;
R is H or an organic radical;
(a) is either zero or one;
(b) is either zero or one;
Rel , when present, is a first electron altering group;
R 2, when present, is a second electron altering group; and
(FG) is a functional group capable of reacting with an amino group of an active agent to
form a releasable linkage, such as a carbamate linkage. Within this formula, polymeric reagents
having the more defined structure are contemplated:
wherein each of POLY1, POLY2, X1, X2, R1, R2, H and (FG) is as previously defined, and R is a
first electron altering group; and R 2 is a second electron altering group.
[0120] Still further exemplary polymeric reagents fall within the following formulae:
wherein, for each structure and in each instance, (n) is independently an integer from 4 to 1500.
[0121] These releasable linkage-providing polymeric reagents can be prepared in
accordance with the procedures set forth in U.S. Patent Application Publication No.
2006/0293499.
[0122] Exemplary conjugates formed using releasable linkage-providing polymeric
reagents include those of the following formulae:
POLY1 is a first water-soluble polymer;
POLY2 is a second water-soluble polymer;
X1 is a first spacer moiety;
X2 is a second spacer moiety;
H is an ionizable hydrogen atom;
R1 is H or an organic radical;
R2 is H or an organic radical;
(a) is either zero or one;
(b) is either zero or one;
Rel , when present, is a first electron altering group;
R 2, when present, is a second electron altering group;
Y is O or S;
Y2 is O or S; and
IL-15 is a residue of an IL-15 moiety.
wherein, for each structure and in each instance, (n) is independently an integer from 4 to 1500,
and IL-15 is a residue of an IL-1 moiety.
[0124] Carboxyl groups represent another functional group that can serve as a point of
attachment on the IL-15 moiety. Structurally, the conjugate will comprise the following:
O
(IL-15)-C-X-POLY
where IL-15 and the adjacent carbonyl group corresponds to the carboxyl-containing IL-15
moiety, X is a linkage, preferably a heteroatom selected from O, N(H), and S, and POLY is a
water-soluble polymer such as PEG, optionally terminating in an end-capping moiety.
[0125] The C(0)-X linkage results from the reaction between a polymeric derivative
bearing a terminal functional group and a carboxyl-containing IL-1 moiety. As discussed above,
the specific linkage will depend on the type of functional group utilized. If the polymer is
end-functionalized or "activated" with a hydroxyl group, the resulting linkage will be a carboxylic
acid ester and X will be O. If the polymer backbone is functionalized with a thiol group, the
resulting linkage will be a thioester and X will be S. When certain multi-arm, branched or forked
polymers are employed, the C(0)X moiety, and in particular the X moiety, may be relatively more
complex and may include a longer linkage structure.
[0126] Water-soluble derivatives containing a hydrazide moiety are also useful for
conjugation at a carbonyl and carboxylic acid. To the extent that the IL-15 moiety does not
contain a carbonyl moiety or a carboxylic acid, one can be added using techniques known to one
of ordinary skill in the art. For example, a carbonyl moiety can be introduced by reducing a
carboxylic acid (e.g., the C-terminal carboxylic acid) and/or by providing glycosylated or glycated
(wherein the added sugars have a carbonyl moiety) versions of the IL-15 moiety. With respect to
IL- 15 moieties containing a carboxylic acid, a PEG-hydrazine reagent can, in the presence of a
coupling agent (e.g., DCC), covalently attach to the IL-15 moiety [e.g., mPEG-OCH2C(0)NHNH 2
+ HOC(0)-(IL-15) results in mPEG-OCH2C(0)NHNHC(0)-IL-15]. Specific examples of
water-soluble derivatives containing a hydrazide moiety, along with the corresponding conjugates,
are provided in Table 2, below. In addition, any water-soluble derivative containing an activated
ester (e.g., a succinimidyl group) can be converted to contain a hydrazide moiety by reacting the
water-soluble polymer derivative containing the activated ester with hydrazine (NH2-NH2) or tertbutyl
carbazate [NH2NHC0 C(CH3 )3]. In the table, the variable (n) represents the number of
repeating monomeric units and "-C(0)-(IL-15)" represents the residue of the IL-15 moiety
following conjugation to the polymeric reagent. Optionally, the hydrazone linkage can be reduced
using a suitable reducing agent. While each polymeric portion [e.g., (OCH2CH2) or
(CH2CH20 )n] presented in Table 2 terminates in a "CH3" group, other groups (such as H and
benzyl) can be substituted therefor.
Polymeric Reagent Corresponding Conjugate
o O
II
II
H3 H2 H2q ) H2 H2-0- H2 - -- H- H2
H3CO-(CH2CH20 CH2CH2- 0 - CH2 C-NH-NH-C(0)-(IL-1 5)
Hydrazone Linkage
mPEG-Hydrazine Reagents
0
II O
H3CO-(CH2CH20 ) CH2CH2- NH- C- H- H2
H3CO-(CH2CH20 )nCH2CH2--NH'C
II
- NH-NH-C(0)-(IL-15)
mPEG-Hydrazine Reagents
Hydrazone Linkage
0 0
II H
H3CO-(CH2CH20) CH2CH2-NH-NH-C-NH-NH 2
H3CO-(CH2CH20 ) CH2CH2-N-NH-C
II
-NH-NH-C(OHIL-1 5)
Hydrazone Linkage
mPEG-Hydrazine Reagents
S S
II
H3Ca(CH 2CH20 )nCH2CH2- NH- C- NH- NH2
H3CO-(CH2CH20) CH2CH2-- NH'C
II
- NH-NH-C(0)-(IL-15)
mPEG-Hydrazine Reagents
Hydrazone Linkage
S S
II H
H3Ca(CH 2CH20) CH2CH2-NH-NH-C-NH-NH2 II
H3CO-(CH2CH20)nCH2CH2--N-NH-C-NH-NH-C(0)-(IL-1 5;
Hydrazone Linkage
mPEG-Hydrazine Reagents
o o 0 0
H Ca(CH2CH 0) CH CH -NH-C
II
-NH-NH-C
II
-NH-NH 2
II
H3CO-(CH2CH20 )nCH2CH2--NH-C-NH-NH-C
II
-NH-NH-C(0)-(IL-15)
mPEG-Hydrazine Reagents
Hydrazone Linkage
0 O
II
H3CO-{CH2CH20)nCH2CH2-0— C- NH-NH2 H3Ca(CH2CH20) CH2CH2 --0-C
II
- NH- NH-C(0)-(IL-1 5)
mPEG-Hydrazine Reagents Hydrazone Linkage
O
0 0
H3CO-(CH2CH20 ) CH2-C-NH-NH2
H3CO-(CH 2CH20 )nCH2- C-NH-NH-C-(IL-15) mPEG-Hydrazine Reagents
C(0)NHNHC(0) Linkage
[0127] Thiol groups contained within the IL-15 moiety can serve as effective sites of
attachment for the water-soluble polymer. In particular, cysteine residues provide thiol groups
when the IL-15 moiety is a protein. The thiol groups in such cysteine residues can then be reacted
with an activated PEG that is specific for reaction with thiol groups, e.g., an N-maleimidyl
polymer or other derivative as described in U.S. Patent No. 5,739,208 and in WO 01/62827. In
addition, a protected thiol may be incoiporated into an oligosaccharide side chain of an activated
glycoprotein, followed by deprotection with a thiol-reactive water-soluble polymer.
[0128] Specific examples of reagents, along with the corresponding conjugate, are
provided in Table 3, below. In the table, the variable (n) represents the number of repeating
monomeric units and "-S-(IL-15)" represents the IL-15 moiety residue following conjugation to
the water-soluble polymer. While each polymeric portion [e.g., (OCH2CH2) or (CH2CH20 ) ]
presented in Table 3 terminates in a "CH3" group, other groups (such as H and benzyl) can be
substituted therefor.
[0129] With respect to SEQ ID NOs: 1 and 2 corresponding to exemplary IL-15 moieties,
it can be seen that there is a cysteine residue at position 125. Thus, an exemplary thiol attachment
sites is the cysteine located at position 125. Although it is preferred not to disrupt any disulfide
bonds, associated with a given IL-15 moiety, it may be possible to attach a polymer within the side
chain of one or more of these cysteine residues and retain a degree of activity. In addition, it is
possible to add a cysteine residue to the IL-15 moiety using conventional synthetic techniques.
See, for example, the procedure described in WO 90/12874 for adding cysteine residues, wherein
such procedure can be adapted for an IL-15 moiety. In addition, conventional genetic engineering
processes can also be used to introduce a cysteine residue into the IL-15 moiety. In some
embodiments, however, it is preferred not to introduce an additional cysteine residue and/or thiol
group.
Table 3
Thiol-Selective Pol meric Rea ents and the IL-15 Moiet Con ugate Formed Therefrom
branched mPEG2 Maleimide Reagent Thioether Linkage
Ό 30] With respect to conjugates formed from water-soluble polymers bearing one or
more maleimide functional groups (regardiess of whether the maleimide reacts with an amine or
thiol group on the IL-1 5 moiety), the corresponding maleamie acid form(s) of the water-soluble
polymer can also react with the IL- 5 moiety. Under certain conditions (e.g., a H of about 7-9
and in the presence of water), the maleimide ring will "open" to form the corresponding maleamie
acid. The maleamie acid, in torn, can react with an amine or thiol group of an IL-1 5 moiety.
Exemplary maleamie acid-based reactions are schematically shown below. POLY represents the
water-soluble polymer, and IL-1 5 represents the IL-1 5 moiety.
[0131] A representative conjugate in accordance with the invention can have the following
structure:
POLY-Lo, 1-C(0 )Z-Y-S-S -(IL- 5)
wherein POLY is a water-soluble polymer, L is an optional linker, Z is a heteroatom selected from
the group consisting of O.NH, and S, and Y is selected from the group consisting of C2-10 alkyi,
C;2 10 substituted aikyl, aryl, and substituted aryl, and IL-1 5 is an IL- 5 moiety. Polymeric
reagents that can be reacted with an IL-1 5 moiety and result in this type of conjugate are described
in U.S. Patent Application Publication No. 2005/0014903.
[0132] As previously indicated, exemplary conjugates of the invention wherein the
water-soluble polymer is in a branched form, will have the branched form of the water-soluble
polymer comprise the following structure:
wherein each (n) is independently an integer having a value of from 2 to 4000.
[0133] Exemplary conjugates having a water-soluble polymer in branched form are
prepared using the following reagent:
thereby forming a conjugate having the following structure:
o
H3C-(OCH2CH2)n-NH-C-0 -CH2 0 O
HC-OCH2CH2 CH2-C-NH-CH 2CH2-NH-C
H3C-(OCH2CH2) -NH-C-0-CH 2
wherein:
(for each structure) each (n) is independently an integer having a value of from 2 to 4000;
and
IL-15 is a residue of IL-15 moiety.
[0134] An additional exemplary conjugate can be formed using a reagent:
thereby forming a conjugate having the following structure:
wherein:
(for each structure) (n) is independently an integer having a value of from 2 to 4000; and
IL-15 is a residue of IL-15 moiety.
[0135] Conjugates can be formed using thiol-selective polymeric reagents in a number of
ways and the invention is not limited in this regard. For example, the IL-15 moiety - optionally
in a suitable buffer (including amine-containing buffers, if desired) ~ is placed in an aqueous
media at a pH of about 7-8 and the thiol-selective polymeric reagent is added at a molar excess.
The reaction is allowed to proceed for about 0.5 to 2 hours, although reaction times of greater than
2 hours (e.g., 5 hours, 10 hours, 12 hours, and 24 hours) can be useful if PEGylation yields are
determined to be relatively low. Exemplary polymeric reagents that can be used in this approach
are polymeric reagents bearing a reactive group selected from the group consisting of maleimide,
sulfone (e.g., vinyl sulfone), and thiol (e.g., functionalized thiols such as an ortho pyridinyl or
"OPSS").
[0136] With respect to polymeric reagents, those described here and elsewhere can be
purchased from commercial sources or prepared from commercially available starting materials.
In addition, methods for preparing the polymeric reagents are described in the literature.
[0137] The attachment between the IL-15 moiety and the non-peptidic water-soluble
polymer can be direct, wherein no intervening atoms are located between the IL-15 moiety and the
polymer, or indirect, wherein one or more atoms are located between the IL-15 moiety and the
polymer. With respect to the indirect attachment, a "spacer moiety" serves as a linker between the
residue of the IL-15 moiety and the water-soluble polymer. The one or more atoms making up the
spacer moiety can include one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen
atoms, and combinations thereof. The spacer moiety can comprise an amide, secondary amine,
carbamate, thioether, and/or disulfide group. Nonlimiting examples of specific spacer moieties
include those selected from the group consisting
of .0-, -S-, -S-S-, -C(O)-, -C(0)-NH-, -NH-C(0)-NH-, -0-C(0)-NH-, -C(S)-, -CH2-, -CH2-CH2-, -
CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-
, -0-CH 2-, -CH2-0-, -0-CH 2-CH2-, -CH2-0-CH 2-, -CH2-CH2-0-, -0-CH 2-CH2-CH2-, -CH2-0-C
H2-CH2-, -CH2-CH2-O-CH2-, -CH2-CH2-CH2-O-, -O-CH2-CH2-CH2-CH2-, -CH2-O-CH2-CH2-CH2
-, -CH2-CH2-0-CH 2-CH2-, -CH2-CH2-CH2-O-CH 2-, -CH2-CH2-CH2-CH2-0 -, -C(0)-NH-CH 2-, -C(
0)-NH-CH 2-CH2-, -CH2-C(0)-NH-CH 2-, -CH2-CH2-C(0)-NH-, -C(0)-NH-CH 2-CH2-CH2-, -CH2-
C(0)-NH-CH 2-CH2-, -CH2-CH2-C(0)-NH-CH 2-, -CH2-CH2-CH2-C(0)-NH-, -C(0)-NH-CH 2-CH2
-CH2-CH2-, -CH2-C(0)-NH-CH 2-CH2-CH2-, -CH2-CH2-C(0)-NH-CH 2-CH2-, -CH2-CH2-CH2-C(
0)-NH-CH -, -CH2-CH2-CH2-C(0)-NH-CH 2-CH2-, -CH2-CH2-CH2-CH2-C(0)-NH-, -C(0 )-0-CH
2-, -CH2-C(0 )-0-CH -, -CH2-CH2-
C(0 )-0-CH 2-, -C(0 )-0-CH 2-CH2-, -NH-C (0)-CH 2-, -CH2-NH-C (0)-CH 2-, -CH2-CH2-NH-C (0 )-
CH2-, -NH-C (0)-CH 2-CH2-, -CH2-NH-C (0)-CH 2-CH2-, -CH2-CH -NH-C (0)-CH 2-CH2-, -C(0)-N
H-CH2-, -C(0)-NH-CH 2-CH2-, -0-C (0)-NH-CH 2-, -0-C (0)-NH-CH 2-CH2-
, -NH-CH 2-, -NH-CH 2-CH2-, -CH2-NH-CH 2-, -CH2-CH2-NH-CH 2-, -C(O)-
CH2-, -C(0)-CH 2-CH2-, -CH2-C(0)-CH 2-, -CH2-CH2-
C(0)-CH 2-, -CH2-CH2-C(0)-CH 2-CH2-, -CH2-CH2-C(0 )-, -CH2-CH2-CH2-C(0)-NH-CH 2-CH2-N
H-, -CH2-CH2-CH2-C(0)-NH-CH 2-CH2-NH-C (0 )-, -CH2-CH2-CH2-C(0)-NH-CH 2-CH2-NH-C (0
)-CH 2-, -CH2-CH2-CH2-C(0)-NH-CH 2-CH2-NH-C (0)-CH 2-CH2-, -0-C (0)-NH-[CH 2]h-(OCH 2CH
2) -, bivalent cycloalkyl group, -0-, -S-, an amino acid, -N(R6)-, and combinations of two or more
of any of the foregoing, wherein R6 is H or an organic radical selected from the group consisting
of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and
substituted aryl, (h) is zero to six, and (j) is zero to 20. Other specific spacer moieties have the
following structures: -C(0)-NH-(CH 2) -6-NH-C (0 )-, -NH-C (0)-NH-(CH 2) -6-NH-C (0 )-,
and -0-C (0)-NH-(CH ) - -NH-C (0 )-, wherein the subscript values following each methylene
indicate the number of methylenes contained in the structure, e.g., (CH2) i-6 means that the
structure can contain 1, 2, 3, 4, 5 or 6 methylenes. Additionally, any of the above spacer moieties
may further include an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomer
units [i.e., -(CH 2CH20 ) - o]. That is, the ethylene oxide oligomer chain can occur before or after
the spacer moiety, and optionally in between any two atoms of a spacer moiety comprised of two
or more atoms. Also, the oligomer chain would not be considered part of the spacer moiety if the
oligomer is adjacent to a polymer segment and merely represent an extension of the polymer
segment.
[0138] Compositions
[0139] The conjugates are typically part of a composition. Generally, the composition
comprises a plurality of conjugates, preferably although not necessarily, each conjugate is
comprised of the same IL-15 moiety (i.e., within the entire composition, only one type of IL-15
moiety is found). In addition, the composition can comprise a plurality of conjugates wherein any
given conjugate is comprised of a moiety selected from the group consisting of two or more
different IL-15 moieties (i.e., within the entire composition, two or more different IL-15 moieties
are found). Optimally, however, substantially all conjugates in the composition (e.g., 85% or
more of the plurality of conjugates in the composition) are each comprised of the same IL-15
moiety.
[0140] The composition can comprise a single conjugate species (e.g., a monoPEGylated
conjugate wherein the single polymer is attached at the same location for substantially all
conjugates in the composition) or a mixture of conjugate species (e.g., a mixture of
monoPEGylated conjugates where attachment of the polymer occurs at different sites and/or a
mixture monPEGylated, diPEGylated and triPEGylated conjugates). The compositions can also
comprise other conjugates having four, five, six, seven, eight or more polymers attached to any
given moiety having IL- 15 activity. In addition, the invention includes instances wherein the
composition comprises a plurality of conjugates, each conjugate comprising one water-soluble
polymer covalently attached to one IL-15 moiety, as well as compositions comprising two, three,
four, five, six, seven, eight, or more water-soluble polymers covalently attached to one IL- 15
moiety.
[0141] With respect to the conjugates in the composition, the composition will satisfy one
or more of the following characteristics at least about 85% of the conjugates in the composition
will have from one to four polymers attached to the IL-15 moiety; at least about 85% of the
conjugates in the composition will have from one to three polymers attached to the IL-15 moiety;
at least about 85% of the conjugates in the composition will have from one to two polymers
attached to the IL-15 moiety; at least about 85% of the conjugates in the composition will have
one polymer attached to the IL-15 moiety; at least about 95% of the conjugates in the composition
will have from one to five polymers attached to the IL-15 moiety; at least about 95% of the
conjugates in the composition will have from one to four polymers attached to the IL-15 moiety;
at least about 95% of the conjugates in the composition will have from one to three polymers
attached to the IL-15 moiety; at least about 95% of the conjugates in the composition will have
from one to two polymers attached to the IL-15 moiety; at least about 95% of the conjugates in the
composition will have one polymer attached to the IL-15 moiety; at least about 99% of the
conjugates in the composition will have from one to five polymers attached to the IL-15 moiety; at
least about 99% of the conjugates in the composition will have from one to four polymers attached
to the IL-15 moiety; at least about 99% of the conjugates in the composition will have from one to
three polymers attached to the IL-15 moiety; at least about 99% of the conjugates in the
composition will have from one to two polymers attached to the IL-15 moiety; and at least about
99% of the conjugates in the composition will have one polymer attached to the IL-15 moiety. It
is understood that a reference to a range of polymers, e.g., "from x to y polymers," contemplates a
number of polymers x to y inclusive (that is, for example, "from one to three polymers"
contemplates one polymer, two polymers and three polymers, "from one to two polymers"
contemplates one polymer and two polymers, and so forth). In addition, it is also contemplated
that given conjugate having two or more polymers attached to the IL-15 moiety will have mixtures
of stable and releasably attached polymers (wherein at least polymer is stably attached to the IL-15
moiety and at least one polymer is releasably attached to the IL-15 moiety).
[0142] In one or more embodiments, it is preferred that the conjugate-containing
composition is free or substantially free of albumin. It is also preferred that the composition is
free or substantially free of proteins that do not have IL-15 activity. Thus, it is preferred that the
composition is 85%, more preferably 95%, and most preferably 99% free of albumin.
Additionally, it is preferred that the composition is 85%, more preferably 95%, and most
preferably 99% free of any protein that does not have IL-15 activity. To the extent that albumin is
present in the composition, exemplary compositions of the invention are substantially free of
conjugates comprising a poly(ethylene glycol) polymer linking a residue of an IL-15 moiety to
albumin.
[0143] Control of the desired number of polymers for any given moiety can be achieved
by selecting the proper polymeric reagent, the ratio of polymeric reagent to the IL-15 moiety,
temperature, pH conditions, and other aspects of the conjugation reaction. In addition, reduction
or elimination of the undesired conjugates (e.g., those conjugates having four or more
attachedpolymers) can be achieved through purification means.
[0144] For example, the polymer-IL-15 moiety conjugates can be purified to obtain/isolate
different conjugated species. Specifically, the product mixture can be purified to obtain an average
of anywhere from one, two, three, four, five or more PEGs per IL-15 moiety, typically one, two or
three PEGs per IL-15 moiety. The strategy for purification of the final conjugate reaction mixture
will depend upon a number of factors, including, for example, the molecular weight of the
polymeric reagent employed, the particular IL-15 moiety, the desired dosing regimen, and the
residual activity and in vivo properties of the individual conjugate(s).
[0145] If desired, conjugates having different molecular weights can be isolated using gel
filtration chromatography and/or ion exchange chromatography. That is to say, gel filtration
chromatography is used to fractionate differently numbered polymer-to- IL-15 moiety ratios (e.g.,
1-mer, 2-mer, 3-mer, and so forth, wherein "1-mer" indicates 1 polymer to IL-15 moiety, "2-mer"
indicates two polymers to IL-15 moiety, and so on) on the basis of their differing molecular
weights (where the difference corresponds essentially to the average molecular weight of the
water-soluble polymer portion). For example, in an exemplary reaction where a 35,000 Dalton
protein is randomly conjugated to a polymeric reagent having a molecular weight of about 20,000
Daltons, the resulting reaction mixture may contain unmodified protein (having a molecular
weight of about 35,000 Daltons), monoPEGylated protein (having a molecular weight of about
55,000 Daltons), diPEGylated protein (having a molecular weight of about 75,000 Daltons), and
so forth.
[0146] While this approach can be used to separate PEG and other polymer-IL- 15 moiety
conjugates having different molecular weights, this approach is generally ineffective for separating
positional isoforms having different polymer attachment sites within the IL-15 moiety. For
example, gel filtration chromatography can be used to separate from each other mixtures of PEG
1-mers, 2-mers, 3-mers, and so forth, although each of the recovered conjugate compositions may
contain PEG(s) attached to different reactive groups (e.g., lysine residues) within the IL-15
moiety.
[0147] Gel filtration columns suitable for carrying out this type of separation include
Superdex™ and Sephadex™ columns available from GE Healthcare (Buckinghamshire, UK).
Selection of a particular column will depend upon the desired fractionation range desired. Elution
is generally carried out using a suitable buffer, such as phosphate, acetate, or the like. The
collected fractions may be analyzed by a number of different methods, for example, (i) absorbance
at 280 m for protein content, (ii) dye-based protein analysis using bovine serum albumin (BSA)
as a standard, (iii) iodine testing for PEG content (Sims et al. (1980) Anal. Biochem, 107 :60-63),
(iv) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), followed by staining
with barium iodide, and (v) high performance liquid chromatography (HPLC).
[0148] Separation of positional isoforms is carried out by reverse phase chromatography
using a reverse phase-high performance liquid chromatography (RP-HPLC) using a suitable
column (e.g., a C18 column or C3 column, available commercially from companies such as
Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange
column, e.g., a Sepharose™ ion exchange column available from GE Healthcare. Either approach
can be used to separate polymer-active agent isomers having the same molecular weight (i.e.,
positional isoforms).
[0149] The compositions are preferably substantially free of proteins that do not have IL-
15 activity. In addition, the compositions preferably are substantially free of all other
noncovalently attached water-soluble polymers. In some circumstances, however, the
composition can contain a mixture of polymer- IL-15 moiety conjugates and unconjugated IL-15
moiety.
[0150] Optionally, the composition of the invention further comprises a pharmaceutically
acceptable excipient. If desired, the pharmaceutically acceptable excipient can be added to a
conjugate to form a composition.
[0151] Exemplary excipients include, without limitation, those selected from the group
consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants,
buffers, acids, bases, amino acids, and combinations thereof.
[0152] A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid,
an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate
excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose,
D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose,
and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and
the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol),
pyranosyl sorbitol, myoinositol, cyclodextrins, and the like.
[0153] The excipient can also include an inorganic salt or buffer such as citric acid,
sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate
monobasic, sodium phosphate dibasic, and combinations thereof.
[0154] The composition can also include an antimicrobial agent for preventing or deterring
microbial growth. Nonlimiting examples of antimicrobial agents suitable for one or more
embodiments of the present invention include benzalkonium chloride, benzethonium chloride,
benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,
phenylmercuric nitrate, thimersol, and combinations thereof.
[0155] An antioxidant can be present in the composition as well. Antioxidants are used to
prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the
preparation. Suitable antioxidants for use in one or more embodiments of the present invention
include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,
hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde
sulfoxylate, sodium metabisulfite, and combinations thereof.
[0156] A surfactant can be present as an excipient. Exemplary surfactants include:
polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (both of
which are available from BASF, Florham Park, NJ); sorbitan esters; lipids, such as phospholipids
such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably
not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; and IL-151ating
agents, such as EDTA, zinc and other such suitable cations.
[0157] Acids or bases can be present as an excipient in the composition. Nonlimiting
examples of acids that can be used include those acids selected from the group consisting of
hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid,
trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and
combinations thereof. Examples of suitable bases include, without limitation, bases selected from
the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium
hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate,
sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and
combinations thereof.
[0158] One or more amino acids can be present as an excipient in the compositions
described herein. Exemplary amino acids in this regard include arginine, lysine and glycine.
[0159] The amount of the conjugate (i.e., the conjugate formed between the active agent
and the polymeric reagent) in the composition will vary depending on a number of factors, but will
optimally be a therapeutically effective dose when the composition is stored in a unit dose
container (e.g., a vial). In addition, the pharmaceutical preparation can be housed in a syringe. A
therapeutically effective dose can be determined experimentally by repeated administration of
increasing amounts of the conjugate in order to determine which amount produces a clinically
desired endpoint.
[0160] The amount of any individual excipient in the composition will vary depending on
the activity of the excipient and particular needs of the composition. Typically, the optimal
amount of any individual excipient is determined through routine experimentation, i.e., by
preparing compositions containing varying amounts of the excipient (ranging from low to high),
examining the stability and other parameters, and then determining the range at which optimal
performance is attained with no significant adverse effects.
[0161] Generally, however, the excipient will be present in the composition in an amount
of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more
preferably from about 5 to about 95% by weight of the excipient, with concentrations less than
30% by weight most preferred.
[0162] These foregoing pharmaceutical excipients along with other excipients are
described in "Remington: The Science & Practice of Pharmacy", 1 th ed., Williams & Williams,
(1995), the "Physician's Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and
Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical
Association, Washington, D.C., 2000.
[0163] The compositions encompass all types of formulations and in particular those that
are suited for injection, e.g., powders or lyophilates that can be reconstituted as well as liquids.
Examples of suitable diluents for reconstituting solid compositions prior to injection include
bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's
solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid
pharmaceutical compositions, solutions and suspensions are envisioned.
[0164] The compositions of one or more embodiments of the present invention are
typically, although not necessarily, administered via injection and are therefore generally liquid
solutions or suspensions immediately prior to administration. The pharmaceutical preparation can
also take other forms such as syrups, creams, ointments, tablets, powders, and the like. Other
modes of administration are also included, such as pulmonary, rectal, transdermal, transmucosal,
oral, intrathecal, intratumorally, peritumorally, intraperitonally, subcutaneous, intra-arterial, and so
forth.
[0165] The invention also provides a method for administering a conjugate as provided
herein to a patient suffering from a condition that is responsive to treatment with conjugate. The
method comprises administering to a patient, generally via injection, a therapeutically effective
amount of the conjugate (preferably provided as part of a pharmaceutical composition). As
previously described, the conjugates can be injected (e.g., intramuscularly, subcutaneously and
parenterally). Suitable formulation types for parenteral administration include ready-for-injection
solutions, dry powders for combination with a solvent prior to use, suspensions ready for injection,
dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid
concentrates for dilution prior to administration, among others.
[0166] The method of administering the conjugate (preferably provides as part of a
pharmaceutical composition) can optionally be conducted so as to localize the conjugate to a
specific area. For example, the liquid, gel and solid formulations comprising the conjugate could
be surgically implanted in a diseased area (such as in a tumor, near a tumor, in an inflamed area,
and near an inflamed area). Conveniently, organs and tissue can also be imaged in order to ensure
the desired location is better exposed to the conjugate.
[0167] The method of administering may be used to treat any condition that can be
remedied or prevented by administration of the conjugate. Those of ordinary skill in the art
appreciate which conditions a specific conjugate can effectively treat. For example, the
conjugates can be used either alone or in combination with other pharmacotherapy to treat patients
suffering from a condition. Exemplary conditions include, without limitation: melanoma, renal
cancer, non-small cell lung cancer, small cell lung cancer, prostate cancer, breast cancer,
hematoligcal cancers, head and neck cancer, ovarian cancer, and colon cancer. Advantageously,
the conjugate can be administered to the patient prior to, simultaneously with, or after
administration of another active agent.
[0168] The actual dose to be administered will vary depending upon the age, weight, and
general condition of the subject as well as the severity of the condition being treated, the judgment
of the health care professional, and conjugate being administered. Therapeutically effective
amounts are known to those skilled in the art and/or are described in the pertinent reference texts
and literature. Generally, a therapeutically effective amount will range from about 0.001 mg to
100 mg, preferably in doses from 0.01 mg/day to 75 mg/day, and more preferably in doses from
0.10 mg/day to 50 mg/day. A given dose can be periodically administered up until, for example,
the clinician determines an appropriate endpoint (e.g., cure, regression, partial regression, and so
forth) is achieved.
[0169] The unit dosage of any given conjugate (again, preferably provided as part of a
pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the
judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be
known by those of ordinary skill in the art or can be determined experimentally using routine
methods. Exemplary dosing schedules include, without limitation, administration once daily,
three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any
combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is
halted.
[01 70] It is to be understood that while the invention has been described in conjunction
with the preferred specific embodiments thereof, that the foregoing description as well as the
examples that follow are intended to illustrate and not limit the scope of the invention. Other
aspects, advantages and modifications within the scope of the invention will be apparent to those
skilled in the art to which the invention pertains.
[0171] All articles, books, patents and other publications referenced herein are hereby
incorporated by reference in their entireties.
EXPERIMENTAL
[0172] The practice of the invention will employ, unless otherwise indicated, conventional
techniques of organic synthesis, biochemistry, protein purification and the like, which are within
the skill of the art. Such techniques are fully explained in the literature. See, for example, J .
March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York:
Wiley-Interscience, 1992), supra.
[0173] In the following examples, efforts have been made to ensure accuracy with respect
to numbers used (e.g., amounts, temperatures, etc.) but some experimental error and deviation
should be taken into account. Unless indicated otherwise, temperature is in degrees C and
pressure is at or near atmospheric pressure at sea level. Each of the following examples is
considered to be instructive to one of ordinary skill in the art for carrying out one or more of the
embodiments described herein.
[0174] An aqueous solution ("stock solution") comprising recombinant IL-1 5 ("rhIL-1 5")
corresponding to the amino acid sequence of SEQ ID NO: 1 for use in the examples.
[0175] SDS-PAGE Analysis Samples were analyzed by sodium dodecyl sulfatepolyacrylamide
gel electrophoresis (SDS-PAGE) using Invitrogen gel electrophoresis system
(XCell SureLock Mini-Cell). Samples were mixed with sample buffer. Then, the prepared
samples were loaded onto a NuPAGE Novex precast gel and run for approximately thirty minutes.
[0176] SEC-HPLC Analysis It is contemplated that size exclusion chromatography
(SEC-HPLC) analysis will be used in connection with characterizing conjugate composition.
When performed, SEC-HPLC analysis can be earned out on an Agilent 100 HPLC system
(Agilent). Samples can be analyzed using a Shodex protein W-804 column (300 x 8 mm,
Phenomenex), and a mobile phase consisting of sodium phosphate and sodium sulfate, pH 7. The
flow rate for the column can be 0.5 ml/min. Eluted protein and PEG-protein conjugates can be
detected using UV at 280nm and 220nm.
[0177] A related characterization technique, reversed phase high-performance liquid
chromatography (RP-HPLC), can also be performed on an Agilent 1100 HPLC system (Agilent).
Samples can be analyzed using a Zorbax 300SB-C3 column (3.5 mh particle size, 150 mm x 3.0
mm, Agilent), and mobile phases consisting of 0.1% triiluoroacetic acid in water (buffer A) and
0.1% trifluoroacetic acid in acetonitrile (buffer B). The flow rate for the column can be 0.3
ml/min. The protein and PEG-protein conjugates can be eluted with a linear gradient over 20
minutes, and were detected using UV at 280nm.
[0178] Generally, purification can be carried out on an SP-HP column using 10 M
sodium citrate (pH 2.7). The pH is lowered to optimize binding of the conjugates to the column.
The conjugates are bound in this buffer and eluted using a 0=500 mM NaCl gradient.
Example 1
Measurement of IL-15 Activity of rIL-15 Based on STAT5 Phosphorylation in CTLL-2 Cells
[0179] One day prior to assay, CTLL-2 cells were split into fresh growth medium (RPMI
1640 + 10% FBS + 10% T-STIM + 2 mM L-glut + 1 M Na-pyruvate). On the day of assay,
cells were pre-incubated in assay medium (RPMI 1640 + 1% FBS + 2 mM L-glut + 1mM Napyruvate)
for at least 4 hours and then placed in a 96-well plate at 50,000 cells/well in the assay
medium.
[0180] Dilutions of the test article were prepared in an appropriate buffer immediately
prior to assay and each dilution of test article was incubated in a separate well of the CTLL-2
cells. The phosphorylation of STAT5 was then determined using the MSD
Phospho(Tyr694)/Total STATa,b Whole Cell Lysate Kit (catalog #K15163D, Meso Seal
Diagnostics, LLC, Gaithersburg, MD).
[0181] rIL-15 obtained from PeproTech (catalog #200-15) demonstrated IL-15 activity by
exhibiting an average pSTAT5 EC50 at 0.20705 ng/mL at 5 minutes (an average over two runs)
and 0.08187 ng/mL at 10 minutes.
Example 2
PEGylation of rIL-15 with Branched mPEG-N-Hydroxysuccinimidyl Derivative, 20kDa
mPEG2-ru-20K-N-Hydroxylsuccinimidyl Derivative, 20kDa, ("mPEG2-ru-20K-NHS")
[0182] mPEG2-ru-20K-NHS, stored at -80 °C under argon, was warmed to ambient
temperature under nitrogen purging. A stock solution (200 mg/mL) of mPEG2-ru-20K-NHS was
prepared in 2 mM HC1, and mPEG2-ru-20K-NHS was added to the rIL-15 with molar ratios of
mPEG2-ru-20K-NHS to rIL-15 ranging from abound 5:1 to 100:1. The final concentration of rlL-
15 in the mixture was 0.5 raG/mL (0.031 mM). Sodium bicarbonate buffer ( 1 M, pH 8.0) was
added to the mixture to reach a final concentration of 100 mM, and conjugation was allowed to
proceed for thirty minutes to provide [mPEG2-ru-20K]-[rIL-15] conjugates. After thirty minutes,
quenching was achieved by adding 1M glycine (pH 6.0) to the reaction mixture to achieve a final
concentration of 100 mM.
[0183] A typical conjugation product profile of [mPEG2-ru-20K]-[rIL-l 5] is provided in
FIG. 1. As shown in FIG. 1, increasing molar ratios of mPEG2-ru-20K-NHS to rIL-15 from
about 5:1 (lane 3), about 10:1 (lane 4), about 25:1 (lane 5), about 50:1 (lane 6), and about 100:1
(lane 7) result in a shift from mono-PEG-IL-15 as major product (lane 3) to di-, tri-PEG-IL-15 and
higher species. The distinct mono-, di-, tri- and higher PEG-IL-15 conjugates are to be purified
and characterized.
[0184] Generally, purification can be carried out on an SP-HP column using 10 mM
sodium citrate (pH 2.7). The pH is lowered to optimize binding of the conjugates to the column.
The conjugates are bound in this buffer and eluted using a 0=500 mM NaCl gradient.
Example 3
PE -C2-fmoc-20K-NHS
mPEG 2-C2-fomc-20K-N-Hydroxysuccinimide Derivative, 20kDa, ("mPEG2-C2-fmoc-
20K-NHS")
[0185] mPEG2-C2-fmoc-20K-NHS, stored at -80 °C under argon, was warmed to ambient
temperature under nitrogen purging. A stock solution (200 mg/mL) of mPEG2-C2-fmoc-
20K-NHS was prepared in 2 mM HC1, and mPEG2-C2-fmoc-20K-NHS was added to the rIL-15
with molar ratios of mPEG -C2-fmoc-20K-NHS to rIL-15 ranging from 5:1 of 100:1. The final
concentration of rIL-15 in the mixture was 0.5 mg/mL (0.031 mM). Sodium bicarbonate buffer ( 1
M, pH 8.0) was added to the mixture to reach a final concentration of 100 mM, and conjugation
was allowed to proceed for thirty minutes to provide [mPEG2-C2-fmoc-20K]-[rIL-15] conjugates.
After thirty minutes, quenching was achieved by adding 1M glycine (pH 6.0) to the reaction
mixture to achieve a final concentration of 100 mM. The pH of the quenched reaction mixture was
then adjusted to 4.0 using glacial acetic acid prior to column chromatography purification and
characterization.
Example 4
PEG latio -20K-NHS
mPEG2-CAC-fmoc-20K-N-Hydroxysuccinimide Derivative, 20kDa, ("mPEG2-CAC-fmoc-
20K-NHS")
[0186] mPEG2-CAC-fmoc-20K-NHS, stored at -80 °C under argon, was warmed to ambient
temperature under nitrogen purging. A stock solution (200 mg/mL) of mPEG2-CAC-fmoc-
20K-NHS was prepared in 2 mM HCl, and mPEG2-CAC-fmoc-20K-NHS was added to the rIL-15
with molar ratios of mPEG2-CAC-fmoc-20K-NHS to rIL-15 ranged from 5:1 of 100:1. The final
concentration of rIL-1 5 in the mixture was 0.5 mg/mL (0.03 mM). Sodium bicarbonate buffer ( 1
M, pH 8.0) was added to the mixture to reach a final concentration of 100 mM, and conjugation
was allowed to proceed for thirty minutes to provide [mPEG2-CAC-fmoc-20K]-[rIL-15]
conjugates. After thirty minutes, quenching was achieved by adding 1M glycine (pH 6.0) to the
reaction mixture to achieve a final concentration of 100 mM. The pH of the quenched reaction
mixture was then adjusted to 4.0 using glacial acetic acid prior to column chromatography
purification and characterization.
Example 5
PEGylation of rIL-15 with Branched mPEG-N-Hydroxysuccinimidyl Derivative, 40kDa
mPEG2-ru-40K-N-HydiOxylsuccinimidyl Derivative, 40kDa, ("mPEG2-ru-40K-NHS")
[0187] mPEG2-ru-40K-NHS, stored at -80 °C under argon, was wanned to ambient
temperature under nitrogen purging. A stock solution (200 mg/mL) of mPEG2-ru-40K-NHS was
prepared in 2 mM HCl, and mPEG2-ru-40K-NHS was added to rIL-15 with molar ratios ranging
from 5:1 of 100:1. The final concentration of rIL-15 in the mixture was 0.5 mg/mL (0.031 mM).
Sodium bicarbonate buffer ( 1 M, pH 8.0) was added to the mixture to reach a final concentration
of 100 mM, and conjugation was allowed to proceed for thirty minutes to provide [mPEG2-ru-
40K-]-[rIL-15] conjugates. After thirty minutes, quenching was achieved by adding 1M glycine
(pH 6.0) to the reaction mixture to achieve a final concentration of 100 mM. The pH of the
quenched reaction mixture was then adjusted to 4.0 using glacial acetic acid prior to column
chromatography purification and characterization.
[0188] A conjugation product profile of [mPEG2-ru-40K]-[rIL-l 5] is provided in FIG. 2.
As shown in FIG. 2, increasing molar ratios of mPEG2-ru-40K-NHS to rIL-15 from about 5:1
(lane 2), about 10:1 (lane 3), about 25:1 (lane 4), about 50:1 (lane 5), and about 100:1 (lane 6)
result in a shift from lower PEGylated products to higher PEGylated products. The distinct
conjugates can be purified and characterized.
Example 6
PE -fmoc-40K-NHS
mPEG2-C2-fomc-40K-N-HydiOxysuccinimide Derivative, 40kDa, ("mPEG2-C2-fmoc-
40K-NHS")
[0189] mPEG2-C2-fmoc-40K-NHS, stored at -80 °C under argon, was warmed to ambient
temperature under nitrogen purging. A stock solution (200 mg/mL) of mPEG2-C2-fmoc-
40K-NHS was prepared in 2 mM HCl, and mPEG2-C2-fmoc-40K-NHS was added to the rIL-15
with molar ratios of mPEG2-C2-fmoc-40K-NHS to rIL-15 ranging from 5:1 of 100:1. The final
concentration of rIL-15 in the mixture was 0.5 mg/mL (0.03 mM). Sodium bicarbonate buffer ( 1
M, pH 8.0) was added to the mixture to reach a final concentration of 100 mM, and conjugation
was allowed to proceed for thirty minutes to provide [mPEG2-C2-fmoc-40K]-[rIL-15] conjugates.
After thirty minutes, quenching was achieved by adding 1M glycine (pH 6.0) to the reaction
mixture to achieve a final concentration of 100 mM. The pH of the quenched reaction mixture was
then adjusted to 4.0 using glacial acetic acid prior to column chromatography purification and
characterization.
[0190] Following this same general approach, conjugates were prepared using lOkDa and
20kDa versions of the mPEG2-C2-fmoc-40K-N-hydroxysuccinimide derivative.
Example 7
PEG latio -CAC-fmoc-40K-NHS
mPEG2-CAC-fmoc-40K-N-Hydroxysuccinimide Derivative, 40kDa, ("mPEG2-CAC-fmoc-
40K-NHS")
[0191] mPEG2-CAC-fmoc-40K-NHS, stored at -80 °C under argon, was warmed to
ambient temperature under nitrogen purging. A stock solution (200 mg/mL) of mPEG2-CACfmoc-
40K-NHS was prepared in 2 mM HCl, and mPEG2-CAC-fmoc-40K-NHS was added to the
rIL-15 with molar ratios of mPEG2-CAC-fmoc-40K-NHS to rIL-15 ranged from 5:1 of 100:1.
The final concentration of rIL-15 in the mixture was 0.5 mg/mL (0.031 mM). Sodium bicarbonate
buffer ( 1 M, pH 8.0) was added to the mixture to reach a final concentration of 100 mM, and
conjugation was allowed to proceed for thirty minutes to provide [mPEG2-CAC-fmoc-40K]-[iTL-
15] conjugates. After thirty minutes, quenching was achieved by adding 1M glycine (pH 6.0) to
the reaction mixture to achieve a final concentration of 100 mM. The pH of the quenched reaction
mixture was then adjusted to 4.0 using glacial acetic acid prior to column chromatography
purification and characterization.
[0192] Following this same general approach, conjugates were prepared using lOkDa and
20kDa versions of the mPEG2-CAC-fmoc-40K-N-hydroxysuccinimide derivative. In these
additional preparations, a reagent :rIL- 15 molar ratio of 100:1 was used and pH adjustment to 2.7
was carried out with hydrochloric acid prior to purification and characterization (rather than
adjustment to pH 4.0 using glacial acetic acid).
Example 8
Measurement of IL-15 Activity of rIL-15 Conjugates
Based on STAT5 Phosphorylation in CTLL-2 Cells
[0193] One day prior to the assay, CTLL-2 cells were split into fresh growth medium
[RPMI 1640 supplemented with 10% FBS, 10% T-cell culture supplement (catalog #3541 15,
Coming, Inc., Tewksbury, MA), 2 mM L-glutamate, and 1mM sodium pyruvate]. On the day of
assay, cells were pre-incubated in assay medium (RPMI 1640 supplemented with 1% FBS, 2 mM
L-glutamate, and 1mM sodium pyruvate) for at least four hours, and then plated in assay medium
in a 96-well plate at 50,000 cells/well. Dilutions of the test article were prepared in an appropriate
buffer immediately prior to assay. Stimulation of CTLL-2 cells was initiated by the transfer of
25X test article solutions to triplicate wells containing CTLL-2 cells. Plates were incubated at
37 °C, 5% C0 2 for 10 minutes, and the reaction was stopped by cell lysis. Detection of phospho-
STAT5 and total STAT5 protein levels in cell lysates was performed using the MSD
Phospho(Tyr694)/Total STATa,b Whole Cell Lysate Kit (catalog #K15163D, Meso Scale
Diagnostics, LLC, Gaithersburg, MD). Following a 10 minute treatment, recombinant human
IL-15 obtained from PeproTech (catalog #200-15) demonstrated IL-15 activity by inducing
STAT5 phosporylation in CTLL-2 cells with an average EC50 of 0.063 +/- 0.028 ng/mL, which
served as the control. Conjugates prepared in accordance with Examples 2, 3 and 5 were tested,
with the results following a 10 minute treatment provided in Table 4.
Table 4
IL-15 Activit of rIL-15 Con u ates Based on STAT5 Phos hor lation in CTLL-2 Cells
""Conjugates with releaseable linkages were tested without an additional evaluation of the extent of any
released polymers within the fraction.
Example 9
Evaluation of Antitumor Activity of rIL-15 Conjugates
in B16F10 Melanoma Tumor
[0194] To test and compare the efficacy of rIL-15 conjugates in an in vivo model, syngenic
tumors were induced in 6-8 week old female C57BL/6 mice by subcutaneously injecting, into post
ventral abdominal region, mouse metastatic B16F10 melanoma cells at a density of 1c 106 cells
mL-1 in 100 L volume of non-serum containing cell culture medium. The mice developed
tumors of approximately 100 mm3 by the end of 6-8 days. The mice were divided into six groups,
each group consisting of 10 animals. Each group was assigned a different test article as shown in
the Table 5.
Table 5
Grou Assi nments and Administration Parameters
[0195] Groups A through D correspond to rIL-15 conjugates that were administered at 0.6
mg/kg body weight by intravenous injection to the mice only once while Group E represents
unconjugated IL-15 administered every other day via intraperitoneal injection. Group F represents
a vehicle control, wherein phosphate buffered saline was administered by intravenous injection.
[0196] Following dose administration, the mice were measured for changes in body weight
and for the growth of subcutaneous metastatic melanoma tumors by digital calipers three times a
week. Daily clinical signs were also monitored. The study end point was reached when the mean
volume of the tumors reach 1500 mm3 (i.e., a mean tumor volume of 1500 mm3) .
[0197] The first group to reach a mean tumor volume of 1500 mm3 was Group F (the
vehicle control group), which occurred on day 13 of the study. All other groups (i.e., Groups A
through E) reached the study endpoint around day 24, thereby indicating efficacy of both IL-15
and the rIL-15 conjugates in inhibiting tumor growth.
[0198] Furthermore, although, the tumor growth delay observed to reach 1000 mm for the
unconjugated IL-15 and rIL-15 conjugates ranged from 1.5 to 4.5 days, the rIL-15 conjugates were
shown to be about three time more potent to unconjugated IL-15 at least because the total dose of
unconjugated IL-15 (administration of 0.3 mg/kg every day for six doses) was three times the
single dose of a conjugated rIL-15 (administration of a single dose of 0.6 mg/kg). See Table 6.
Table 6
Tumor Growth Measures Associated with Grou s A Through F
[0199] In addition to the increased potency of the rIL-15 conjugates, there were no
significant clinical signs, such as a decrease in percent body weight. In the plot provided as
FIG. 3, the percent body weight change following administration of each of Groups A through F
is shown.
Example 10
Receptor Affinities of rIL-15 Conjugates for IL-15R
[0200] The affinities of IL-15 and rIL-1 5 conjugates were measured using Surface
Plasmon Resonance ("SPR"). Briefly, the surface of a Biacore CM5 sensor chip was activated
using a : 1 mixture of NHS:EDC to generate active NHS esters. Goat anti-human Fc antibody
was covalently attached to the surface by injecting it for five minutes in 10 mM sodium acetate
(pH 4). There were approximately 8000 RU of antibodies bound to the surface. Any remaining
NHS ester was then quenched with ethanolamine
[0201] At the initiation of each injection cycle, IL-1 5-Ra-Fc was captured on a sensor chip
channel by a five minute injection step in PBSP. Typically, 150-200 RU of receptors were bound
on the surface.
[0202] rIL-15 conjugates were diluted to 10 mM in PBS (containing 0.05% Tween 20 and
0.1 mg/ml BSA). A series of 3-fold dilutions were made and injected onto a sensor chip which
waa coated with IL-15Ra. The affinities were measured by determining the ka and k rates
separately, and the ratio between kd and ka was used to calculate the Kd values.
[0203] As shown in Table 7, IL- 15 has an affinity to IL-Ra of 3.8 pM. This affinity
decreases to 43 pM with a mono-PEG species and to 183 pM with the di- and tri-PEG species (all
species prepared in accordance with Example 2). The effect is greater with a 40kDa PEG; the
affinity decreases to 2-4 nM with mono- and di- and tri- species and to 9.4 nM with the tri-PEG
species (all species prepared in accordance with Example 5).
Table 7
Affinities of IL-15 and its conjugates to IL-15Ra, as measured by Surface Plasmon
Resonance
SEQUENCE LISTING
SEQ I D NO: l
10 20 30 40 50 60
NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH
70 80 90 100 110
DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS
SEQ I D NO : 2
10 20 30 40 50 60
NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH
70 80 90 100 110
DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS
SEQ I D NO : 3
10 20 30 40 50 60
MRISKPHLRS ISIQCYLCLL LNSHFLTEAG IHVFILGCFS AGLPKTEANW VNVISDLKKI
70 80 90 100 110 120
EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN
130 140 150 160
SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS

What is claimed is:
1. A conjugate comprising a residue of an IL-15 moiety covalently attached to a
water-soluble polymer.
2. The conjugate of claim 1, wherein the IL-15 moiety covalently attached to the
water-soluble polymer is covalently attached via a releaseable linkage.
3. The conjugate of claim 1, wherein the IL-15 moiety covalently attached to the
water-soluble polymer is covalently attached via a stable linkage.
4. The conjugate of any one of claims 1, 2 and 3, wherein the water-soluble polymer is a
branched water-soluble polymer.
5. The conjugate of any one of claims 1, 2, 3 and 4, wherein the water-soluble polymer is a
polymer selected from the group consisting of poly(alkylene oxide), poly(vinyl pyrrolidone),
poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).
6. The conjugate of claim 5, wherein the water-soluble polymer is a poly(alkylene oxide).
7. The conjugate of claim 6, wherein the poly(alkylene oxide) is a poly(ethylene glycol).
8. The conjugate of claim 7, wherein the poly(ethylene glycol) is terminally capped with
an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy,
alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.
9. The conjugate of any one of claims 1, 2, 3, 4, 5, 6 and 7, wherein the water-soluble
polymer has a weight-average molecular weight in a range of from about 500 Daltons to about
100,000 Daltons.
10. The conjugate of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein the
conjugate is covalently attached at an amine group of the residue of the IL-15 moiety.
11. The conjugate of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein one, two,
three or four water-soluble polymers are attached to the residue of the IL-15 moiety.
12. The conjugate of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein one, two or
three water-soluble polymers are attached to the residue of the IL-15 moiety.
13. The conjugate of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein one or two
water-soluble polymers are attached to the residue of the IL-15 moiety.
14. The conjugate of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein one
water-soluble polymer is attached to the residue of the IL-15 moiety.
15. A conjugate comprising a residue of an IL-15 moiety covalently attached to a
water-soluble polymer, wherein the water-soluble polymer, prior to being covalently attached, is a
polymeric reagent bearing an N-hydroxysuccinimidyl group.
16. A pharmaceutical composition comprising a conjugate of any one of claims 1 through
15 and a pharmaceutically acceptable excipient.
17. A method comprising administering to an individual a pharmaceutical composition of
claim 16.
18. A method for making a conjugate comprising contacting, under conjugation
conditions, an IL-15 moiety with a polymeric reagent.