DUAL VARIABLE REGION ANTIBODY-LIKE BINDING PROTEINS
HAVING CROSS-OVER BINDING REGION ORIENTATION
FIELD OF THE INVENTION
The invention relates to antibody-like binding proteins comprising four
polypeptide chains that form four antigen binding sites, wherein each pair of
polypeptides forming the antibody-like binding protein possesses dual variable
domains having a cross-over orientation. The invention also relates to methods for
making such antigen-like binding proteins.
BACKGROUND OF THE INVENTION
Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific
antibodies having binding specificities for two different antigens can be produced
using recombinant technologies and are projected to have broad clinical applications.
It is well known that complete IgG antibody molecules are Y-shaped molecules
comprising four polypeptide chains: two heavy chains and two light chains. Each
light chain consists of two domains, the N-terminal domain being known as the
variable or VL domain (or region) and the C-terminal domain being known as the
constant (or C domain (constant kappa (CK) or constant lambda (CX) domain). Each
heavy chain consists of four or five domains, depending on the class of the antibody.
The N-terminal domain is known as the variable (or VH) domain (or region), which is
followed by the first constant (or CHI) domain, the hinge region, and then the second
and third constant (or C and C ) domains. In an assembled antibody, the VL and
Hdomains associate together to form an antigen binding site. Also, the CL and CHI
domains associate together to keep one heavy chain associated with one light chain.
The two heavy-light chain heterodimers associate together by interaction of the CH2
and CH3 domains and interaction between the hinge regions on the two heavy chains.
It is known that proteolytic digestion of an antibody can lead to the production
of antibody fragments (Fab and Fab2). Such fragments of the whole antibody can
exhibit antigen binding activity. Antibody fragments can also be produced
recombinantly. Fv fragments, consisting only of the variable domains of the heavy
and light chains associated with each other may be obtained. These Fv fragments are
monovalent for antigen binding. Smaller fragments such as individual variable
domains (domain antibodies or dABs; Ward et ah, 1989, Nature 341(6242): 544-46),
and individual complementarity determining regions or CDRs (Williams et ah, 1989,
Proc. Natl. Acad. Set U.S.A. 86(14): 5537-41) have also been shown to retain the
binding characteristics of the parent antibody, although most naturally occurring
antibodies generally need both a VHand VLto retain full binding potency.
Single chain variable fragment (scFv) constructs comprise a VHand a VL
domain of an antibody contained in a single polypeptide chain wherein the domains
are separated by a flexible linker of sufficient length (more than 12 amino acid
residues), that forces intramolecular interaction, allowing self-assembly of the two
domains into a functional epitope binding site (Bird et ah, 1988, Science 242(4877):
423-26). These small proteins (MW -25,000 Da) generally retain specificity and
affinity for their antigen in a single polypeptide and can provide a convenient building
block for larger, antigen-specific molecules.
An advantage of using antibody fragments rather than whole antibodies in
diagnosis and therapy lies in their smaller size. They are likely to be less
immunogenic than whole antibodies and more able to penetrate tissues. A
disadvantage associated with the use of such fragments is that they have only one
antigen binding site, leading to reduced avidity. In addition, due to their small size,
they are cleared very fast from the serum, and hence display a short half-life.
It has been of interest to produce bispecific antibodies (BsAbs) that combine
the antigen binding sites of two antibodies within a single molecule, and therefore,
would be able to bind two different antigens simultaneously. Besides applications for
diagnostic purposes, such molecules pave the way for new therapeutic applications,
e.g., by redirecting potent effector systems to diseased areas (where cancerous cells
often develop mechanisms to suppress normal immune responses triggered by
monoclonal antibodies, like antibody-dependent cellular cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC)), or by increasing neutralizing or
stimulating activities of antibodies. This potential was recognized early on, leading to
a number of approaches for obtaining such bispecific antibodies. Initial attempts to
couple the binding specificities of two whole antibodies against different target
antigens for therapeutic purposes utilized chemically fused heteroconjugate molecules
(Staerz et ah, 1985, Nature 314(6012): 628-31).
Bispecific antibodies were originally made by fusing two hybridomas, each
capable of producing a different immunoglobulin (Milstein et ah, 1983, Nature
305(5934): 537-40), but the complexity of species (up to ten different species)
produced in cell culture made purification difficult and expensive (George et al.,
1997, THE ANTIBODIES 4 : 99-141 (Capra et al, ed., Harwood Academic Publishers)).
Using this format, a mouse IgG2a and a rat IgG2b antibody were produced together in
the same cell (e.g., either as a quadroma fusion of two hybridomas, or in engineered
CHO cells). Because the light chains of each antibody associate preferentially with
the heavy chains of their cognate species, three major species of antibody are
assembled: the two parental antibodies, and a heterodimer of the two antibodies
comprising one heavy/light chain pair of each, associating via their Fc portions. The
desired heterodimer can be purified from this mixture because its binding properties
to Protein A are different from those of the parental antibodies: rat IgG2b does not
bind to Protein A, whereas the mouse IgG2a does. Consequently, the mouse-rat
heterodimer binds to Protein A but elutes at a higher pH than the mouse IgG2a
homodimer, and this makes selective purification of the bispecific heterodimer
possible (Lindhofer et al, 1995, J. Immunol. 155(1): 219-25). The resulting
bispecific heterodimer is fully non-human, hence highly immunogenic, which could
have deleterious side effects (e.g., "HAMA" or "HARA" reactions), and/or neutralize
the therapeutic. There remained a need for engineered bispecifics with superior
properties that can be readily produced in high yield from mammalian cell culture.
Despite the promising results obtained using heteroconjugates or bispecific
antibodies produced from cell fusions as cited above, several factors made them
impractical for large scale therapeutic applications. Such factors include: rapid
clearance of heteroconjugates in vivo, the laboratory intensive techniques required for
generating either type of molecule, the need for extensive purification of
heteroconjugates away from homoconjugates or mono-specific antibodies, and the
generally low yields obtained.
Genetic engineering has been used with increasing frequency to design,
modify, and produce antibodies or antibody derivatives with a desired set of binding
properties and effector functions. A variety of recombinant methods have been
developed for efficient production of BsAbs, both as antibody fragments (Carter et
al., 1995, J. Hematother. 4(5): 463-70; Pluckthun et al., 1997, Immunotechnology
3(2): 83-105; Todorovska et al., 2001, J. Immunol. Methods 248(1-2): 47-66) and full
length IgG formats (Carter, 2001, J. Immunol. Methods 248(1-2): 7-15).
Combining two different scFvs results in BsAb formats with minimal
molecular mass, termed sc-BsAbs or Ta-scFvs (Mack et al., 1995, Proc. Natl. Acad.
Sci. U.S.A. 92(15): 7021-25; Mallender et al, 1994, J. Biol. Chem. 269(1): 199-206).
BsAbs have been constructed by genetically fusing two scFvs to a dimerization
functionality such as a leucine zipper (Kostelny et al, 1992, J. Immunol. 148(5):
1547-53; de Kruif et al, 1996, J. Biol. Chem. 271(13): 7630-34).
Diabodies are small bivalent and bispecific antibody fragments. The
fragments comprise a VHconnected to a VL on the same polypeptide chain, by using a
linker that is too short (less than 12 amino acid residues) to allow pairing between the
two domains on the same chain. The domains are forced to pair intermolecularly with
the complementary domains of another chain and create two antigen-binding sites.
These dimeric antibody fragments, or "diabodies," are bivalent and bispecific
(Holliger et al, 1993, Proc. Natl. Acad. Sci. U.S.A. 90(14): 6444-48). Diabodies are
similar in size to a Fab fragment. Polypeptide chains of VHand VL domains joined
with a linker of between 3 and 12 amino acid residues form predominantly dimers
(diabodies), whereas with a linker of between 0 and 2 amino acid residues, trimers
(triabodies) and tetramers (tetrabodies) predominate. In addition to the linker length,
the exact pattern of oligomerization seems to depend on the composition as well as
the orientation of the variable domains (Hudson et al, 1999, J. Immunol. Methods
23 1(1-2): 177-89). The predictability of the final structure of diabody molecules is
very poor.
Although sc-BsAb and diabody-based constructs display interesting clinical
potential, it was shown that such non-covalently associated molecules are not
sufficiently stable under physiological conditions. The overall stability of a scFv
fragment depends on the intrinsic stability of the VL and VH domains as well as on the
stability of the domain interface. Insufficient stability of the VH-VL interface of scFv
fragments has often been suggested as a main cause of irreversible scFv inactivation,
since transient opening of the interface, which would be allowed by the peptide linker,
exposes hydrophobic patches that favor aggregation and therefore instability and poor
production yield (W5rn et al, 2001, J. Mol. Biol. 305(5): 989-1010).
An alternative method of manufacturing bispecific bivalent antigen-binding
proteins from VHand VL domains is described in U.S. Patent No. 5,989,830. Such
double head and dual Fv configurations are obtained by expressing a bicistronic
vector, which encodes two polypeptide chains. In the Dual-Fv configuration, the
variable domains of two different antibodies are expressed in a tandem orientation on
two separate chains (one heavy chain and one light chain), wherein one polypeptide
chain has two times a VH in series separated by a peptide linker (V -linker-Vm) and
the other polypeptide chain consists of complementary VL domains connected in
series by a peptide linker (Vu-linker-VL2). In the cross-over double head
configuration, the variable domains of two different antibodies are expressed in a
tandem orientation on two separate polypeptide chains (one heavy chain and one light
chain), wherein one polypeptide chain has two times a VH in series separated by a
peptide linker (V -linker-V ) and the other polypeptide chain consists of
complementary VL domains connected in series by a peptide linker in the opposite
orientation (VL2-linker-Vu). Molecular modeling of the constructs suggested the
linker size to be long enough to span 30-40 A (15-20 amino acid residues).
Increasing the valency of an antibody is of interest as it enhances the
functional affinity of that antibody due to the avidity effect. Polyvalent protein
complexes (PPC) with an increased valency are described in U.S. Patent Application
Publication No. US 2005/0003403 Al. PPCs comprise two polypeptide chains
generally arranged laterally to one another. Each polypeptide chain typically
comprises three or four "v-regions," which comprise amino acid sequences capable of
forming an antigen binding site when matched with a corresponding v-region on the
opposite polypeptide chain. Up to about six "v-regions" can be used on each
polypeptide chain. The v-regions of each polypeptide chain are connected linearly to
one another and may be connected by interspersed linking regions. When arranged in
the form of the PPC, the v-regions on each polypeptide chain form individual antigen
binding sites. The complex may contain one or several binding specificities.
A strategy was proposed by Carter et al. (Ridgway et al, 1996, Protein Eng.
9(7): 617-21; Carter, 2011, J Immunol. Methods 248(1-2): 7-15) to produce a Fc
heterodimer using a set of "knob-into-hole" mutations in the C domain of Fc. These
mutations lead to the alteration of residue packing complementarity between the Cm
domain interface within the structurally conserved hydrophobic core so that formation
of the heterodimer is favored as compared with homodimers, which achieves good
heterodimer expression from mammalian cell culture. Although the strategy led to
higher heterodimer yield, the homodimers were not completely suppressed (Merchant
et al, 1998, Nat. Biotechnol. 16(7): 677-81.
Gunasekaran et al. explored the feasibility of retaining the hydrophobic core
integrity while driving the formation of Fc heterodimer by changing the charge
complementarity at the Cm domain interface (Gunasekaran et al., 2010, J. Biol.
Chem. 285(25): 19637-46). Taking advantage of the electrostatic steering
mechanism, these constructs showed efficient promotion of Fc heterodimer formation
with minimum contamination of homodimers through mutation of two pairs of
peripherally located charged residues. In contrast to the knob-into-hole design, the
homodimers were evenly suppressed due to the nature of the electrostatic repulsive
mechanism, but not totally avoided.
Davis et al. describe an antibody engineering approach to convert Fc
homodimers into heterodimers by interdigitating -strand segments of human IgG and
IgA CH3 domains, without the introduction of extra interchain disulfide bonds (Davis
et al, 2010, Protein Eng. Des. Sel. 23(4): 195-202). Expression of SEEDbody (Sb)
fusion proteins by mammalian cells yields Sb heterodimers in high yield that are
readily purified to eliminate minor by-products.
U.S. Patent Application Publication No. US 2010/331527 Al describes a
bispecific antibody based on heterodimerization of the CH3 domain, introducing in one
heavy chain the mutations H95R and Y96F within the CH3 domain. These amino acid
substitutions originate from the CH3 domain of the IgG3 subtype and will
heterodimerize with an IgGl backbone. A common light chain prone to pair with
every heavy chain is a prerequisite for all formats based on heterodimerization though
the CH3 domain. A total of three types of antibodies are therefore produced: 50%
having a pure IgGl backbone, one-third having a pure H95R and Y96F mutated
backbone, and one-third having two different heavy chains (bispecific). The desired
heterodimer can be purified from this mixture because its binding properties to
Protein A are different from those of the parental antibodies: IgG3-derived CH3
domains do not bind to Protein A, whereas the IgGl does. Consequently, the
heterodimer binds to Protein A, but elutes at a higher pH than the pure IgGl
homodimer, and this makes selective purification of the bispecific heterodimer
possible.
U.S. Patent No. 7,612,181 describes a Dual-Variable-Domain IgG (DVD-IgG)
bispecific antibody that is based on the Dual-Fv format described in U.S. Patent No.
5,989,830. A similar bispecific format was also described in U.S. Patent Application
Publication No. US 2010/0226923 Al. The addition of constant domains to
respective chains of the Dual-Fv (Cm-Fc to the heavy chain and kappa or lambda
constant domain to the light chain) led to functional bispecific antibodies without any
need for additional modifications (i.e., obvious addition of constant domains to
enhance stability). Some of the antibodies expressed in the DVD-Ig/TBTI format
show a position effect on the second (or innermost) antigen binding position (Fv2).
Depending on the sequence and the nature of the antigen recognized by the Fv2
position, this antibody domain displays a reduced affinity to its antigen (i.e., loss of
on-rate in comparison to the parental antibody). One possible explanation for this
observation is that the linker between VLI and VL2 protrudes into the CDR region of
Fv2, making the Fv2 somewhat inaccessible for larger antigens.
The second configuration of a bispecific antibody fragment described in U.S.
Patent No. 5,989,830 is the cross-over double head (CODH), having the following
orientation of variable domains expressed on two chains:
VLI-linker-VL2, for the light chain, and
Vm-linker-Vm, for the heavy chain
The '830 patent discloses that a bispecific cross-over double-head antibody fragment
(construct GOSA.E) retains higher binding activity than a Dual-Fv (see page 20, lines
20-50 of the '830 patent), and further discloses that this format is less impacted by the
linkers that are used between the variable domains (see page 20-21 of the '830 patent).
SUMMARY OF THE INVENTION
The invention provides an antibody-like binding protein comprising four
polypeptide chains that form four antigen binding sites, wherein two polypeptide
chains have a structure represented by the formula:
and two polypeptide chains have a structure represented by the formula:
VH2-L3-VHI-L 4-CHI-FC [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2, CH3 immunoglobulin heavy
chain constant domains;
Li, L2, L3, and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
The invention also provides an antibody-like binding protein comprising two
polypeptide chains that form two antigen binding sites, wherein a first polypeptide
chain has a structure represented by the formula:
and a second polypeptide chain has a structure represented by the formula:
VH2-L3-VHI-L4-CHI [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Li, L2, L , and L4 are amino acid linkers;
and wherein the first and second polypeptides form a cross-over light chain-heavy
chain pair.
The invention further provides a method of making an antibody-like binding
protein comprising four polypeptide chains that form four antigen binding sites,
comprising identifying a first antibody variable domain that binds a first target antigen
and a second antibody variable domain that binds a second target antigen, each
containing a VL, and a ; assigning either the light chain or the heavy chain as
template chain; assigning the VL of the first antibody variable domain or the second
antibody variable domain as VLI ; assigning a VL2, a VHI, and a VH2 according to
formulas [I] and [II] below:
VH2-L3-VHI-L 4-CHI-FC [II]
determining maximum and minimum lengths for Li, L2, L3, and L4; generating the
polypeptide structures of formulas I and II; selecting polypeptide structures of
formulas I and II that bind the first target antigen and the second target antigen when
combined to form the antibody-like binding protein;
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2, CH3 immunoglobulin heavy
chain constant domains; and
Li, L2, L , and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
The invention further provides a method of making an antibody-like binding
protein comprising four polypeptide chains that form four antigen binding sites,
comprising identifying a first antibody variable domain that binds a first target antigen
and a second antibody variable domain that binds a second target antigen, each
containing a V L, and a ; assigning either the light chain or the heavy chain as
template chain; assigning the V L of the first antibody variable domain or the second
antibody variable domain as VLI ; assigning a VL2, a VHI, and a VH2 according to
formulas [I] and [II] below:
VH2-L3-VHI-L 4-CHI [II]
determining maximum and minimum lengths for Li, L2, L3, and L4; generating
polypeptide structures of formulas I and II; selecting polypeptide structures of
formulas I and II that bind the first target antigen and the second target antigen when
combined to form the antibody-like binding protein;
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain; and
Li, L2, L , and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
Specific embodiments of the invention will become evident from the
following more detailed description of certain embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Schematic representation of the antigen binding domains Fvl and Fv2
within the dual V region configuration and arrangement of their respective peptide
linkers L L and L H in the TBTI format.
Figure 2. Schematic diagram (2D) of the antigen binding domains Fvl (anti-IL4) and
Fv2 (anti-IL13) within the cross-over dual variable (CODV) configuration and the
arrangement of their respective peptide linkers.
Figure 3. Schematic representation of the Fv anti-IL4 and Fab anti-IL13 showing one
possible spatial arrangement obtained by protein-protein docking of Fv of anti-IL4
and the Fv of anti-IL13.
Figure 4. Assessment of tetravalent and bispecific binding ability of the CODV
protein in a BIACORE assay by injecting the two antigens sequentially or
simultaneously over a DVD-Ig protein-coated chip. The maximal signal observed by
sequential injection can be obtained by co-injection of both antigens, demonstrating
saturation of all binding sites.
Figure 5. Schematic diagram (2D) of the antigen binding domains within the CODV
configuration and arrangement of their respective peptide linker L L (LI and L2) and L H
(L3 and L 4 ) . In panel A, the light chain is kept in a "linear or template" alignment,
whereas the heavy chain is in the "cross-over" configuration. In panel B, the heavy
chain is kept in a "linear or template" alignment and the light chain is in the "cross
over" configuration.
Figure 6. Schematic representation of CODV-Ig design based on whether the light
chain or heavy chain is used as "template."
Figure 7. Comparison of TBTI/DVD-Ig or CODV-Ig molecules incorporating antiIL4
and anti-IL13 sequences.
Figure 8. Comparison of CODV-Fab and B-Fab formats in a cytotoxic assay using
NALM-6 cells.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides antibody-like binding proteins comprising four
polypeptide chains that form four antigen binding sites, wherein each pair of
polypeptides forming an antibody-like binding protein possesses dual variable
domains having a cross-over orientation. The invention also provides methods for
making such antigen-like binding proteins.
Computer modeling predicted that the cross-over double-head (CODH) design
of U.S. Patent No. 5,989,830 would yield a complex in which both binding sites face
in the opposite direction, without the restraints suggested for the Dual-Fv
configuration of U.S. Patent No. 7,612,181. In particular, computer modeling
indicated that the length of the amino acid linkers between the variable domains was
not critical for the CODH design, but was important for permitting full access to both
antigen binding sites in the Dual-Fv design. As with the DVD-Ig/TBTI format,
antibody-like binding protein constructs were prepared in which constant domains
were attached to the CODH configuration to form antibody-like binding proteins
comprising four polypeptide chains that form four antigen binding sites, wherein each
pair of polypeptides forming an antibody-like binding protein possesses dual variable
domains having a cross-over orientation (i.e., CODH-Ig). CODH-Ig molecules are
expected to possess significantly improved stability as compared with CODH
molecules (as DVD-Ig/TBTI possessed improved stability over Dual-Fv molecules).
In order to test the above hypothesis, a CODH-Ig molecule was prepared using
the anti-IL4 and anti-IL13 antibody sequences described in U.S. Patent Application
Publication No. US 2010/0226923 Al. The CODH-Ig molecule differed from the
CODH molecule of US 2010/0226923 with respect to the lengths of amino acid
linkers separating the variable domains on the respective polypeptide chains. The
CODH-Ig molecules were expressed in cells following transient transfection and were
then purified by Protein A chromatography. Although their size-exclusion
chromatography (SEC) profiles showed aggregation levels of 5-10%, none of the
CODH-Ig molecules were functional, and thus none of the CODH-Ig molecules was
able to bind all of its target antigens. The lack of antigen binding activity may have
been due to a perturbed dimerization of the Fv-regions of the heavy and light chains
due to unsuitable linker lengths compromising correct paratope formation. As a
result, a protocol was developed to identify suitable amino acid linkers for insertion
between the two variable domains and the second variable domain and constant
domain on both the heavy and light polypeptide chains of an antibody-like binding
protein. This protocol was based on protein-protein docking of homology and
experimental models of the FvIL4 and FvIL13 regions, respectively, inclusion of the
Fc 1 domain the model, and construction of appropriate linkers between the FvIL4 and
FvIL13 regions and between the Fv and constant Fcl regions.
Standard recombinant DNA methodologies are used to construct the
polynucleotides that encode the polypeptides which form the antibody-like binding
proteins of the invention, incorporate these polynucleotides into recombinant
expression vectors, and introduce such vectors into host cells. See e.g., Sambrook et
al, 2001, MOLECULAR CLONING: A LABORATORYMANUAL (Cold Spring Harbor
Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be
performed according to manufacturer's specifications, as commonly accomplished in
the art, or as described herein. Unless specific definitions are provided, the
nomenclature utilized in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and commonly used
in the art. Similarly, conventional techniques may be used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment
of patients.
1. General Definitions
As utilized in accordance with the present disclosure, the following terms,
unless otherwise indicated, shall be understood to have the following meanings.
Unless otherwise required by context, singular terms shall include pluralities and
plural terms shall include the singular.
The term "polynucleotide" as used herein refers to single-stranded or doublestranded
nucleic acid polymers of at least 10 nucleotides in length. In certain
embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides
or deoxyribonucleotides or a modified form of either type of nucleotide. Such
modifications include base modifications such as bromuridine, ribose modifications
such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications
such as phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and
phosphoroamidate. The term "polynucleotide" specifically includes single-stranded
and double-stranded forms of DNA.
An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or
synthetic origin or some combination thereof, which by virtue of its origin the isolated
polynucleotide: (1) is not associated with all or a portion of a polynucleotide in which
the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to
which it is not linked in nature, or (3) does not occur in nature as part of a larger
sequence.
An "isolated polypeptide" is one that: (1) is free of at least some other
polypeptides with which it would normally be found, (2) is essentially free of other
polypeptides from the same source, e.g., from the same species, (3) is expressed by a
cell from a different species, (4) has been separated from at least about 50 percent of
polynucleotides, lipids, carbohydrates, or other materials with which it is associated in
nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a
polypeptide with which the "isolated polypeptide" is associated in nature, (6) is
operably associated (by covalent or noncovalent interaction) with a polypeptide with
which it is not associated in nature, or (7) does not occur in nature. Such an isolated
polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of
synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is
substantially free from polypeptides or other contaminants that are found in its natural
environment that would interfere with its use (therapeutic, diagnostic, prophylactic,
research or otherwise).
The term "human antibody" as used herein includes antibodies having variable
and constant regions substantially corresponding to human germline immunoglobulin
sequences. In some embodiments, human antibodies are produced in non-human
mammals, including, but not limited to, rodents, such as mice and rats, and
lagomorphs, such as rabbits. In other embodiments, human antibodies are produced
in hybridoma cells. In still other embodiments, human antibodies are produced
recombinantly.
Naturally occurring antibodies typically comprise a tetramer. Each such
tetramer is typically composed of two identical pairs of polypeptide chains, each pair
having one full-length "light" chain (typically having a molecular weight of about 25
kDa) and one full-length "heavy" chain (typically having a molecular weight of about
50-70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any
immunoglobulin polypeptide having sufficient variable domain sequence to confer
specificity for a target antigen. The amino-terminal portion of each light and heavy
chain typically includes a variable domain of about 100 to 110 or more amino acids
that typically is responsible for antigen recognition. The carboxy -terminal portion of
each chain typically defines a constant domain responsible for effector function.
Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin
polypeptide includes a variable domain (VH) and three constant domains (CHI, Cm,
and CH3), wherein the VHdomain is at the amino-terminus of the polypeptide and the
CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin
polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the
VL domain is at the amino-terminus of the polypeptide and the CL domain is at the
carboxyl-terminus.
Human light chains are typically classified as kappa and lambda light chains,
and human heavy chains are typically classified as mu, delta, gamma, alpha, or
epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,
respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2,
IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2.
IgA is similarly subdivided into subclasses including, but not limited to, IgAl and
IgA2. Within full-length light and heavy chains, the variable and constant domains
typically are joined by a "J" region of about 12 or more amino acids, with the heavy
chain also including a "D" region of about 10 more amino acids. See, e.g.,
FUNDAMENTALIMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is
incorporated by reference in its entirety for all purposes. The variable regions of each
light/heavy chain pair typically form an antigen binding site. The variable domains of
naturally occurring antibodies typically exhibit the same general structure of relatively
conserved framework regions (FR) joined by three hypervariable regions, also called
complementarity determining regions or CDRs. The CDRs from the two chains of
each pair typically are aligned by the framework regions, which may enable binding
to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light
and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2,
CDR2, FR3, CDR3, and FR4.
The term "native Fc" as used herein refers to a molecule comprising the
sequence of a non-antigen-binding fragment resulting from digestion of an antibody
or produced by other means, whether in monomeric or multimeric form, and can
contain the hinge region. The original immunoglobulin source of the native Fc is
preferably of human origin and can be any of the immunoglobulins, although IgGl
and IgG2 are preferred. Native Fc molecules are made up of monomeric polypeptides
that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds)
and non-covalent association. The number of intermolecular disulfide bonds between
monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class
(e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2). One
example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of
an IgG. The term "native Fc" as used herein is generic to the monomeric, dimeric,
and multimeric forms.
The term "Fc variant" as used herein refers to a molecule or sequence that is
modified from a native Fc but still comprises a binding site for the salvage receptor,
FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the
salvage receptor, are known in the art. Thus, the term "Fc variant" can comprise a
molecule or sequence that is humanized from a non-human native Fc. Furthermore, a
native Fc comprises regions that can be removed because they provide structural
features or biological activity that are not required for the antibody-like binding
proteins of the invention. Thus, the term "Fc variant" comprises a molecule or
sequence that lacks one or more native Fc sites or residues, or in which one or more
Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond
formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity
upon expression in a selected host cell, (4) glycosylation, (5) interaction with
complement, (6) binding to an Fc receptor other than a salvage receptor, or (7)
antibody-dependent cellular cytotoxicity (ADCC).
The term "Fc domain" as used herein encompasses native Fc and Fc variants
and sequences as defined above. As with Fc variants and native Fc molecules, the
term "Fc domain" includes molecules in monomeric or multimeric form, whether
digested from whole antibody or produced by other means.
The term "antibody-like binding protein" as used herein refers to a nonnaturally
occurring (or recombinant) molecule that specifically binds to at least one
target antigen, and which comprises four polypeptide chains that form four antigen
binding sites, wherein two polypeptide chains have a structure represented by the
formula:
and two polypeptide chains have a structure represented by the formula:
VH2-L3-VHI-L 4-CHI-FC [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2, CH3 immunoglobulin heavy
chain constant domains;
Li, L2, L , and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II
form a cross-over light chain-heavy chain pair. The term "antibody-like binding
protein" as used herein also refers to a non-naturally occurring (or recombinant)
molecule that specifically binds to at least one target antigen, and which comprises
two polypeptide chains that form two antigen binding sites, wherein a first
polypeptide chain has a structure represented by the formula:
and a second polypeptide chain has a structure represented by the formula:
VH2-L3-VHI-L 4-CHI [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Li, L2, L , and L4 are amino acid linkers;
and wherein the first and second polypeptides form a cross-over light chain-heavy
chain pair. A "recombinant" molecule is one that has been prepared, expressed,
created, or isolated by recombinant means.
One embodiment of the invention provides antibody-like binding proteins
having biological and immunological specificity to between one and four target
antigens. Another embodiment of the invention provides nucleic acid molecules
comprising nucleotide sequences encoding polypeptide chains that form such
antibody-like binding proteins. Another embodiment of the invention provides
expression vectors comprising nucleic acid molecules comprising nucleotide
sequences encoding polypeptide chains that form such antibody-like binding proteins.
Yet another embodiment of the invention provides host cells that express such
antibody-like binding proteins (i.e., comprising nucleic acid molecules or vectors
encoding polypeptide chains that form such antibody-like binding proteins).
The term "swapability" as used herein refers to the interchangeability of
variable domains within the CODV format and with retention of folding and ultimate
binding affinity. "Full swapability" refers to the ability to swap the order of both V
and VH2 domains, and therefore the order of VLI and VL2 domains, in a CODV-Ig
(i.e., to reverse the order) or CODV-Fab while maintaining full functionality of the
antibody-like binding protein as evidenced by the retention of binding affinity.
Furthermore, it should be noted that the designations VH and VLwithin a particular
CODV-Ig or CODV-Fab refer only to the domain's location on a particular protein
chain in the final format. For example, V and VH2 could be derived from VLI and
VL2 domains in parent antibodies and placed into the V and V positions in the
antibody-like binding protein. Likewise, VLI and VL2 could be derived from Vm and
V domains in parent antibodies and placed in the Vm and Vm positions in the
antibody-like binding protein. Thus, the VHand VL designations refer to the present
location and not the original location in a parent antibody. VH and VLdomains are
therefore "swappable."
An "isolated" antibody-like binding protein is one that has been identified and
separated and/or recovered from a component of its natural environment.
Contaminant components of its natural environment are materials that would interfere
with diagnostic or therapeutic uses for the antibody-like binding protein, and may
include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the antibody-like binding protein will be purified: (1) to
greater than 95% by weight of antibody as determined by the Lowry method, and
most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least
15 residues of N-terminal or internal amino acid sequence by use of a spinning cup
sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing
conditions using Coomassie blue or, preferably, silver stain. Isolated antibody-like
binding proteins include the antibody-like binding protein in situ within recombinant
cells since at least one component of the antibody-like binding protein's natural
environment will not be present.
The terms "substantially pure" or "substantially purified" as used herein refer
to a compound or species that is the predominant species present (i.e., on a molar
basis it is more abundant than any other individual species in the composition). In
some embodiments, a substantially purified fraction is a composition wherein the
species comprises at least about 50% (on a molar basis) of all macromolecular species
present. In other embodiments, a substantially pure composition will comprise more
than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the
composition. In still other embodiments, the species is purified to essential
homogeneity (contaminant species cannot be detected in the composition by
conventional detection methods) wherein the composition consists essentially of a
single macromolecular species.
The term "antigen" or "target antigen" as used herein refers to a molecule or a
portion of a molecule that is capable of being bound by an antibody-like binding
protein, and additionally is capable of being used in an animal to produce antibodies
capable of binding to an epitope of that antigen. A target antigen may have one or
more epitopes. With respect to each target antigen recognized by an antibody-like
binding protein, the antibody-like binding protein is capable of competing with an
intact antibody that recognizes the target antigen. A "bivalent" antibody-like binding
protein, other than a "multispecific" or "multifunctional" antibody-like binding
protein, is understood to comprise antigen binding sites having identical antigenic
specificity.
A bispecific or bifunctional antibody typically is an artificial hybrid antibody
having two different heavy chain/light chain pairs and two different binding sites or
epitopes. Bispecific antibodies may be produced by a variety of methods including,
but not limited to, fusion of hybridomas or linking of F(ab') fragments.
A F (ab) fragment typically includes one light chain and the VHand CHI
domains of one heavy chain, wherein the VH-CHI heavy chain portion of the F(ab)
fragment cannot form a disulfide bond with another heavy chain polypeptide. As
used herein, a F (ab) fragment can also include one light chain containing two variable
domains separated by an amino acid linker and one heavy chain containing two
variable domains separated by an amino acid linker and a CHI domain.
A F (ab') fragment typically includes one light chain and a portion of one
heavy chain that contains more of the constant region (between the CHI and CH2
domains), such that an interchain disulfide bond can be formed between two heavy
chains to form a F (ab')2 molecule.
The phrases "biological property," "biological characteristic," and the term
"activity" in reference to an antibody-like binding protein of the invention are used
interchangeably herein and include, but are not limited to, epitope affinity and
specificity, ability to antagonize the activity of the antigen target (or targeted
polypeptide), the in vivo stability of the antibody-like binding protein, and the
immunogenic properties of the antibody-like binding protein. Other identifiable
biological properties or characteristics of an antibody-like binding protein include, for
example, cross-reactivity, (i.e., with non-human homologs of the antigen target, or
with other antigen targets or tissues, generally), and ability to preserve high
expression levels of protein in mammalian cells. The aforementioned properties or
characteristics can be observed or measured using art-recognized techniques
including, but not limited to ELISA, competitive ELISA, surface plasmon resonance
analysis, in vitro and in vivo neutralization assays, and immunohistochemistry with
tissue sections from different sources including human, primate, or any other source
as the need may be.
The term "immunologically functional immunoglobulin fragment" as used
herein refers to a polypeptide fragment that contains at least the CDRs of the
immunoglobulin heavy or light chains from which the polypeptide fragment was
derived. An immunologically functional immunoglobulin fragment is capable of
binding to a target antigen.
A "neutralizing" antibody-like binding protein as used herein refers to a
molecule that is able to block or substantially reduce an effector function of a target
antigen to which it binds. As used herein, "substantially reduce" means at least about
60%, preferably at least about 70%, more preferably at least about 75%, even more
preferably at least about 80%, still more preferably at least about 85%, most
preferably at least about 90% reduction of an effector function of the target antigen.
The term "epitope" includes any determinant, preferably a polypeptide
determinant, capable of specifically binding to an immunoglobulin or T-cell receptor.
In certain embodiments, epitope determinants include chemically active surface
groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or
sulfonyl groups, and, in certain embodiments, may have specific three-dimensional
structural characteristics and/or specific charge characteristics. An epitope is a region
of an antigen that is bound by an antibody or antibody-like binding protein. In certain
embodiments, an antibody-like binding protein is said to specifically bind an antigen
when it preferentially recognizes its target antigen in a complex mixture of proteins
and/or macromolecules. In preferred embodiments, an antibody-like binding protein
is said to specifically bind an antigen when the equilibrium dissociation constant is <
10 8 M, more preferably when the equilibrium dissociation constant is < 10 9 M, and
most preferably when the dissociation constant is < 10 10 M.
The dissociation constant (¾) of an antibody-like binding protein can be
determined, for example, by surface plasmon resonance. Generally, surface plasmon
resonance analysis measures real-time binding interactions between ligand (a target
antigen on a biosensor matrix) and analyte (an antibody-like binding protein in
solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia
Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by
immobilizing the analyte (antibody-like binding protein on a biosensor matrix) and
presenting the ligand (target antigen). The term as used herein refers to the
dissociation constant of the interaction between a particular antibody-like binding
protein and a target antigen.
The term "specifically binds" as used herein refers to the ability of an
antibody-like protein or an antigen-binding fragment thereof to bind to an antigen
containing an epitope with an Kd of at least about 1x 10 6 M, 1x 10 M, 1x 10 8 M,
1x 10 9 M, 1x 10 10 M, 1x 10 11 M, 1x 10 12 M, or more, and/or to bind to an epitope
with an affinity that is at least two-fold greater than its affinity for a nonspecific
antigen.
The term "linker" as used herein refers to one or more amino acid residues
inserted between immunoglobulin domains to provide sufficient mobility for the
domains of the light and heavy chains to fold into cross over dual variable region
immunoglobulins. A linker is inserted at the transition between variable domains or
between variable and constant domains, respectively, at the sequence level. The
transition between domains can be identified because the approximate size of the
immunoglobulin domains are well understood. The precise location of a domain
transition can be determined by locating peptide stretches that do not form secondary
structural elements such as beta-sheets or alpha-helices as demonstrated by
experimental data or as can be assumed by techniques of modeling or secondary
structure prediction. The linkers described herein are referred to as which is
located on the light chain between the N-terminal VLI and VL2 domains; L2, which is
also on the light chain is located between the VL2 and C-terminal CL domains. The
heavy chain linkers are known as L3, which is located between the N-terminal VH2
and VHI domains; and L4, which is located between the VHI and C -Fc domains. The
linkers Li, L2, L3, and L4 are independent, but they may in some cases have the same
sequence and/or length.
The term "vector" as used herein refers to any molecule (e.g., nucleic acid,
plasmid, or virus) that is used to transfer coding information to a host cell. The term
"vector" includes a nucleic acid molecule that is capable of transporting another
nucleic acid to which it has been linked. One type of vector is a "plasmid," which
refers to a circular double-stranded DNA molecule into which additional DNA
segments may be inserted. Another type of vector is a viral vector, wherein additional
DNA segments may be inserted into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are introduced (e.g., bacterial
vectors having a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the
genome of a host cell upon introduction into the host cell and thereby are replicated
along with the host genome. In addition, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such vectors are referred to
herein as "recombinant expression vectors" (or simply, "expression vectors"). In
general, expression vectors of utility in recombinant DNA techniques are often in the
form of plasmids. The terms "plasmid" and "vector" may be used interchangeably
herein, as a plasmid is the most commonly used form of vector. However, the
invention is intended to include other forms of expression vectors, such as viral
vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated
viruses), which serve equivalent functions.
The term "operably linked" is used herein to refer to an arrangement of
flanking sequences wherein the flanking sequences so described are configured or
assembled so as to perform their usual function. Thus, a flanking sequence operably
linked to a coding sequence may be capable of effecting the replication, transcription,
and/or translation of the coding sequence. For example, a coding sequence is
operably linked to a promoter when the promoter is capable of directing transcription
of that coding sequence. A flanking sequence need not be contiguous with the coding
sequence, so long as it functions correctly. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be considered "operably
linked" to the coding sequence.
The phrase "recombinant host cell" (or "host cell") as used herein refers to a
cell into which a recombinant expression vector has been introduced. A recombinant
host cell or host cell is intended to refer not only to the particular subject cell, but also
to the progeny of such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences, such progeny may
not, in fact, be identical to the parent cell, but such cells are still included within the
scope of the term "host cell" as used herein. A wide variety of host cell expression
systems can be used to express the antibody-like binding proteins of the invention,
including bacterial, yeast, baculoviral, and mammalian expression systems (as well as
phage display expression systems). An example of a suitable bacterial expression
vector is pUC19. To express an antibody-like binding protein recombinantly, a host
cell is transformed or transfected with one or more recombinant expression vectors
carrying DNA fragments encoding the polypeptide chains of the antibody-like binding
protein such that the polypeptide chains are expressed in the host cell and, preferably,
secreted into the medium in which the host cells are cultured, from which medium the
antibody-like binding protein can be recovered.
The term "transformation" as used herein refers to a change in a cell's genetic
characteristics, and a cell has been transformed when it has been modified to contain a
new DNA. For example, a cell is transformed where it is genetically modified from
its native state. Following transformation, the transforming DNA may recombine
with that of the cell by physically integrating into a chromosome of the cell, or may
be maintained transiently as an episomal element without being replicated, or may
replicate independently as a plasmid. A cell is considered to have been stably
transformed when the DNA is replicated with the division of the cell. The term
"transfection" as used herein refers to the uptake of foreign or exogenous DNA by a
cell, and a cell has been "transfected" when the exogenous DNA has been introduced
inside the cell membrane. A number of transfection techniques are well known in the
art. Such techniques can be used to introduce one or more exogenous DNA
molecules into suitable host cells.
The term "naturally occurring" as used herein and applied to an object refers to
the fact that the object can be found in nature and has not been manipulated by man.
For example, a polynucleotide or polypeptide that is present in an organism (including
viruses) that can be isolated from a source in nature and that has not been
intentionally modified by man is naturally-occurring. Similarly, "non-naturally
occurring" as used herein refers to an object that is not found in nature or that has
been structurally modified or synthesized by man.
As used herein, the twenty conventional amino acids and their abbreviations
follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty
conventional amino acids; unnatural amino acids such as -, -disubstituted amino
acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may
also be suitable components for the polypeptide chains of the antibody-like binding
proteins of the invention. Examples of unconventional amino acids include: 4-
hydroxyproline, -carboxyglutamate, -,,-trimethyllysine, --acetyllysine, Ophosphoserine,
N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-
hydroxylysine, --methylarginine, and other similar amino acids and imino acids
(e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand
direction is the amino terminal direction and the right-hand direction is the carboxylterminal
direction, in accordance with standard usage and convention.
Naturally occurring residues may be divided into classes based on common
side chain properties:
(1) hydrophobic: Met, Ala, Val, Leu, e, Phe, Trp, Tyr, Pro;
(2) polar hydrophilic: Arg, Asn, Asp, Gin, Glu, His, Lys, Ser, Thr ;
(3) aliphatic: Ala, Gly, e, Leu, Val, Pro;
(4) aliphatic hydrophobic: Ala, He, Leu, Val, Pro;
(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(6) acidic: Asp, Glu;
(7) basic: His, Lys, Arg;
(8) residues that influence chain orientation: Gly, Pro;
(9) aromatic: His, Trp, Tyr, Phe; and
(10) aromatic hydrophobic: Phe, Trp, Tyr.
Conservative amino acid substitutions may involve exchange of a member of
one of these classes with another member of the same class. Conservative amino acid
substitutions may encompass non-naturally occurring amino acid residues, which are
typically incorporated by chemical peptide synthesis rather than by synthesis in
biological systems. These include peptidomimetics and other reversed or inverted
forms of amino acid residues. Non-conservative substitutions may involve the
exchange of a member of one of these classes for a member from another class.
A skilled artisan will be able to determine suitable variants of the polypeptide
chains of the antibody-like binding proteins of the invention using well-known
techniques. For example, one skilled in the art may identify suitable areas of a
polypeptide chain that may be changed without destroying activity by targeting
regions not believed to be important for activity. Alternatively, one skilled in the art
can identify residues and portions of the molecules that are conserved among similar
polypeptides. In addition, even areas that may be important for biological activity or
for structure may be subject to conservative amino acid substitutions without
destroying the biological activity or without adversely affecting the polypeptide
structure.
The term "patient" as used herein includes human and animal subjects.
A "disorder" is any condition that would benefit from treatment using the
antibody-like binding proteins of the invention. "Disorder" and "condition" are used
interchangeably herein and include chronic and acute disorders or diseases, including
those pathological conditions that predispose a patient to the disorder in question.
The terms "treatment" or "treat" as used herein refer to both therapeutic
treatment and prophylactic or preventative measures. Those in need of treatment
include those having the disorder as well as those prone to have the disorder or those
in which the disorder is to be prevented.
The terms "pharmaceutical composition" or "therapeutic composition" as used
herein refer to a compound or composition capable of inducing a desired therapeutic
effect when properly administered to a patient.
The term "pharmaceutically acceptable carrier" or "physiologically acceptable
carrier" as used herein refers to one or more formulation materials suitable for
accomplishing or enhancing the delivery of an antibody-like binding protein.
The terms "effective amount" and "therapeutically effective amount" when
used in reference to a pharmaceutical composition comprising one or more antibody
like binding proteins refer to an amount or dosage sufficient to produce a desired
therapeutic result. More specifically, a therapeutically effective amount is an amount
of an antibody-like binding protein sufficient to inhibit, for some period of time, one
or more of the clinically defined pathological processes associated with the condition
being treated. The effective amount may vary depending on the specific antibody-like
binding protein that is being used, and also depends on a variety of factors and
conditions related to the patient being treated and the severity of the disorder. For
example, if the antibody-like binding protein is to be administered in vivo, factors
such as the age, weight, and health of the patient as well as dose response curves and
toxicity data obtained in preclinical animal work would be among those factors
considered. The determination of an effective amount or therapeutically effective
amount of a given pharmaceutical composition is well within the ability of those
skilled in the art.
One embodiment of the invention provides a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a therapeutically effective
amount of an antibody-like binding protein.
2. Antibody-like Binding Proteins
In one embodiment of the invention, the antibody-like binding proteins
comprise four polypeptide chains that form four antigen binding sites, wherein two
polypeptide chains have a structure represented by the formula:
V -L -VL^-C L [I]
and two polypeptide chains have a structure represented by the formula:
VH2-L3-VHI -L4-CHI-FC [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2, CH3 immunoglobulin heavy
chain constant domains;
Li, L2, L , and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
In another embodiment of the invention, the antibody-like binding proteins
comprise two polypeptide chains that form two antigen binding sites, wherein a first
polypeptide chain has a structure represented by the formula:
and a second polypeptide chain has a structure represented by the formula:
V H2-L 3-VHI-L 4-CHI [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Li, L2, L3, and L4 are amino acid linkers;
and wherein the first and second polypeptides form a cross-over light chain-heavy
chain pair.
The antibody-like binding proteins of the invention may be prepared using
domains or sequences obtained or derived from any human or non-human antibody,
including, for example, human, murine, or humanized antibodies.
In some antibody-like binding proteins of the invention, the length of L 3 is at
least twice the length of . In other antibody-like binding proteins of the invention,
the length of L4 is at least twice the length of L2. In some antibody-like binding
proteins of the invention, the length of Li is at least twice the length of L3. In other
antibody-like binding proteins of the invention, the length of L 2 is at least twice the
length of L4.
In some antibody-like binding proteins of the invention, Li is 3 to 12 amino
acid residues in length, L 2 is 3 to 14 amino acid residues in length, L 3 is 1to 8 amino
acid residues in length, and L4 is 1 to 3 amino acid residues in length. In other
antibody-like binding proteins, Li is 5 to 10 amino acid residues in length, L2 is 5 to 8
amino acid residues in length, L is 1 to 5 amino acid residues in length, and L4 is 1 to
2 amino acid residues in length. In a preferred antibody-like binding protein, Li is 7
amino acid residues in length, L2 is 5 amino acid residues in length, L is 1 amino acid
residues in length, and L4 is 2 amino acid residues in length.
In some antibody-like binding proteins of the invention, Li is 1 to 3 amino
acid residues in length, L2 is 1 to 4 amino acid residues in length, L is 2 to 15 amino
acid residues in length, and L4 is 2 to 15 amino acid residues in length. In other
antibody-like binding proteins, Li is 1 to 2 amino acid residues in length, L2 is 1 to 2
amino acid residues in length, L3 is 4 to 12 amino acid residues in length, and L4 is 2
to 12 amino acid residues in length. In a preferred antibody-like binding protein, Li is
1 amino acid residue in length, L2 is 2 amino acid residues in length, L3 is 7 amino
acid residues in length, and L4 is 5 amino acid residues in length.
In some antibody-like binding proteins of the invention, Li, L3, or L4 may be
equal to zero. However, in antibody-like binding proteins wherein L3, or L4 is
equal to zero, the corresponding transition linker between the variable region and
constant region or between the dual variable domains on the other chain cannot be
zero. In some embodiments, Li is equal to zero and L3 is 2 or more amino acid
residues, L3 is equal to zero and Li is equal to 1 or more amino acid residues, or L4 is
equal to 0 and L2 is 3 or more amino acid residues.
In some antibody-like binding proteins of the invention, at least one of the
linkers selected from the group consisting of L2, L3, and L4 contains at least one
cysteine residue.
Examples of suitable linkers include a single glycine (Gly) residue; a diglycine
peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues
(Gly-Gly-Gly-Gly; SEQ ID NO: 25); a peptide with five glycine residues (Gly-Gly-
Gly-Gly-Gly; SEQ ID NO: 26); a peptide with six glycine residues (Gly-Gly-Gly-
Gly-Gly-Gly; SEQ ID NO: 27); a peptide with seven glycine residues (Gly-Gly-Gly-
Gly-Gly-Gly-Gly; SEQ ID NO: 28); a peptide with eight glycine residues (Gly-Gly-
Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 29). Other combinations of amino acid
residues may be used such as the peptide Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 30) and
the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 31). Other
suitable linkers include a single Ser, and Val residue; the dipeptide Arg-Thr, Gin-Pro,
Ser-Ser, Thr-Lys, and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 52), Thr-Val-Ala-
Ala-Pro (SEQ ID NO: 53), Gln-Pro-Lys-Ala-Ala (SEQ ID NO: 54), Gln-Arg-Ile-Glu-
Gly (SEQ ID NO: 55); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 48), Arg-Thr-Val-
Ala-Ala-Pro-Ser (SEQ ID NO: 49), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 50),
and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: 51). The examples listed above are
not intended to limit the scope of the invention in any way, and linkers comprising
randomly selected amino acids selected from the group consisting of valine, leucine,
isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine, glycine, and proline have been shown to be suitable in the
antibody-like binding proteins of the invention (see Example 12).
The identity and sequence of amino acid residues in the linker may vary
depending on the type of secondary structural element necessary to achieve in the
linker. For example, glycine, serine, and alanine are best for linkers having maximum
flexibility. Some combination of glycine, proline, threonine, and serine are useful if a
more rigid and extended linker is necessary. Any amino acid residue may be
considered as a linker in combination with other amino acid residues to construct
larger peptide linkers as necessary depending on the desired properties.
In some antibody-like binding proteins of the invention, VLI comprises the
amino acid sequence of SEQ ID NO: 1; VL2 comprises the amino acid sequence of
SEQ ID NO: 3; V comprises the amino acid sequence of SEQ ID NO: 2; and VH2
comprises the amino acid sequence of SEQ ID NO: 4.
In some embodiments of the invention, the antibody-like binding protein is
capable of specifically binding one or more antigen targets. In preferred
embodiments of the invention, the antibody-like binding protein is capable of
specifically binding at least one antigen target selected from the group consisting of
B7.1, B7.2, BAFF, BlyS, C3, C5, CCL1 1 (eotaxin), CCL15 (MIP-ld), CCL17
(TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC,
CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL3 (MIP-la),
CCL4 (MIP-lb), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CD3, CD19,
CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-cadherin), Chitinase, CSF 1
(M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12
(SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, IL15,
IL17, IL18, ILIA, IL1B, IL1F10, ILl , IL2, IL4, IL6, IL7, IL8, IL9, IL12/23, IL22,
IL23, IL25, IL27, IL35, ITGB4 (b 4 integrin), LEP (leptin), MHC class II, TLR2,
TLR4, TLR5, TNF, TNF-a, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll
like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5/CCXCR1), DNGR-
1(CLEC91), and HMGB1. In other embodiments of the invention, the antibody-like
binding protein is capable of inhibiting the function of one or more of the antigen
targets.
In some embodiments of the invention, the antibody-like binding protein is
bispecific and capable of binding two different antigen targets or epitopes. In a
preferred embodiment of the invention, the antibody-like binding protein is bispecific
and each light chain-heavy chain pair is capable of binding two different antigen
targets or epitopes. In a more preferred embodiment, the antibody-like binding
protein is capable of binding two different antigen targets that are selected from the
group consisting of IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and
HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-
18, IL-la and IL- , TNFa and IL12/23, TNFa and IL-12p40, TNFa and IL- ,
TNFa and IL-23, and IL17 and IL23. In an even more preferred embodiment, the
antibody-like binding protein is capable of binding the antigen targets IL4 and IL13.
In some embodiments of the invention, the antibody-like binding protein
specifically binds IL4 with an on-rate of 2.97 E+07 and an off-rate of 3.30 E-04 and
specifically binds IL13 with an on-rate of 1.39 E+06 and an off-rate of 1.63 E-04. In
other embodiments of the invention, the antibody-like binding protein specifically
binds IL4 with an on-rate of 3.16 E+07 and an off-rate of 2.89 E-04 and specifically
binds IL13 with an on-rate of 1.20 E+06 and an off-rate of 1.12 E-04.
In one embodiment of the invention, an antibody-like binding protein
comprising four polypeptide chains that form four antigen binding sites is prepared by
identifying a first antibody variable domain that binds a first target antigen and a
second antibody variable domain that binds a second target antigen, each containing a
V L, and a ; assigning either the light chain or the heavy chain as template chain;
assigning the V L of the first antibody variable domain or the second antibody variable
domain as VLI ; assigning a VL2, a VHI , and a VH2 according to formulas [I] and [II]
below:
VH2-L3-VHI-L 4 -CHI-FC [II]
determining maximum and minimum lengths for Li, L2, L3, and L4; generating
polypeptide structures of formulas I and II; selecting polypeptide structures of
formulas I and II that bind the first target antigen and the second target antigen when
combined to form the antibody-like binding protein;
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2, CH3 immunoglobulin heavy
chain constant domains; and
Li, L2, L3, and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
In another embodiment of the invention, an antibody-like binding protein
comprising four polypeptide chains that form four antigen binding sites is prepared by
identifying a first antibody variable domain that binds a first target antigen and a
second antibody variable domain that binds a second target antigen, each containing a
V L, and a ; assigning either the light chain or the heavy chain as template chain;
assigning the V L of the first antibody variable domain or the second antibody variable
domain as VLI ; assigning a VL2, a VHI , and a VH2 according to formulas [I] and [II]
below:
VH2-L 3-VHI-L 4 -CH I [II]
determining maximum and minimum lengths for Li, L2, L3, and L4 ; generating
polypeptide structures of formulas I and II; selecting polypeptide structures of
formulas I and II that bind the first target antigen and the second target antigen when
combined to form the antibody-like binding protein;
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain; and
Li, L2, L3, and L4 are an amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
In other embodiments of the invention, an antibody-like binding protein in
which the first antibody variable domain and the second antibody variable domain are
the same is prepared.
One embodiment of the invention provides a method for making an antibody
like binding protein, comprising expressing in a cell one or more nucleic acid
molecules encoding polypeptides having structures represented by the formulas [I]
and [II] below:
VH2-L3-VHI-L 4-CHI-FC [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2, CH3 immunoglobulin heavy
chain constant domains; and
Li, L2, L , and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II
form a cross-over light chain-heavy chain pair.
Another embodiment of the invention provides a method for making an
antibody-like binding protein, comprising expressing in a cell one or more nucleic
acid molecules encoding polypeptides having structures represented by the formulas
[I] and [II] below:
VH2-L3-VHI-L 4-CHI [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain; and
Li, L2, L , and L4 are amino acid linkers;
and wherein the polypeptide of formula I and the polypeptide of formula II form a
cross-over light chain-heavy chain pair.
3. Uses for Antibody-like Binding Proteins
The antibody-like binding proteins of the invention can be employed in any
known assay method, such as competitive binding assays, direct and indirect
sandwich assays, and immunoprecipitation assays for the detection and quantitation of
one or more target antigens. The antibody-like binding proteins will bind the one or
more target antigens with an affinity that is appropriate for the assay method being
employed.
For diagnostic applications, in certain embodiments, antibody-like binding
proteins can be labeled with a detectable moiety. The detectable moiety can be any
one that is capable of producing, either directly or indirectly, a detectable signal. For
example, the detectable moiety can be a radioisotope, such as H, "C, P, S,
Tc, In, or Ga; a fluorescent or chemiluminescent compound, such as fluorescein
isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,
-galactosidase, or horseradish peroxidase.
The antibody-like binding proteins of the invention are also useful for in vivo
imaging. An antibody-like binding protein labeled with a detectable moiety can be
administered to an animal, preferably into the bloodstream, and the presence and
location of the labeled antibody in the host assayed. The antibody-like binding
protein can be labeled with any moiety that is detectable in an animal, whether by
nuclear magnetic resonance, radiology, or other detection means known in the art.
The invention also relates to a kit comprising an antibody-like binding protein
and other reagents useful for detecting target antigen levels in biological samples.
Such reagents can include a detectable label, blocking serum, positive and negative
control samples, and detection reagents.
4. Antibody-Like Binding Protein Therapeutic Compositions and
Administration Thereof
Therapeutic or pharmaceutical compositions comprising antibody-like binding
proteins are within the scope of the invention. Such therapeutic or pharmaceutical
compositions can comprise a therapeutically effective amount of an antibody-like
binding protein, or antibody-like binding protein-drug conjugate, in admixture with a
pharmaceutically or physiologically acceptable formulation agent selected for
suitability with the mode of administration.
Acceptable formulation materials preferably are nontoxic to recipients at the
dosages and concentrations employed.
The pharmaceutical composition can contain formulation materials for
modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity,
clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release,
adsorption, or penetration of the composition. Suitable formulation materials include,
but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine,
or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or
sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates,
phosphates, or other organic acids), bulking agents (such as mannitol or glycine),
chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing
agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropylbeta-
cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates
(such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or
immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents,
hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight
polypeptides, salt-forming counterions (such as sodium), preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol,
methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),
solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols
(such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such
as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate
80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents
(such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides -
preferably sodium or potassium chloride - or mannitol sorbitol), delivery vehicles,
diluents, excipients and/or pharmaceutical adjuvants (see, e.g., REMINGTON'S
PHARMACEUTICAL SCIENCES (18th Ed., A.R. Gennaro, ed., Mack Publishing Company
1990), and subsequent editions of the same, incorporated herein by reference for any
purpose).
The optimal pharmaceutical composition will be determined by a skilled
artisan depending upon, for example, the intended route of administration, delivery
format, and desired dosage. Such compositions can influence the physical state,
stability, rate of in vivo release, and rate of in vivo clearance of the antibody-like
binding protein.
The primary vehicle or carrier in a pharmaceutical composition can be either
aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for
injection can be water, physiological saline solution, or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions for parenteral
administration. Neutral buffered saline or saline mixed with serum albumin are
further exemplary vehicles. Other exemplary pharmaceutical compositions comprise
Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can
further include sorbitol or a suitable substitute. In one embodiment of the invention,
antibody-like binding protein compositions can be prepared for storage by mixing the
selected composition having the desired degree of purity with optional formulation
agents in the form of a lyophilized cake or an aqueous solution. Further, the
antibody-like binding protein can be formulated as a lyophilizate using appropriate
excipients such as sucrose.
The pharmaceutical compositions of the invention can be selected for
parenteral delivery. Alternatively, the compositions can be selected for inhalation or
for delivery through the digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the skill of the art.
The formulation components are present in concentrations that are acceptable
to the site of administration. For example, buffers are used to maintain the
composition at physiological pH or at a slightly lower pH, typically within a pH range
of from about 5 to about 8.
When parenteral administration is contemplated, the therapeutic compositions
for use in this invention can be in the form of a pyrogen- free, parenterally acceptable,
aqueous solution comprising the desired antibody-like binding protein in a
pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which an antibody-like binding protein is
formulated as a sterile, isotonic solution, properly preserved. Yet another preparation
can involve the formulation of the desired molecule with an agent, such as injectable
microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or
polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained
release of the product which can then be delivered via a depot injection. Hyaluronic
acid can also be used, and this can have the effect of promoting sustained duration in
the circulation. Other suitable means for the introduction of the desired molecule
include implantable drug delivery devices.
In one embodiment, a pharmaceutical composition can be formulated for
inhalation. For example, an antibody-like binding protein can be formulated as a dry
powder for inhalation. Antibody-like binding protein inhalation solutions can also be
formulated with a propellant for aerosol delivery. In yet another embodiment,
solutions can be nebulized.
It is also contemplated that certain formulations can be administered orally. In
one embodiment of the invention, antibody-like binding proteins that are administered
in this fashion can be formulated with or without those carriers customarily used in
the compounding of solid dosage forms such as tablets and capsules. For example, a
capsule can be designed to release the active portion of the formulation at the point in
the gastrointestinal tract when bioavailability is maximized and pre-systemic
degradation is minimized. Additional agents can be included to facilitate absorption
of the antibody-like binding protein. Diluents, flavorings, low melting point waxes,
vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders
can also be employed.
Another pharmaceutical composition can involve an effective quantity of
antibody-like binding proteins in a mixture with non-toxic excipients that are suitable
for the manufacture of tablets. By dissolving the tablets in sterile water, or another
appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients
include, but are not limited to, inert diluents, such as calcium carbonate, sodium
carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as
starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic
acid, or talc.
Additional pharmaceutical compositions of the invention will be evident to
those skilled in the art, including formulations involving antibody-like binding
proteins in sustained- or controlled-delivery formulations. Techniques for
formulating a variety of other sustained- or controlled-delivery means, such as
liposome carriers, bio-erodible microparticles or porous beads and depot injections,
are also known to those skilled in the art. Additional examples of sustained-release
preparations include semipermeable polymer matrices in the form of shaped articles,
e.g. films, or microcapsules. Sustained release matrices can include polyesters,
hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-Lglutamate,
poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-
hydroxybutyric acid. Sustained-release compositions can also include liposomes,
which can be prepared by any of several methods known in the art.
Pharmaceutical compositions of the invention to be used for in vivo
administration typically must be sterile. This can be accomplished by filtration
through sterile filtration membranes. Where the composition is lyophilized,
sterilization using this method can be conducted either prior to, or following,
lyophilization and reconstitution. The composition for parenteral administration can
be stored in lyophilized form or in a solution. In addition, parenteral compositions
generally are placed into a container having a sterile access port, for example, an
intravenous solution bag or vial having a stopper pierceable by a hypodermic injection
needle.
Once the pharmaceutical composition has been formulated, it can be stored in
sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or
lyophilized powder. Such formulations can be stored either in a ready-to-use form or
in a form (e.g., lyophilized) requiring reconstitution prior to administration.
The invention also encompasses kits for producing a single-dose
administration unit. The kits can each contain both a first container having a dried
protein and a second container having an aqueous formulation. Also included within
the scope of this invention are kits containing single and multi-chambered pre-filled
syringes (e.g., liquid syringes and lyosyringes).
The effective amount of an antibody-like binding protein pharmaceutical
composition to be employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will appreciate that the
appropriate dosage levels for treatment will thus vary depending, in part, upon the
molecule delivered, the indication for which the antibody-like binding protein is being
used, the route of administration, and the size (body weight, body surface, or organ
size) and condition (the age and general health) of the patient. Accordingly, the
clinician can titer the dosage and modify the route of administration to obtain the
optimal therapeutic effect. A typical dosage can range from about 0.1 g g to up to
about 100 mg/kg or more, depending on the factors mentioned above. In other
embodiments, the dosage can range from 0.1 g/kg up to about 100 mg/kg; or 1 g/kg
up to about 100 mg/kg; or 5 g/kg, 10 g/kg, 15 g/kg, 20 g/kg, 25 g/kg, 30 g/kg,
35 g/kg, 40 g/kg, 45 g/kg, 50 g/kg, 55 g/kg, 60 g/kg, 65 g/kg, 70 g/kg, 75
g/kg, up to about 100 mg/kg.
Dosing frequency will depend upon the pharmacokinetic parameters of the
antibody-like binding protein in the formulation being used. Typically, a clinician
will administer the composition until a dosage is reached that achieves the desired
effect. The composition can therefore be administered as a single dose, as two or
more doses (which may or may not contain the same amount of the desired molecule)
over time, or as a continuous infusion via an implantation device or catheter. Further
refinement of the appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by them. Appropriate
dosages can be ascertained through use of appropriate dose-response data.
The route of administration of the pharmaceutical composition is in accord
with known methods, e.g., orally; through injection by intravenous, intraperitoneal,
intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular,
intraarterial, intraportal, or intralesional routes; by sustained release systems; or by
implantation devices. Where desired, the compositions can be administered by bolus
injection or continuously by infusion, or by implantation device.
The composition can also be administered locally via implantation of a
membrane, sponge, or other appropriate material onto which the desired molecule has
been absorbed or encapsulated. Where an implantation device is used, the device can
be implanted into any suitable tissue or organ, and delivery of the desired molecule
can be via diffusion, timed-release bolus, or continuous administration.
5. Examples
The Examples that follow are illustrative of specific embodiments of the
invention, and various uses thereof. They are set forth for explanatory purposes only,
and should not be construed as limiting the scope of the invention in any way.
Example 1. Design and Engineering of Bispecific Cross-over Dual Variable
Region Antibody-like Binding Proteins
The cross-over dual variable region in an Fv format was described in U .S.
Patent No. 5,989,830 and was referred to as a cross-over double head (CODH)
configuration. Molecular modeling predicted that the cross-over double-head
(CODH) design results in a complex with both binding sites facing in opposite
directions, without the restraints suggested for the Dual-Fv configuration. The CODH
Fv format was examined to determine whether it could be converted into complete
antibody-like molecules by adding a CL domain to the light chain and an Fc region to
the heavy chain. A similar conversion was successful for the corresponding dual
variable domains (DVD-Ig) and TBTI as described in U .S. Patent No. 7,612,181 and
International Publication No. WO 2009/052081. The arrangement of the variable
regions in the CODH format is shown in the structures below, which indicate the
amino to carboxyl orientation of the peptide chains:
(a) light chain: NH2-VL i-Linker-VL2-COOH
(b) heavy chain: NH2-VH2-Linker-V Hi-COOH
The amino to carboxyl terminal arrangement of the variable regions in (a) and
(b) above can be distinguished from the arrangement in the Dual-Fv configuration
shown in (c) and (d) below:
(c) light chain: NH2-VL i-Linker-V L2-COOH
(d) heavy chain: NH2-Vm -Linker-VH2-COOH
The main difference to note is the distinct placement of the corresponding
light chain and heavy chain variable regions (VHI/VLI and V H2 L2) with respect to
each other in the two dual variable region configurations. The corresponding VLI and
VHI domains were both at the N-terminus of the light and heavy chains in dual
variable region configuration. In contrast, in the cross-over configuration, one half of
one pair of an antibody variable region was separated spatially within the protein
chain in the cross-over configuration. In the cross-over configuration, the VL i domain
would be at the N-terminus of the protein light chain but the pairing VHI domain is at
the C-terminus of the cross-over configuration heavy chain. The spatial relationship
between VLI and VHI found in the dual variable region configuration is the
arrangement found in natural antibodies.
One potential disadvantage of the dual Fv configuration is that the linker LL
separating the two variable regions protrudes into the antigen binding site of the Fv2
domain (see Figure 1). This protrusion may interfere with antigen binding and result
in a perturbed accessibility of Antigen 2 to Fv2. This perturbed accessibility or
interference may prevent antigen binding. In addition, this interference might be
more pronounced where the size of Antigen 2 is larger. Indeed, it has been
documented in U.S. Patent No. 7,612,181 that the binding affinity and neutralization
ability of a DVD-Ig molecule depends on which antigen specificity is presented at the
N-terminus or C-terminus. See U.S. Patent No. 7,612,181, Example 2.
Therefore, to create more stable antibody-like binding proteins that are not
subject to loss of antigen affinity as compared to the parental antibody, cross-over
dual variable region molecules having a CL domain on the light chain and an Fc
region on the heavy chain were designed and constructed. The polypeptides that form
these antibody-like proteins have the structures shown below, in which the amino to
carboxyl terminal orientation of the polypeptide chains is indicated:
(e) light chain: NH2-VLi-Linker-VL2-C L-COOH
( ) heavy chain: NH2-VH2-Linker-VHi-C Hi-Fc-COOH
To evaluate whether this bispecific antibody-like protein design would bind to
two different antigens, two previously generated and humanized variable regions from
antibodies specific for IL4 (parental humanized anti-IL4) and IL13 (parental
humanized anti-IL13) were used to construct the bispecific antibody-like molecules
shown in Table 1. Sequencing of the mouse antibodies and the humanization process
were described in International Publication No. WO 2009/052081 (TBTI). Briefly,
amino acid sequences of the variable heavy and light chains of the murine anti-IL13
clone B-B13 and the murine anti-IL4 clone 8D4-8 were determined by amino acid
sequencing. The murine sequences were humanized and then back-translated into
nucleotide sequences as described in Example 5 of International Publication No. WO
2009/052081, which is incorporated herein by reference in its entirety. The parental
humanized anti-IL4 VHand VL, and parental humanized anti-IL13 VHand VL
sequences were combined and arranged as shown in Table 1. The shorthand codes in
column one of Table 1 were created to simplify discussion of these antibody-like
binding proteins. The antibody-like binding proteins differ in the size of the linker
inserted between the two variable regions as shown in Table 1. DNA molecules
encoding the polypeptides shown in Table 1 were generated from the back-translated
parental anti-IL4 and anti-IL13 antibodies. CHI, CL, and Fc domains were obtained
from IGHG1 (GenBank Accession No. 569F4) and IGKC (GenBank Accession No.
Q502W4).
Table 1. Cross-over Double Head Immunoglobulins
The protein combinations shown in Table 2 were expressed by transient
transfection and purified by Protein A chromatography. In each case, size exclusion
chromatography revealed less than 12% aggregation, with most having less than 7%
aggregation; but none of the cross-over double head immunoglobulins were found to
display any ability to bind either IL4 or IL13. However, no functional antibody-like
binding could be detected and the reasons for this lack of activity could not be
ascertained. It was previously predicted that this arrangement would show superior
stability over the dual variable region domain antibodies described in U.S. Patent No.
7,612,181 and International Publication No. WO 2009/052081.
Table 2. Binding of CODH-Ig to IL4 and IL13
Protein Combination Aggregation IL4 IL13
binding binding
anti-IL13 VH-(G4S)-anti-IL4 VH-Cm -Fc 5.4% ND* ND
anti-IL4 VL-(G4S)-anti-IL13 VL-CL
anti-IL13 VH -(G4S)-anti-IL4 VH - Cm -Fc 6.3% ND ND
anti-IL4 VL -(G4S)2-anti-IL13 VL -CL
anti-IL13 VH -(G4S)2-anti-IL4 VH - Cm- 11.5% ND ND
Fc
anti-IL4 VL -(G4S)-anti-IL13 VL -CL
anti-IL13 VH -(G4S)2-anti-IL4 VH - Cm - 10.1% ND ND
Fc
anti-IL4 VL -(G4S)2-anti-IL13 VL -CL
anti-IL4 VH -(G4S)-anti-IL13 VH - CHi-Fc 2.7% ND ND
anti-IL13 VL -(G4S)-anti-IL4 VL -CL
anti-IL4 VH -(G4S)-anti-IL13 VH - Cm -Fc 3.6% ND ND
anti-IL13 VL -(G4S)2-anti-IL4 VL -CL
anti-IL4 VH -(G4S)2-anti-IL13 VH - Cm- 2.9% ND ND
Fc
anti-IL13 VL -(G4S)-anti-IL4 VL -CL
anti-IL4 VH -(G4S)2-anti-IL13 VH - Cm- 10.8% ND ND
Fc
anti-IL13 VL -(G4S)2-anti-IL4 VL -CL
ND means none detected
Example 2. Design of CODV-Ig Proteins by Molecular Modeling
To obtain fully functional antibody-like proteins utilizing the cross-over
double head configuration that are amendable to incorporation of the Fc and CLI
domains, a molecular modeling protocol was developed for the inclusion and
evaluation of different linkers between the constant and variable domains and
between the dual variable domains on both the heavy and light chains. The question
was whether the addition of unique linkers between each constant/variable domain
interface and between the two variable/variable domain interfaces on both the heavy
and light chains would allow proper protein folding to occur and produce functional
antibody-like molecules in the cross-over dual variable region configuration (see
Figure 2). In other words, a total of four independent and unique linkers were
evaluated (see Figure 2). This molecular modeling protocol was based on proteinprotein
docking of homology models and experimental models of the FVIL4 and FVILI3
regions, respectively, in combination with appropriate linkers between the FVIL4 and
FVILI3 regions and between the Fv and constant or Fc regions.
The independent linkers were assigned unique names as follows: Li refers to
the linker between N-terminal V L and the C-terminal V L on the light chain; L2 refers
to the linker between the C-terminal V L and CL on the light chain; L refers to the
linker between N-terminal V H and the C-terminal V H on the heavy chain; L4 refers to
the linker between the C-terminal V H and CHI (and Fc) on the heavy chain. It should
be noted that the designations V H and V L refer only to the domain's location on a
particular protein chain in the final format. For example, VHI and V H2 could be
derived from VLI and V L2 domains in parent antibodies and placed into the VHI and
VH2 positions in a CODV-Ig. Likewise, VLI and VL2 could be derived from V and
VH2 domains in parent antibodies and placed into the V and VH2 positions in a
CODV-Ig. Thus, V H and V L designations refer to present location and not the original
location in a parent antibody.
In more detail, a homology model of FVIL4 was constructed on PDB entries
1YLD (light chain) and 1IQW (heavy chain). The FVIL4 dimer was recomposed on an
in-house crystal structure of the IL13/anti-IL13 FabiLi3 complex and optimized. In
order to obtain an estimate of the volume required by IL4 when bound to FVIL4, the
crystal structure of IL4 (lRCB.pdb) was docked to the homology model of FVIL4.
Next, twenty -two putative models of the complex were generated that merited further
consideration.
In parallel, the homology model of FVIL4 was docked to FVILI3 extracted from
an in-house crystal structure of the IL13/FabiLi3 complex. One superior solution was
found that permitted construction of relatively short linkers while showing no steric
interference for antigen binding and placement of the constant domains as was the
case for dual variable region immunoglobulins (see Figure 3). In this arrangement
FVIL4 (VLI ) was placed at the N-terminus of the light chain, followed by FVILI3 (VL2)
and Fc (CLI) on the light chain C-terminus. On the heavy chain, FVILI3 (Vm) was
placed N-terminally, followed by FVIL4 (VHI) and the constant regions (CHI - CH2 -
CH3) .
As shown in Table 3, the models of the light chain suggested that the linker Li
between the VLI and VL2 domains and the linker L2 between the VL2 and CLI domains
should be between one to three and zero to two glycine residues long, respectively.
Models of the heavy chain suggested that the linker L3 between the VH2 and VHI
domains and the linker L4 between the VHI and CHI domains should be between two
to six and four to seven glycine residues long, respectively (see Table 3 and Figure 2).
In this example, glycine was used as a prototypical amino acid for the linkers but
other amino acid residues may also serve as linkers. The structural stability of the
proposed models was verified by optimization of the linker conformations,
minimization, and molecular dynamics calculations. Systematic combination between
four light chain and six heavy chain constructs resulted in 24 possible cross-over dual
variable region bispecific anti-IL4 and anti-IL13 antibody-like binding proteins (see
Table 4).
Table 3. Proposed Linker Lengths
Table 4. CODV-Ig for Expression
*A short hand code was devised to represent the associated structures. Codes
beginning with HC represent the adjacent heavy chain and codes beginning with LC
represent the adjacent light chains.
In Table 4, the prefix "anti" is not included but it is intended to mean that IL13 refers
to anti-IL13 and IL4 refers to anti-IL4.
Example 3. Generation of CODV-Ig Expression Plasmids
Nucleic acid molecules encoding the variable heavy and light chains of the six
heavy chains and four light chains described in Table 4 were generated by gene
synthesis at Geneart (Regensburg, Germany). The variable light chain domains were
fused to the constant light chain (IGKC, GenBank Accession No. Q502W4) by
digestion with the restriction endonucleases ApaLI and BsiWI and subsequently
ligated into the ApaLI/BsiWI sites of the episomal expression vector pFF, an
analogon of the pTT vector described by Durocher et ah, (2002, Nuc Acids Res.
30(2): E9), creating the mammalian expression plasmid for expression of the light
chains.
The variable heavy chain domains were fused to the "Ted" variant of the
human constant heavy chain (IGHG1, GenBank Accession No. 569F4), or
alternatively, to a 6x His tagged CHI domain from the human constant IGHG1 in order
to create a bispecific Fab. Next, the VH domain was digested with the restriction
endonucleases ApaLI and Apal and then fused to the IGHG1 or His tagged CHI
domain respectively, by ligation into the ApaLI/Apal sites of the episomal expression
vector pFF, creating the mammalian expression plasmids for expression of the heavy
chains (IgGl or Fab respectively).
Example 4. Expression of CODV-Ig
The expression plasmids encoding the heavy and light chains of the
corresponding constructs were propagated in E. coli DH5a cells. Plasmids used for
transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega
Kit.
HEK 293 -FS cells growing in Freestyle Medium (Invitrogen) were transfected
with indicated LC and HC plasmids encoding the heavy chains and light chains
shown in Table 4 using 293fectin (Invitrogen) transfection reagent as described by the
manufacturer. After 7 days, cells were removed by centrifugation and the supernatant
was passed over a 0.22 filter to remove particles.
CODV-IgGl constructs were purified by affinity chromatography on Protein
A columns (HiTrap Protein A HP Columns, GE Life Sciences). After elution from
the column with 100 mM acetate buffer and 100 mM NaCl, pH 3.5, the CODV-IgGl
constructs were desalted using HiPrep 26/10 Desalting Columns, formulated in PBS
at a concentration of 1 mg/mL and filtered using a 0.22 membrane.
Bispecific CODV Fab constructs were purified by IMAC on HiTrap IMAC
HP Columns (GE Life Sciences). After elution from the column with a linear
gradient (Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 50 - 500 mM
imidazole, pH 7.4), the protein containing fractions were pooled and desalted using
HiPrep 26/10 Desalting Columns, formulated in PBS at a concentration of 1 mg/mL
and filtered using a 0.22 membrane.
Protein concentration was determined by measurement of absorbance at 280
nm. Each batch was analyzed by SDS-PAGE under reducing and non-reducing
conditions to determine the purity and molecular weight of each subunit and of the
monomer.
A Nunc F96-MaxiSorp-Immuno plate was coated with goat anti-Human IgG
(Fc specific) [NatuTec A80-104A]. The antibody was diluted to 10 g/ml in
carbonate coating buffer (50 mM sodium carbonate, pH 9.6) and dispensed at 50 
per well. The plate was sealed with adhesive tape, and stored overnight at 4° C. The
plate was washed three times with Wash buffer (PBS, pH 7.4 and 0.1% Tween20).
150 of blocking solution (1% BSA / PBS) was dispensed into each well to cover
the plate. After 1 hour at room temperature, the plate was washed three times with
Wash buffer. 100 of sample or standards (in a range from 1500 ng/ml to 120
ng/ml) were added and allowed to sit for 1 hour at room temperature. The plate was
washed three times with Wash buffer. 100 of goat anti-Human IgG-FC - HRP
conjugate [NatuTec A80-104P-60] diluted 1:10.000 were added using incubation
solution (0.1% BSA, PBS, pH 7.4, and 0.05% Tween20). After 1 hour incubation at
room temperature, the plate was washed three times with Wash buffer. 100 of
ABTS substrate (10 mg ABTS tablet (Pierce 34026) in 0.1M Na2HP0 4, 0.05 M citric
acid solution, pH 5.0). Addition of 10 of 30% ¾ / 10 ml Substrate buffer prior
to use) were dispensed to each well, and the color was allowed to develop. After the
color developed (approximately 10 to 15 minutes), 50 of 1% SDS solution were
added to stop the reaction. The plate was read at A4 0 5 .
Example 5. Characterization of CODV-Ig Variants
To determine whether the CODV-Ig antibody-like protein heavy and light
chains were pairing and folding properly, the aggregation level was measured by
analytical size-exclusion chromatography (SEC). Analytical SEC was performed on
assembled pairs using an explorer 10 (GE Healthcare) equipped with a
TSKgel G3000SWXL column (7.8 mm x 30 cm) and TSKgel SWXL guard column
(Tosoh Bioscience). The analysis was run at 1 ml/min using 250 mM NaCl, 100 mM
Na-phosphate, pH 6.7, with detection at 280 nm. 30 ΐ of protein sample (at 0.5-1
mg/ml) were applied onto the column. For estimation of the molecular size, the
column was calibrated using a gel filtration standard mixture (MWGF-1000, SIGMA
Aldrich). Data evaluation was performed using UNICORN software v5.1 1.
Table 5 shows the results of the first set of 24 different CODV-Ig molecules
made using the anti-IL4 and anti-IL13 variable region combinations described in
Table 4. The codes assigned in Table 4 represent the adjacent structures shown in
Table 4. For the pairs of light chain and heavy chains where protein was produced,
aggregation levels were measured using SEC. The results are shown in Table 5 where
LC4 (Li = 1; L2 = 2) was most successful in pairing with all six heavy chains. LC4
corresponds to the structure IL4 VL-(Gly)-IL13 VL-(Gly2)-Cu having linker Li equal
to 1, where a single amino acid residue separated the two VL domains of the dual
variable region light chain. In addition, LC4 had L2 equal to 2, which contained a
Gly-Gly dipeptide linker between the central VL and the C-terminal CHI .
Table 5. Levels of Aggregation Among Pairs of Heavy and Light Chains
*ND indicates that no protein was produced
Where CODV-Ig molecules were produced, a single-concentration BIACORE
experiment at intermediate IL13 and IL4 concentrations was performed to verify
binding to target antigens. CODV-Ig antibody-like molecules corresponding to the
LC4:HC4 and LC4:HC6 combinations described in Table 4 were chosen for
assessment of a full kinetic analysis using surface plasmon resonance.
As depicted in Table 5, most of the CODV-Ig molecules could not be
produced at all or only as aggregates (up to 90%). The heavy chain/light chain
combinations giving rise to acceptable aggregation levels (5-10%) after one
chromatography step were the ones combined with the light chain IL4 VL-(Gly)-ILl 3
VL-(Gly2)-Cu. The light chain was the most finicky chain within these CODV-Ig
variants and served as platform to accept different heavy chains with different linker
compositions.
1. Kinetic Analysis
Two pairs of heavy and light chains were selected for full kinetic analysis.
Recombinant human IL13 and IL4 was purchased from Chemicon (USA). Kinetic
characterization of purified antibodies was performed using surface plasmon
resonance technology on a BIACORE 3000 (GE Healthcare). A capture assay using a
species specific antibody (e.g., human-Fc specific MAB 1302, Chemicon) for capture
and orientation of the investigated antibodies was used. The capture antibody was
immobilized via primary amine groups ( 11000 RU) on a research grade CM5 chip
(GE Life Sciences) using standard procedures. The analyzed antibody was captured
at a flow rate of 10 / with an adjusted RU value that would result in maximal
analyte binding of 30 RU. Binding kinetics were measured against recombinant
human IL4 and IL13 over a concentration range between 0 to 25 nM in HBS EP (10
mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005 % Surfactant P20) at a
flow rate of 30 ΐ/min. Chip surfaces were regenerated with 10 mM glycine, pH 2.5.
Kinetic parameters were analyzed and calculated in the BIAevaluation program
package v4. 1 using a flow cell without captured antibody as reference.
Table 6 below shows the comparison of kinetics of the parental BB13 (anti-
IL13) and 8D4 (anti-IL4) antibodies (expressed as IgGs) with the respective domains
within the CODV-Ig format (Table 4, Codes LC4:HC4 and LC4:HC6). As shown in
Table 6, the CODV-Ig constructs did not exhibit decreased binding properties against
the corresponding antigens, when compared to the parental anti-IL13 and anti-IL4
antibodies. The loss in the on-rate observed in the DVD-Ig/TBTI format using the
same Fv sequences did not occur with the CODV-Ig configuration. The opposite
facing binding sites should allow for binding large antigens or bridging different cells
with a bispecific antibody-like configuration, and would also be suitable for a wider
selection of parental antibodies. A further advantage of the CODV-Ig was that no
linker residues protrude into the antigen binding site and reduce accessibility of the
antigen.
Table 6. Kinetic Analysis of LC4:HC4 and LC4:HC6
LC4:HC4 CODV-Ig with IL4 3.16E+07 2.89E-04 9.14E-12
LC4:HC4 CODV-IG with IL13 1.20E+06 1.12E-04 9.34E-11
LC4:HC6 CODV-Ig with IL4 2.97E+07 3.30E-04 l.llE-1 1
LC4:HC6 CODV-IG with IL13 1.39E+06 1.63E-04 1.18E-10
2. Co-injection of IL4 and IL13 for Demonstration of Additive Antigen
Binding b CODV-Ig
To investigate additive binding of both antigens, a wizard-driven co-inject
method was applied in which one antigen was injected immediately followed by the
other antigen after a lag time (IL4 then IL13 and vice versa). The resulting binding
level could be compared to that achieved with a 1:1 mixture of both antigens in the
same concentration. In order to show additive binding of both the IL4 and IL13
antigens by the CODV-Ig molecules, a BIACORE experiment was performed with a
CODV-Ig combination [HC4:LC4] by co-injection of both antigens in three separate
analysis cycles (see Figure 4). The co-injection was done with 3.125 nM IL4/25 nM
IL13 (and vice versa) and with a 1:1 mixture of 3.125 nM IL4 and 25 nM IL13. A coinjection
of HBS-EP buffer was done as a reference. At a time point of 800 seconds,
an identical binding level of 63 RU was achieved after injection of the antigen
mixture or co-injection of the antigens, regardless of the co-injection sequence. When
CODV-Ig protein had been saturated by the first antigen (IL4), the second antigen
(IL13) was injected and a second binding signal was observed. This observation was
reproduced when the antigen injection sequence was reversed. This demonstrates the
additive and non-inhibition of binding of both antigens by the CODV-Ig. Therefore,
the CODV-Ig construct was able to bind both antigens simultaneously (i.e., exhibit
bispecificity) saturating all binding sites (i.e., exhibited tetravalency).
Example 6. Tolerance of Linker Lengths for CODV-Ig
The tolerance for linkers of various lengths was evaluated by constructing
CODV-Ig molecules having different combinations of linker lengths for Li, L2 on the
light chain and L 3 and L4 on the heavy chain. CODV-Ig constructs were generated
with heavy chain linkers L3 and L4 varying between 1 through 8 residues for L3 and
either 0 or 1 residues for L4. The heavy chain contained anti-IL4 as the N-terminal
binding domain and anti-IL13 as the C-terminal binding domain followed by Cm-Fc.
The light chain linkers Li and L2 were varied from 3 to 12 residues for Li and from 3
to 14 residues for L2. The light chain contained anti-IL13 as the N-terminal binding
domain and anti-IL4 as the C-terminal binding domain followed by CLI.
1. Characterization of CODV-Ig Variants
Determination of aggregation level was by analytical size-exclusion
chromatography (SEC). Analytical SEC was performed using an explorer 10
(GE Healthcare) equipped with a TSKgel G3000SWXL column (7.8 mm x 30 cm)
and TSKgel SWXL guard column (Tosoh Bioscience). The analysis was run at 1
ml/min using 250 mM NaCl, 100 mM Na-phosphate pH 6.7, with detection at 280
nm. 30 of protein sample (at 0.5-1 mg/ml) was applied onto the column. For
estimation of the molecular size, the column was calibrated using a gel filtration
standard mixture (MWGF-1000, SIGMA Aldrich). Data evaluation was performed
using UNICORN software v5. 11.
Recombinant human IL13 and IL4 were purchased from Chemicon (USA).
Recombinant human TNF-a was purchased from Sigma Aldrich (H8916-10 g),
recombinant human IL- (201-LB/CF), recombinant human IL-23 (1290-IL/CF),
recombinant human EGFR (344 ER), and recombinant human HER2 ( 1129-ER-50)
were purchased from R&D Systems.
Kinetic binding analysis by Biacore was performed as follows. Surface
plasmon resonance technology on a Biacore 3000 (GE Healthcare) was used for
detailed kinetic characterization of purified antibodies. A capture assay using a
species-specific antibody (e.g., human-Fc specific MAB 1302, Chemicon) for capture
and orientation of the investigated antibodies was used. For determination of IL4 and
IL13 binding kinetics, the corresponding CODV Fabs as in Example 10, Table 12
were captured using the anti-human Fab capture Kit (GE Healthcare). The capture
antibody was immobilized via primary amine groups ( 11000 RU) on a research grade
CM5 chip (GE Life Sciences) using standard procedures. The analyzed antibody was
captured at a flow rate of 10 / with an adjusted RU value that would result in
maximal analyte binding of 30 RU. Binding kinetics were measured against
recombinant human IL4 and IL13 over a concentration range between 0 to 25 nM in
HBS EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant
P20) at a flow rate of 30 / . Chip surfaces were regenerated with 10 mM
glycine pH 2.5. Kinetic parameters were analyzed and calculated in the
BIAevaluation program package v4. 1 using a flow cell without captured antibody as a
reference.
Binding affinities of CODV-Ig, CODV-Fab, and TBTI against EGFR and
HER2 were measured using a Proteon XPR36 protein interaction array system
(Biorad). The antigens were immobilized by amine reactive coupling on GLC sensor
chips (Biorad). Dilution series of the bispecific antibody variants in PBSET buffer
(Biorad) were analyzed in parallel in one-shot kinetics mode with double referencing.
Data were analyzed using Proteon Manager Software v3.0 (Biorad) with either
Langmuir 1:1 model with mass transfer or bivalent analyte model.
Table 7 summarizes the results for yield, aggregation (as measured by size
exclusion chromatography), and binding affinity for CODV-Ig having different size
combinations of linkers. The results revealed that CODV-Ig molecules in which L2
was zero generally could not be produced, or where protein was produced, there was a
high level of aggregation (see Batch ID Nos. 101, 102, 106-1 11, and 132-137 in Table
7). Therefore, in contrast to the molecular modeling prediction from Example 2,
where L2 equal to zero was within the acceptable range, these results indicate that the
VL2-CL transition (or L2) requires a linker of at least one residue (see Table 7).
Table 7. Optimization of Linker Sizes for CODV-Ig
Alig;nment on the heavy chain must be IL1 3VH-L3-IL4VH-L4-CHI-FC
n.p. means the construct was not producible.
In addition, the CODV-Ig linker lengths described above were found to be
more sensitive to increases in 1 amino acid residue than increases in 2 amino acid
residues. For example, while Batch ID Nos. 103 and 104 differ by 1 amino acid
residue in L2, Batch ID No. 103 shows 6 fold more aggregation and Batch ID No. 104
displays less aggregation and twice the yield. In contrast, Batch ID Nos. 104 and 105,
which differ by two residues in L2, displayed similar profiles with respect to yield,
aggregation, and binding.
Example 7. Heavy Chain as Template Chain for CODV-Ig
In examples 1 through 5, the optimal short linker sizes on the light chain
suggested that the light chain was serving as a template by remaining in a linear
arrangement and that larger linkers were required on the heavy chain in order for the
heavy chain to fold properly into the cross-over configuration to conform to the
template light chain (see Figure 5, Panel A). Whether the short linkers specifically
placed on the heavy chain to maintain a liner arrangement on the heavy chain
rendered the heavy chain the "template" chain, and whether the pattern would repeat
itself and larger linkers would be required to allow the non-template chain to fold
properly and accommodate the now template heavy chain was evaluated next (see
Figure 5, Panel B).
Figure 6 illustrates these principles of CODV-Ig design based on having either
the light chain or the heavy chain as the "template." To evaluate the generic nature of
this concept, CODV-Ig constructs were generated with heavy chain linkers L and L4
varying between 1 through 8 residues for L and either 0 or 1 residues for L4. The
heavy chain contained anti-IL4 as the N-terminal binding domain and anti-IL13 as the
C-terminal binding domain followed by Cm-Fc. The light chain linkers Li and L2
were varied from 3 to 12 residues for Li and from 3 to 14 residues for L2. The light
chain contained anti-IL13 as the N-terminal binding domain and anti-IL4 as the Cterminal
binding domain followed by CLI.
Table 8 summarizes the results for yield, aggregation (as measured by size
exclusion chromatography), and binding affinity for CODV-Ig having different size
combinations of linkers and where the heavy chain is maintained in a linear
arrangement as the template chain and the light chain is allowed to fold in a cross
over configuration. The results revealed that CODV-Ig molecules in which L4 was
zero generally could not be produced, or where protein was produced, there was a
high level of aggregation (similar to molecules in which L2 was equal to zero) (see
Batch ID Nos. 207-209, 211-212, 219-224, 231-236, 243-252, and 263-266 in Table
8). One exception was Batch ID No. 210, in which Li was 7, L2 was 5, L was 2, and
L4 was zero. This arrangement produced a sufficient amount of protein and had an
acceptable level of aggregation and binding, which suggested that some combination
of linker sizes could be found to compensate for a zero length linker at L4 in some
circumstances.
Table 8. Optimization of Linker Sizes with Heavy Chain as Template
208 IL4 x IL13 5 5 2 0 10.2 18.4 10 77
209 IL4 x IL13 7 3 2 0 16.2 22.2 5 47
210 IL4 x IL13 7 5 2 0 14.7 9.7 4 47
211 IL4 x IL13 10 3 2 0 2.1 12.8 7 53
212 IL4 x IL13 10 5 2 0 7.0 36.3 10 29
213 IL4 x IL13 5 3 2 2 4.0 13.2 5 27
214 IL4 x IL13 5 5 2 2 8.0 7.9 10 53
215 IL4 x IL13 7 3 2 2 14.9 11.5 4 50
216 IL4 x IL13 7 5 2 2 7.5 3.6 1 1 40
217 IL4 x IL13 10 3 2 2 2.4 4.4 8 79
218 IL4 x IL13 10 5 2 2 4.6 6.6 4 36
219 IL4 x IL13 3 6 3 0 2.1 51.8 8 7 1
220 IL4 x IL13 3 10 3 0 3.9 59.4 1 42
221 IL4 x IL13 3 14 3 0 1.9 57.6 35 8 1
222 IL4 x IL13 6 6 3 0 4.0 11.8 7 53
223 IL4 x IL13 6 10 3 0 10.3 16.6 6 23
224 IL4 x IL13 6 14 3 0 5.1 13.5 9 52
225 IL4 x IL13 3 6 3 2 2.8 71.6 6 68
226 IL4 x IL13 3 10 3 2 7.3 65.8 6 64
227 IL4 x IL13 3 14 3 2 1.6 53.6 7 39
228 IL4 x IL13 6 6 3 2 4.0 19.1 7 44
229 IL4 x IL13 6 10 3 2 2.2 15.4 3 14
230 IL4 x IL13 6 14 3 2 4.0 16.2 6 76
231 IL4 x IL13 5 3 5 0 n.p. - - -
232 IL4 x IL13 5 5 5 0 0.6 24.9 8 70
233 IL4 x IL13 7 3 5 0 0.4 15.1 3 113
234 IL4 x IL13 7 5 5 0 1.3 30.7 3 122
235 IL4 x IL13 10 3 5 0 0.1 11.3 2 82
236 IL4 x IL13 10 5 5 0 0.4 18.6 1 1 112
237 IL4 x IL13 5 3 5 2 2.1 45.3 8.1 101.0
238 IL4 x IL13 5 5 5 2 0.6 45.4 9.3 67.2
239 IL4 x IL13 7 3 5 2 n.p. - - -
240 IL4 x IL13 7 5 5 2 1.6 31.7 4 65
241 IL4 x IL13 10 3 5 2 0.2 14.7 7 119
242 IL4 x IL13 10 5 5 2 1.1 17.6 10 37
243 IL4 x IL13 3 6 6 0 1.6 54.3 5 50
244 IL4 x IL13 3 10 6 0 1.5 63.9 10 10
245 IL4 x IL13 3 14 6 0 1.0 61.5 10 69
246 IL4 x IL13 6 6 6 0 1.1 16.2 6 57
247 IL4 x IL13 6 10 6 0 4.7 27.9 2 4 1
248 IL4 x IL13 6 14 6 0 0.9 18.1 10 79
249 IL4 x IL13 10 6 6 0 0.3 8.7 3 87
250 IL4 x IL13 10 8 6 0 0.7 21.3 8 53
251 IL4 x IL13 12 6 6 0 1.3 9.7 8 70
252 IL4 x IL13 12 8 6 0 1.3 11.7 7 85
253 IL4 x IL13 3 6 6 2 5.1 66.8 6 66
254 IL4 x IL13 3 10 6 2 2.4 62.4 6 80
255 IL4 x IL13 3 14 6 2 2.0 72.1 2 60
256 IL4 x IL13 6 6 6 2 2.0 32.4 4 8 1
257 IL4 x IL13 6 10 6 2 1.9 29.8 7 30
258 IL4 x IL13 6 14 6 2 2.5 24.6 5 70
259 IL4 x IL13 10 6 6 2 1.4 16.4 8 7 1
260 IL4 x IL13 10 8 6 2 0.8 16.6 10 7 1
261 IL4 x IL13 12 6 6 2 1.2 12.3 5 265
262 IL4 x IL13 12 8 6 2 1.1 13.2 4 111
263 IL4 x IL13 10 6 8 0 2.4 10.8 2 74
264 IL4 x IL13 10 8 8 0 0.8 8.0 7 22
265 IL4 x IL13 12 6 8 0 1.0 9.5 8 66
266 IL4 x IL13 12 8 8 0 2.0 9.3 3 69
267 IL4 x IL13 10 6 8 2 1.4 15.0 9 170
268 IL4 x IL13 10 8 8 2 1.0 12.9 4 52
269 IL4 x IL13 12 6 8 2 1.2 8.8 5 66
270 IL4 x IL13 12 8 8 2 2.4 11.7 3 72
* Alignment on the ight chain must be IL 13VL- LI-IL4VL-L2-CLI
The results from Tables 7 and 8 clearly show that linkers are required between
the variable and constant domains to allow optimal folding. Only in rare
arrangements was a linker equal to zero tolerated (see Batch ID Nos. 103-105, in
which Li (LC) was zero, and Batch No. 210, in which L4 was zero). However, in
each case, the corresponding transition linker between the variable region and
constant region on the other chain could not be zero.
The results above indicated that the combinations Li=7, L 2=5, L 3=l, and L4=2
were a good starting point for optimizing a new CODV-Ig in which the heavy chain is
the template. The ranges in Table 9 were shown to be reasonable ranges for
successfully engineering a new CODV-Ig from two parent antibodies.
Table 9. Ranges of Linker Sizes for Either LC or HC as Template
Example 8. Universal Applicability of CODV-Ig Format
To evaluate the suitability of the CODV-Ig format for engineering new
antibody-like binding proteins the variable regions from numerous existing human
and humanized antibodies having specificity for insulin-like growth factor 1 receptor
(IGFlR(l)), a second antibody to insulin-like growth factor 1 receptor (IGF1R(2)),
human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor
(EGFR), tumor necrosis factor - alpha (TNFa), Interleukin 12 and 23 (IL- 12/23) and
interleukin lbeta (IL- ) were incorporated into the CODV-Ig format (see Table 10).
Table 10. Descriptive Codes for Heavy and Light Chains Used in Bispecific
*A short hand code was devised to represent the associated structures. Codes
beginning with HC represent the adjacent heavy chain and codes beginning with LC
represent the adjacent light chains.
The antibody variable regions from known human and humanized antibodies
were used to test the universal applicability of the CODV-Ig format in designing
bispecific antibody-like binding proteins. In addition, the possibility of positional
effects with regard to the placement of certain antibody variable regions either Nterminal
or C-terminal on either the heavy chain or the light chain was examined.
Based on the design of CODV-Ig molecules having a linker composition of Li= 7,
L2= 5, L = 1, and L4=2, different sequences of antibodies were introduced into the
CODV-Ig format.
Activities of bispecific antibodies or derivatives against ILl and TNFa were
determined by using commercially available HEK-Blue TNFa/ILl reporter cells
(InvivoGen). To determine antibody activities against TNFa and ILl , the cytokines
were pre-incubated for 1hour with different concentrations of the antibodies and
added to 50,000 HEK Blue TNFa/ILl cells. Cytokine mediated induction of SEAP
was measured after 24 hours in the culture supernatant with the QUANTI-Blue assay
(InvivoGen).
As shown in Table 11, all constructs showed good to excellent protein yield
and acceptable levels of aggregation (see, in particular, Batch ID Nos. 301 and 302 in
Table 11). The measured affinity for each antibody variable domain was within the
published or expected affinity. In cases where affinity was assessed, no positional
effects were detected. In summary, as shown in the following tables, no positional
effects were seen with any of the antibody variable domains used or with the use of
these domains on either antibody chain.
Table 11. Universal Use of CODV-Ig Format for Bispecific Antibody-like Binding Proteins
*n.m.= not measurable by Biacore.
1 - Heavy chain and light chains corresponding o codes can be found in Table 10.
Example 9. Retention of Parental Antibody Affinity in CODV-Ig Format
The identical antibody sequences for anti-IL4 and anti-IL13 were incorporated
into either the TBTI/DVD-Ig or CODV-Ig formats for a direct comparison of these
configurations, the positioning of the linkers, and affinities of the resulting molecules. As
shown in Figure 7, the parental affinity of each of the antibodies was maintained in the
CODV format. As shown in the upper panel of Figure 7, when the variable regions were
placed in the TBTI/DVD-Ig format, a drop in affinity of the IL4 antibody positioned at
the inner Fv2 position was manifested as a reduction of the on-rate of antibody binding to
the antigen. In contrast, there was no loss in affinity for the CODV-Ig format as
compared to the parental antibodies (see Figure 7, lower panel).
Example 10. Adaptability of CODV-Ig to Fab Format
The ability of the CODV-Ig format to provide fragments such as Fab fragments
was evaluated next. Two different variable heavy chains were fused to each other
through linker L3 and elongated C-terminally by linker L4. This VH complex was then
fused to the CHI domain of IGHG1 (GenBank Accession No. Q569F4) harboring Cterminally
the five amino acid sequence DKTHT (SEQ ID NO: 60) from the hinge region
followed by six histidine residues. Two different variable light chains were fused to each
other in a cross-over configuration to the corresponding heavy chain through linker Li
and extended C-terminally by linker L2 and subsequently fused to the constant kappa
chain (IGKC, GenBank Accession No. Q502W4).
Fab fragments were expressed by transient transfection as described previously.
Seven days post-transfection, cells were removed by centrifugation, 10 % vol/vol 1M
Tris-HCl, pH 8,0, was added and the supernatant was passed over a 0.22 filter to
remove particles. The Fab proteins were captured using HisTrap High Performance
columns (GE Healthcare) and eluted via imidazole gradient. The protein containing
fractions were pooled and desalted using PD-10 or Sephadex columns. Concentrated and
sterile filtered (0.22 ) protein solutions were adjusted to 1 mg/ml and kept at 4°C until
use.
Immediate advantages were observed in that the Fab-like molecules in a CODV
orientation showed no tendency to aggregate and retained the affinities of the parental
antibodies (see Table 12). Binding protein constructs from Batch ID Nos. 401-421
directly compared antibody-like proteins in which antibody variable regions were
arranged as in CODV-Ig molecules with the heavy chain as the template (401, 402, 406,
and 407), CODV Fab-like fragments (402, 408, 413, 418, and 421), four domain
antibody-like molecules in TBTI/DVD-Ig format (404, 409, 414, and 419), and CODV-Ig
with no linkers (405, 410, 415, and 420). As shown in Table 12, the results of this
comparison indicated that there is more likely to be a loss in affinity as compared to the
parent antibodies when the variable region is incorporated into a TBTI or DVD-Ig
format. In contrast, both the CODV-Ig and CODV-Ig Fab-like formats were better able
to maintain parental affinities. The results further confirmed that CODV-Ig molecules
require linkers between the variable regions and between the variable regions and the
constant domains (see Table 12).
Table 12. Use of CODV-Ig Format for Fab-like Fragments
Example 11. Substitution of Variable Domains within CODV-Ig and CODV-Fab
To characterize the CODV format in a T-cell engaging approach, bispecific
CODV Fab-like binding proteins (CODV-Fab) having a TCR binding site (CD3epsilon)
and a CD19 binding site were generated and compared to a bispecific Fab derived from
the TBTI/DVD-Ig format (B-Fab). To investigate the importance of the orientation of the
binding sites (TCR x CD19 vs. CD19 x TCR), both orientations were evaluated for each
of the binding proteins.
The binding proteins were characterized in a cytotoxic assay using NALM-6
(CD 19 expressing) cells as target cells and primary human T-cells as effector cells. CD3
positive cells were isolated from freshly prepared human PBMC's. Effector and target
cells were mixed at a ratio of 10: 1 and incubated for 20 hours with the indicated
concentrations of bispecific binding proteins (see Figure 8). Apoptotic target cells were
determined in a FACS-based assay using 7-Aminoactinomycin staining.
The B-Fab format in the configuration CD3-CD19 (1060) was shown to be active
in inducing T-cell mediated cytotoxicity towards NALM-6 cells with an EC50 of 3.7
ng/ml. A similarly high activity was observed for the CD19-CD3 CODV-Fab ( 1109)
with an EC50 of 3.2 ng/ml (see Figure 8).
A swap of the configuration of the B-Fab molecule (Fab of the TBTI / DVD-Ig
format) to a CD19-CD3 orientation resulted in a significant loss of activity (see Figure 8).
The swapped B-Fab molecule showed no activity at concentrations that were maximal for
both orientations of CODV-Ig Fab and the other orientation of B-Fab. For the CODV-Ig
Fabs and one orientation of B-Fab, a maximum response was observed (ranging between
1 and 100 ng/ml). For the CD19-CD3 orientation of B-Fab, even at the maximal
concentration (30 g/ml), the optimal cytotoxic response was not reached. In sharp
contrast, a change in the orientation of the domains in the CODV-Fab towards CD3-
CD19 ( 1108), resulted in a molecule with significant activity in this assay (see Figure 8).
Although domain swapping in the CODV-Fab also reduced the induction of T-cell
mediated cytotoxicity (increase of EC50 by factor -100), this effect was far less
pronounced than was seen in the B-Fab format and the molecule was able to induce
cytotoxicity up to the maximal level. The data were representative and obtained from
three independent experiments.
Example 12. Influence of Amino Acid Sequence Identity on CODV-Ig Linkers
The optimized construct corresponding to Batch ID 204 (see Example 7 and Table
8) was chosen to investigate the influence of linker composition on the linkers Li to L4.
Linker lengths were set at 7, 5, 1, and 2 residues in length for Ll L2, L3, and L4,
respectively (see Table 13). Test sequences were derived from naturally occurring
linkers at the transitions between natural antibody VHand CHI domains or between
antibody Fv and CL domains of kappa or lambda light chains. The candidate sequences
were ASTKGPS (SEQ ID NO: 48), which is derived from the VH and C domain
transition, RTVAAPS (SEQ ID NO: 49) and GQPKAAP (SEQ ID NO: 50), which were
derived from the Fv and CL domain transitions of kappa and lambda light chains,
respectively. Furthermore, one construct was generated with an arbitrary linker
composition to show that any sequence can be potentially used in linkers Li to L4. This
linker composition was obtained by randomly distributing the amino acids valine,
leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine, glycine, and proline at the 15 positions of the four linkers. The
aromatic amino acids phenylalanine, tyrosine, and tryptophan, as well as the amino acids
methionine and cysteine were deliberately excluded to avoid potential increases in
aggregation.
A three-dimensional model of the construct for Batch ID No. 204 was generated
to assure suitability or refine the choices of linker composition. Thus, serine was chosen
for linker L3 as positively and negatively charged residues are observed nearby in the
three-dimensional model. The residues in linker L4 were selected to be compatible with
solvent exposure of these positions as suggested by the model. Similarly, no problems
were anticipated or predicted for the linker compositions of Li and L2. Threedimensional
models of selected proposals for linker composition were constructed.
As shown in Table 12, linker composition may have a dramatic influence on
yield. Sequences that were derived from lambda chain on Li (comparing Batch ID Nos.
505-507 with Batch ID Nos. 501-503) were more productive protein generators (up to 8
fold increase). Indeed, the linkers based on random generation also produced good
yields, as shown in Table 13, Batch ID No. 508. Therefore, linker composition should be
one parameter considered during CODV-Ig optimization.
Table 13. Effect of Linker Composition on CODV-Ig
Activities of bispecfic antibodies or derivatives against cytokines IL4 and IL13
were determined in commercially available HEK-Blue IL-4/IL-13 reporter cells
(InvivoGen). HEK-Blue IL-4/IL-13 cells are designed to monitor the activation of the
STAT6 pathway by IL-4 or IL13. Stimulation of the cells with either cytokine results in
production of the reporter gene secreted embryonic alkaline phosphatase (SEAP), which
can be measured in the culture supernatant with the QUANTI-Blue assay. To test
antibody activities against IL4 or IL13, the cytokines were pre-incubated for 1 hour with
different concentrations of the antibodies and added to 50,000 HEK-Blue IL-4/IL-13
cells. Cytokine-mediated induction of SEAP was measured after 24 hours incubation in
the cell culture supernatant with the QUANTI-Blue assay (InvivoGen).
Example 13. Introduction of Cysteines into CODV-Ig Linkers
Published data suggest that the stability of antibodies and antibody-derived
proteins can be increased by the introduction of non-natural disulfide bridges (see
Wozniak-Knopp et al., 2012, "Stabilisation of the Fc Fragment of Human IgGl by
Engineered Intradomain Disulfide Bonds," PLoS ONE 7(1): e30083). To examine
whether the equivalent Fc fragment derived from a human IgGl antibody and engineered
into a CODV-Ig molecule can be stabilized by the introduction of inter- and intra-chain
disulfide bridges, the equivalent Fc positions of the CODV-Ig construct Batch ID No.
204 (from Example 7) were mutated to cysteine residues, and the mutant proteins were
overproduced, purified, and characterized (see Table 14).
As shown in Table 14, each of the mutated CODV-Ig molecules containing
additional cysteine residues had melting temperatures that were the same as the melting
temperature for the CODV-Ig construct Batch ID No. 204.
In addition, two simultaneous cysteines were introduced at Kabat positions 100
for the light chain and 44 for the heavy chain on each of the variable domains as
described in Brinkmann et al, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 7538-42. These
positions have been shown to be structurally conserved within antibody folds, and
therefore tolerable of cysteine substitution without interfering with the integrity of the
individual domains.
As shown in Table 15, CODV and CODV-Ig constructs in which cysteine
residues were introduced at Kabat positions 100 for the light chain and 44 for the heavy
chain on each of the variable domains had higher melting temperatures than the CODV
and CODV-Ig constructs in which cysteine residues were not introduced at these
positions (see, e.g., Batch ID Nos. 704 and 706 and Batch ID Nos. 713 and 714).
1. Thermostability Measurements of CODV and TBTI Variants
Melting points (Tm) of CODV and TBTI variants were determined using
differential scanning fluorimetry (DSF). Samples were diluted in D-PBS buffer
(Invitrogen) to a final concentration of 0.2 g/l and added to 21of a 40x concentrated
solution of SYPRO-Orange dye (Invitrogen) in D-PBS in white semi-skirt 96-well plates.
All measurements were done in duplicate using a MyiQ2 real time PCR instrument
(Biorad). Tm values were extracted from the negative first derivative of the melting
curves using iQ5 Software v2. 1.
Next the effect of introducing cysteine residues directly into the linkers or within
the variable region was examined. In this example, Batch ID No. 204 (from Example 7
and Table 8) was used as the model CODV-Ig binding protein, and cysteine was
substituted for glycine in Ll L3, or the variable region on the basis of the threedimensional
model. As shown in Table 16 below, the results indicate how the
introduction of cysteine pairs would affect yield and aggregation. The envisioned
mutations were all modeled to ascertain that disulfide bonds were properly formed and
correct geometry was maintained for the linkers and their environment in the models.
Nevertheless, Batch ID No. 808 showed good yield and little aggregation, thus
suggesting that a proper cysteine bridge could be formed.
While the invention has been described in terms of various embodiments, it is
understood that variations and modifications will occur to those skilled in the art.
Therefore, it is intended that the appended claims cover all such equivalent variations that
come within the scope of the invention as claimed. In addition, the section headings used
herein are for organizational purposes only and are not to be construed as limiting the
subject matter described.
Each embodiment herein described may be combined with any other embodiment
or embodiments unless clearly indicated to the contrary. In particular, any feature or
embodiment indicated as being preferred or advantageous may be combined with any
other feature or features or embodiment or embodiments indicated as being preferred or
advantageous, unless clearly indicated to the contrary.
All references cited in this application are expressly incorporated by reference
herein.
Table 14. Effect of Disulfide Bridge Stabilization on CODV-Ig
Table 15. Effect of Disulfide Bridge Stabilization on CODV-Ig
Table 16A. Effect of Disulfide Bridge Stabilization on CODV-Ig
Table 16B. Effect of Disulfide Bridge Stabilization on CODV-Ig
WHAT IS CLAIMED IS:
Claim 1. An antibody-like binding protein comprising four polypeptide chains that
form four antigen binding sites, wherein two polypeptide chains have a structure
represented by the formula:
VLI-L -VL -L -CL [I]
and two polypeptide chains have a structure represented by the formula:
VH2-L3-VHI -L4-CHI-FC [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
V 2 a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2, CH3 immunoglobulin heavy chain
constant domains; and
Li, L2, L3, and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
Claim 2. The antibody-like binding protein of claim 1, wherein:
Li is 3 to 1 amino acid residues in length;
L2 is 3 to 14 amino acid residues in length;
L3 is 1 to 8 amino acid residues in length; and
L4 is 1 to 3 amino acid residues in length.
Claim 3. The antibody-like binding protein of either claim 1 or 2, wherein:
Li is 5 to 10 amino acid residues in length;
L2 is 5 to 8 amino acid residues in length;
L3 is 1 to 5 amino acid residues in length; and
L4 is 1 to 2 amino acid residues in length.
Claim 4. The antibody-like binding protein of any one of claims 1-3, wherein:
Li is 7 amino acid residues in length;
L2 is 5 amino acid residues in length;
L3 is 1 amino acid residues in length; and
L4 is 2 amino acid residues in length.
Claim 5. The antibody-like binding protein of claim 1, wherein:
Li is 1 to 3 amino acid residues in length;
L2 is 1 to 4 amino acid residues in length;
L3 is 2 to 15 amino acid residues in length; and
L4 is 2 to 15 amino acid residues in length.
Claim 6. The antibody-like binding protein of either claim 1 or 5, wherein:
Li is 1 to 2 amino acid residues in length;
L2 is 1 to 2 amino acid residues in length;
L3 is 4 to 12 amino acid residues in length; and
L4 is 2 to 12 amino acid residues in length.
Claim 7. The antibody-like binding protein of any one of claims 1, 5, or 6, wherein:
Li is 1 amino acid residue in length;
L2 is 2 amino acid residues in length;
L3 is 7 amino acid residues in length; and
L4 is 5 amino acid residues in length.
Claim 8. The antibody-like binding protein of claim 1, wherein Li is 0 amino acid
residues in length and L3 is 2 or more amino acid residues in length.
Claim 9. The antibody-like binding protein of claim 1, wherein L3 is 0 amino acid
residues in length and Li is 1 or more amino acid residues in length.
Claim 10. The antibody- like binding protein of claim 1, wherein L4 is 0 amino acid
residues in length and L2 is 3 or more amino acid residues in length.
Claim 11. The antibody- like binding protein of any one of claims 1-10, wherein the
binding protein is capable of specifically binding one or more antigen targets.
Claim 12. The antibody- like binding protein of any one of claims 1-1 1, wherein the
one or more antigen targets is selected from the group consisting of B7. 1, B7.2, BAFF,
BlyS, C3, C5, CCL1 1 (eotaxin), CCL15 (MIP-ld), CCL17 (TARC), CCL19 (MIP-3b),
CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2/eotaxin-2),
CCL25 (TECK), CCL26 (eotaxin-3), CCL3 (MIP-la), CCL4 (MIP-lb), CCL5
(RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CD3, CD19, CD20, CD24, CD40, CD40L,
CD80, CD86, CDH1 (E-cadherin), Chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3
(GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A,
FCER2, HER2, IGFIR, IL-1, IL-12, IL13, IL15, IL17, IL18, ILIA, ILIB, ILIFIO, ILip,
IL2, IL4, IL6, IL7, IL8, IL9, IL12/23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b 4
integrin), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNFa, TNFSF4 (OX40
ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREMl, TSLP, TWEAK, XCR1
(GPR5/CCXCR1), DNGR-1(CLEC91), and HMGB1.
Claim 13. The antibody- like binding protein of any one of claims 1-12, wherein the
binding protein is bispecific and capable of binding two different antigen targets.
Claim 14. The antibody- like binding protein of any one of claims 1-13, wherein the
two different antigen targets are selected from the group consisting of IL4 and IL13,
IGFIR and HER2, IGFIR and EGFR, EGFR and HER2, BK and IL13, PDL-1 and
CTLA-4, CTLA4 and MHC class II, IL-12 and IL-1 8, IL-1 a and IL- , TNFa and
IL12/23, TNFa and IL-12p40, TNFa and ILip, TNFa and IL-23, and IL17 and IL23.
Claim 15. The antibody-like binding protein of any one of claims 1-14, wherein the
binding protein is capable of inhibiting the function of one or more of the antigen targets.
Claim 16. The antibody-like binding protein of any one of claims 1-15, wherein at
least one of the linkers selected from the group consisting of Ll L2, L3, and L4 contains at
least one cysteine residue.
Claim 17. An antibody-like binding protein comprising two polypeptide chains that
form two antigen binding sites, wherein a first polypeptide chain has a structure
represented by the formula:
and a second polypeptide chain has a structure represented by the formula:
VH2-L3-VHI-L 4-CHI [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain; and
Li, L2, L3, and L4 are amino acid linkers;
and wherein the first and second polypeptides form a cross-over light chain-heavy chain
pair.
Claim 18. The antibody-like binding protein of claim 17, wherein:
Li is 3 to 1 amino acid residues in length;
L2 is 3 to 14 amino acid residues in length;
L3 is 1 to 8 amino acid residues in length; and
L4 is 1 to 3 amino acid residues in length.
Claim 19. The antibody-like binding protein of either claim 17 or 18, wherein:
Li is 5 to 10 amino acid residues in length;
L2 is 5 to 8 amino acid residues in length;
L3 is 1 to 5 amino acid residues in length; and
L4 is 1 to 2 amino acid residues in length.
Claim 20. The antibody-like binding protein of any one of claims 17-19, wherein:
Li is 7 amino acid residues in length;
L2 is 5 amino acid residues in length;
L3 is 1 amino acid residues in length; and
L4 is 2 amino acid residue in length.
Claim 21. The antibody-like binding protein of claim 17, wherein:
Li is 1 to 3 amino acid residues in length;
L2 is 1 to 4 amino acid residues in length;
L3 is 2 to 15 amino acid residues in length; and
is 2 to 15 amino acid residues in length.
Claim 22. The antibody-like binding protein of either claim 17 or 21, wherein:
Li is 1 to 2 amino acid residues in length;
L2 is 1 to 2 amino acid residues in length;
L3 is 4 to 12 amino acid residues in length; and
is 2 to 12 amino acid residues in length.
Claim 23. The antibody-like binding protein of any one of claims 17, 21,
wherein:
Li is 1 amino acid residue in length;
L2 is 2 amino acid residues in length;
L3 is 7 amino acid residues in length; and
L4 is 5 amino acid residues in length.
Claim 24. The antibody-like binding protein of claim 17, wherein Li is 0 amino acid
residues in length and L3 is 2 or more amino acid residues in length.
Claim 25. The antibody-like binding protein of claim 17, wherein L3 is 0 amino acid
residues in length and Li is 1 or more amino acid residues in length.
Claim 26. The antibody-like binding protein of claim 17, wherein L4 is 0 amino acid
residues in length and L2 is 3 or more amino acid residues in length.
Claim 27. The antibody-like binding protein of any one of claims 17-26, wherein the
binding protein is capable of specifically binding one or more antigen targets.
Claim 28. The antibody-like binding protein of any one of claims 17-27, wherein the
one or more antigen targets is selected from the group consisting of B7. 1, B7.2, BAFF,
BlyS, C3, C5, CCL1 1 (eotaxin), CCL15 (MIP-ld), CCL17 (TARC), CCL19 (MIP-3b),
CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2/eotaxin-2),
CCL25 (TECK), CCL26 (eotaxin-3), CCL3 (MIP-la), CCL4 (MIP-lb), CCL5
(RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CD3, CD19, CD20, CD24, CD40, CD40L,
CD80, CD86, CDH1 (E-cadherin), Chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3
(GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A,
FCER2, HER2, IGFIR, IL-1, IL-12, IL13, IL15, IL17, IL18, ILIA, ILIB, ILIFIO, ILip,
IL2, IL4, IL6, IL7, IL8, IL9, IL12/23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b 4
integrin), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNFa, TNFSF4 (OX40
ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREMl, TSLP, TWEAK, XCR1
(GPR5/CCXCR1), DNGR-1(CLEC91), and HMGB1.
Claim 29. The antibody-like binding protein of any one of claims 17-28, wherein the
binding protein is bispecific and capable of binding two different antigen targets.
Claim 30. The antibody-like binding protein of any one of claims 17-29, wherein the
two different antigen targets are selected from the group consisting of IL4 and IL13,
IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and
CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-l and IL- , TNFa and
IL12/23, TNFa and IL-12p40, TNFa and ILip, TNFa and IL-23, and IL17 and IL23.
Claim 31. The antibody-like binding protein of any one of claims 17-30, wherein the
binding protein is capable of inhibiting the function of one or more of the antigen targets.
Claim 32. The antibody- like binding protein of any one of claims 17-31, wherein at
least one of the linkers selected from the group consisting of Ll L2, L3, and L4 contains at
least one cysteine residue.
Claim 33. An isolated nucleic acid molecule comprising a nucleotide sequence
encoding the antibody-like binding protein of any one of claims 1-32.
Claim 34. An expression vector comprising the nucleic acid molecule of claim 33.
Claim 35. An isolated host cell comprising the nucleic acid molecule of claim 33 or
the expression vector of claim 34.
Claim 36. The host cell of claim 35, wherein the host cell is a mammalian cell or an
insect cell.
Claim 37. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier and a therapeutically effective amount of the antibody-like binding protein of any
one of claims 1-32.
Claim 38. A method for making the antibody-like binding protein of any one of
claims 1-16, comprising expressing in a cell one or more nucleic acid molecules encoding
polypeptides having structures represented by the formulas [I] and [II] below:
VH2-L3-VHI -L4-CHI-FC [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
V 2 a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2, CH3 immunoglobulin heavy chain
constant domains; and
Li, L2, L3, and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
Claim 39. A method for making the antibody-like binding protein of any one of
claims 17-32, comprising expressing in a cell one or more nucleic acid molecules
encoding polypeptides having structures represented by the formulas [I] and [II] below:
VH2-L3-VHI-L 4-CHI [II]
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain; and
Li, L2, L3, and L4 are amino acid linkers;
and wherein the polypeptide of formula I and the polypeptide of formula II form a cross
over light chain-heavy chain pair.
Claim 40. A method of making an antibody-like binding protein comprising four
polypeptide chains that form four antigen binding sites, comprising:
(a) identifying a first antibody variable domain that binds a first target antigen
and a second antibody variable domain that binds a second target antigen, each
containing a VL, and a VH
(b) assigning either the light chain or the heavy chain as template chain;
(c) assigning the VL of the first antibody variable domain or the second
antibody variable domain as VLI ;
(d) assigning a VL2, a VHI , and a VH2 according to formulas [I] and [II] below:
VLI-L -VL -L -CL [I]
VH2-L3-VHI -L4-CHI-FC [II]
(e) determining maximum and minimum lengths for Ll L2, L3, and L4;
(f) generating polypeptide structures of formulas I and II;
(g) selecting polypeptide structures of formulas I and II that bind the first
target antigen and the second target antigen when combined to form the antibody-like
binding protein;
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain;
Fc is the immunoglobulin hinge region and CH2 H3 immunoglobulin heavy chain
constant domains; and
Li, L2, L3, and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
Claim 4 1. The method of claim 40, wherein the first antibody variable domain and
the second antibody variable domain are the same.
Claim 42. A method of making an antibody-like binding protein comprising four
polypeptide chains that form four antigen binding sites, comprising:
(a) identifying a first antibody variable domain that binds a first target antigen
and a second antibody variable domain that binds a second target antigen, each
containing a VL, and a VH;
(b) assigning either the light chain or the heavy chain as template chain;
(c) assigning the VL of the first antibody variable domain or the second
antibody variable domain as VLI ;
(d) assigning a VL2, a VHi, and a VH2 according to formulas [I] and [II] below:
(e) determining maximum and minimum lengths for Ll L2, L3, and L4;
(f) generating polypeptide structures of formulas I and II;
(g) selecting polypeptide structures of formulas I and II that bind the first
target antigen and the second target antigen when combined to form the antibody-like
binding protein;
wherein:
VLI is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VHI is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CHI is the immunoglobulin CHI heavy chain constant domain; and
Li, L2, L3, and L4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
Claim 43. The method of claim 42, wherein the first antibody variable domain and
the second antibody variable domain are the same.