BRPI0707824A2 - antigen-binding protein, and methods of neutralizing tyrosine kinase receptor activation, inhibiting angiogenesis, reducing tumor growth and producing an antigen-binding protein - Google Patents

Abstract

ANTIGEN BINDING PROTEIN AND METHODS FOR NEUTRALIZING THE ACTIVATION OF A THYROSINE KINASE RECEPTOR, ANGIOGENESIS INHIBITION, REDUCING TUMOR GROWTH ANTIBODIES AND UNDERSTANDING A ANTIGENE PROTEIN PROTECTION single domain link sites. Antibodies can be bivalent or multivalent, and can be bispecific. The invention is also directed to monospecific and bispecific antibodies, which bind to mPDGFR <244>. Antibodies can be administered alone or in combination with antiangiogenic or antineoplastic drugs.

Description

"ANTIGEN-BINDING PROTEIN AND METHODS DEENUTRALIZATION OF A TYROSINKINASE RECEIVER, ANGIOGENESIS INHIBITION, TUMOR GROWTH REDUCTION AND PRODUCTION OF ANTIGEN-BINDING PROTEIN"

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority for application US 60 / 773,994, filed February 15, 2006, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention is directed to novel antibodies comprising single domain binding sites. Antibodies may be bivalent or multivalent, and may be specific or multispecific. Bispecific and multispecific antibodies that bind surface receptor proteins are also described. When administered to a single individual or in combination with angiogenic or antineoplastic drugs, antibodies may be used to inhibit tumor growth as well as inhibit hyperproliferative diseases.

BACKGROUND

Bispecific antibodies (BsAb) are immunoglobulin (Ig) -based molecules that bind to two different epitopes on the same or distinct antigens. Recent laboratory and clinical studies have shown that BsAb can have significant applications in cancer therapy, for example targeting tumor cells with cytotoxic agents such as effector cells, radionuclides, drugs and toxins (Weineret al., 1997, Cancer Immunol. Immunother. 45: 190-2; van Spriel et al., 2000, Immunol Today 21: 391-7; Segai et al., 2000, J. Immunol. Methods 248: Ι-○), or simultaneously marking two different tumor targets (or epitopes) in order to improve the biological activities of individual antibody therapies (Lu et al., 1999, J. Immunol. Methods 230: 159-71; Luet al., 2001, Cancer Res. 61: 7002-8; Lu et al., 2002, J. Immunol. Methods 267: 213-26). A major obstacle in the development of BsAb-based therapeutic agents is the difficulty in producing materials of sufficient quality and quantity for clinical studies via traditional methods, including hybrid hybridoma and chemical conjugation (Carter et al., 1995, J. Hemotherapy 4: 463-70. ). Coexpression of two different sets of IgG light and heavy chains may result in a variety of heavy and light chain pairs, only one of which is the desired functional heterodimeric (Suresh et al., 1986, Methods Enzymol. 121: 210- 28). On the other hand, chemical cross-linking of two IgGs or their fragments is often inefficient and can lead to loss of antibody activity (Zhu et al., 1994, Cancer Lett. 86: 127-34). In both methods, purification of BsAb from unnatural species, such as homodimers and heterodimers with mismatched light and heavy chain Ig chains produced by the hybrid hybridoma, and multimeric aggregates resulting from chemical conjugation, is often difficult and the yield is generally low ( Cao et al., 1998, Bioconj. Chem. 9: 635-44).

To improve efficiency, various recombinant methods have been developed for the production of BsAb as both antibody fragments (Carter et al., 1995; Pluckthun et al., 1997, Immunotechnology 3: 83-105; Todorovska et al., 2001, J. Methods 248: 47-66) as full length IgG formates (Carter, 2001, J. Immunol.Methods 248: 7-15). For example, the production of homogeneous full-length IgG-type BsAb is achieved by so-called "we-in-hole" engineering for efficient heterodimerization of the Ch3 domain (Ridgway et al., 1996, Protein Eng. 9: 617 Merchant et al., 1998, Nat.Biotech 16: 677-81) and fusing two single-chain Fv (scFv) of different specificities at either the N or C terminus of a full-length IgG molecule (Zhuang et al., 2000, Protein Eng. 13: 361-7; Coloma and Morrison, 1997, Nat. Biotechnol. 15: 159-63). BsAbs are also constructed by genetically fusing single-stranded Fv (scFv) or Fab fragments with or without the use of flexible linkers (Mallender et al., 1994, J. Biol. Chem. 269: 199-206; Mack et al., 1995 Proc Natl Acad Sci USA 92: 7021-5 Zapata et al 1995 Protein Eng 8: 1057-62 via a leucine zipper dimerization device (Kostelny et al 1992). J. Immunol 148: 1547-53; from Kruifet al., 1996, J. Biol. Chem. 271: 7630-4), Ig CL / CHI domains (Muller et al., 1998, FEBS Lett. 422: 259-64); poriacibody (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA. 90: 6444-8; Zhuet al., 1996, Bio / Technology (NY) 14: 192-6); Fab-scFv fusion (Lu et al., 2002; Schoonjans et al., 2000, J. Immunol. 165: 7050-7); and mini-antibody formats (Pack et al., 1992, Biochemistry 31: 1579-84; Pack et al., 1993, Bio / Technology 11; 1271-7). In most cases, these recombinant approaches result in the production of divalent bispecific antibody molecules that are monovalent in each of the target antigens. In addition, examples of IgG bispecific antibodies comprising functional Fc domains are limited.

SUMMARY OF THE INVENTION

The invention provides novel bispecific antibodies comprising single variable domain antigen (sVD) binding sites. Antibodies of the invention may also include antigen binding sites which comprise Fvs antibody binding sites in addition to VD antigen binding sites.

Antibodies of the invention may be specific for any antigen. In one embodiment, an antibody of the invention binds to a cell surface antigen, which may be a receptor tyrosine kinase (including, but not limited to, PDGFR, VEGFR1, VEGFR2, EGFR). In another embodiment, an antibody of the invention binds a ligand from a cell surface receptor. In a preferred embodiment, an antibody of the invention has receptor neutralizing activity. The invention further provides pharmaceutical compositions of the antibodies.

The present invention provides methods for inhibiting activation of one or more receptor tyrosine kinases. Tumor growth in a mammal can be treated or inhibited by administering to the mammal an effective amount of an antibody present. Antibodies present may be co-administered with antibodies that bind to another cell surface antigen (including, for example, RTKs) or cytokines (including, for example, RTK ligands). In certain embodiments, the methods also comprise administering to the mammal an antineoplastic agent or treatment, including, for example, a chemotherapeutic agent and / or radiation. In another embodiment, the invention provides a method for treating a non-cancer hyperproliferative disease, e.g., psoriasis, in a mammal.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic diagram showing examples of single domain antibody-based bispecific antigen binding proteins of the invention. The single domain (designated VH2 in the drawing) may be incorporated by fusion to the N-terminus or C-terminus of other antibody domains.

Figure 2 shows expression and purification of mPDGFRα specific Fabs. Fabs were expressed in E. coli host HB2151 cells, purified by affinity chromatography and assayed by SDS-PAGE. A) "Standard" mPDGFRa specific Fabs having heavy (Vh-ChI) and light (Vl-Cl) chains of similar molecular weight. B) Fab fragments 1F2 and 1F9 missing V1 domains and a standard Fab control in the presence or absence of DTT. Molecular weight markers are expressed in kilodaltons. Figure 3 shows binding and blocking of Fabspurified tests. A) Quantitative binding ELISA of purified Fabs in murine PDGFRα. Various amounts of Fabs were first incubated in a 96-well plate pre-coated with PDGFRa-Fc fusion protein followed by incubation with a rabbit HRPanti-anti-human-κ antibody conjugate. Plate - bound HRP antibody was quantified by the addition of peroxidase substrate, and the absorbance was read at A450 nm. B) Inhibition of binding of mPDGFRα to PDGF-AA immobilized by purified Fabs. Various amounts of Fabs were incubated with a fixed amount of mPDGFRa / Fc in solution. The mixtures were incubated in PDGF-AA coated 96-well plates followed by the anti-human-Fc-HRP-antibody conjugate. Bound mPDGFRα was then quantified by the addition of peroxidase substrate, and absorbance was read at A450 nm. The data shown represent the mean ± standard deviation of samples in duplicate.

Figure 4 depicts ebivalent monovalent Fabs structures of the invention.

Figure 5 shows the expression and purification of bivalent mPDGFRα specific Fabs. Fabs were expressed in affinity chromatography purified E. coli host HB2151 cells and assayed by SDS-PAGE. Divalent 1F2-2H Fab is compared to a standard Fab (5C5) under non-reducing conditions. Molecular weight markers are expressed in kilodaltons.

Figure 6 shows binding and blocking tests of purified antibodies. A) Quantitative binding assay of purified anti-mPDGFRα IF-monovalent and bivalent Fab antibodies. Various amounts of antibodies were incubated in a 96-well plate coated with mPDGFRa / Fc, followed by incubation with an anti-human-κ antibody-HRP conjugate. The plate - bound HRP Antibody was then quantified by the addition of peroxidase substrate, and the absorbance was read at 450 nm. B) Inhibition of binding of mPDGFRα to immobilized PDGF-AA by purified Fabs antibodies. Various amounts of antibodies were incubated with a fixed amount of mPDGFRa / Fc and the mixtures were transferred to PDGF-AA pre-coated plates. After washing, the plates were then incubated with an anti-human HRP-Fc antibody conjugate. Connected mPDGFR was quantified by the addition of peroxidase substrate, and absorbance was read at A450 nm. The data shown represent the mean ± standard deviation of duplicate samples. 2B4 is an antibody directed against decamound VEGFR2 (Flkl).

Figure 7 provides a schematic representation of full-length IF2-based IgGs. IF 2 Vh is expressed as a fusion protein in CL and / or CH, providing tetravalent (panel A; MW ~ 150,000) or bivalent (panel B and C: MW ~ 125,000) antibodies.

Figure 8 shows expression and purification of anti-mPDGFRα antibodies. In the right panel, antibodies were treated with DTT before electrophoresis.

Figure 9 shows ligand binding and blocking assays for purified antibodies. A) quantitative binding, purified anti-mPDGFRα antibodies. Various amounts of antibodies were incubated in a 96-well plate pre-coated with mPDGFRa / Fc, followed by incubation with an anti-human κ-HRP antibody conjugate. The plate-bound HRP antibody was quantified by the addition of peroxidase substrate, and the absorbance was read at A450 nm. B) Inhibition of binding of immobilized mPDGFRα to PDGF-AA by purified antibodies. Various amounts of antibodies were incubated with a fixed amount of mPDGFRa / Fc and transferred to PDGF-AA pre-coated plates. The plates were washed and incubated with a bound antibody-anti-human HRP-Fc.mPDGFRα conjugate was then quantified by the addition of deperoxidase substrate, and the absorbance was read at ≤ 450 nm. Data represent mean ± standard deviation of duplicate samples. 2B4 is a mouse VEGFR2 (Flkl) directed antibody.

Figure 10 provides a schematic representation of selected IF2-based tetravalent bispecific antibodies that bind mPDGFRa and flk-1. A) IgG-like antibody designated IF2 / scFv2B4.Vh of IF2 is expressed as a fusion to CL, and 2B4 is expressed as a scFv fusion to CH. Β) The IgG type antibody designated IF2-2B4. IF2 Vh is expressed as an amino terminus fusion of 2B4 V1.

Figure 11 shows expression and purification of specific IF2-based antibodies. Antibodies were expressed on mammalian cells, purified by affinity chromatography, and analyzed by SDS-PAGE. Antibodies were treated with / without (±) DTT prior to electrophoresis. Molecular weight markers are expressed in kilodaltons.

Figure 12 shows the quantitative binding tests of anti-mPDGFRα anti-Flk-1 bispecific antibodies. A) Various amounts of antibodies were first incubated in a 96-well plate pre-coated with mPDGFRa / Fc, followed by incubation with an anti-human-κ HRP-antibody conjugate. B) Various amounts of antibodies were first incubated in a Flk-1 / Fc pre-coated 96-well plate, followed by incubation with an anti-human-k-antibody HRP conjugate. The data shown represent the mean ± standard deviation of samples in duplicate.

Figure 13 shows anti-FlP-1 anti-mPDGFRα specific antibody blockade tests. A) Inhibition of demPDGFRα binding to PDGF-AA immobilized by purified bispecific antibodies. B) Inhibition of Flk-I binding to VEGF165 immobilized by purified bispecific antibodies. In both tests, various amounts of antibodies were incubated with a fixed amount of receptor (mPDGFRa / Fc or Flk-1) in solution and transferred to ligand pre-coated plates (PDGF-AA or VEGF165). The plates were then incubated with an anti-human HRP-Fc antibody conjugate. Bound mPDGFRα was then quantified by the addition of peroxidase substrate, and the absorbance was A450 nm. The data shown represent the mean ± standard deviation of duplicate samples.

Figure 14 shows inhibition of PDGF and VEGF stimulated receptor phosphorylation by bispecific IF2-2B4IgG. EEnd.l cells were first incubated with various antibodies at 37 ° C for 30 min, followed by stimulation with VEGF or PDGF at 37 ° C for 15 min. Following cell isolation, receptors were immunoprecipitated from the delisated cell supernatant by incubation with an anti-mPDGFRa or an anti-mVEGFR2 antibody, followed by ProA / G Sepharose beads. Precipitated receptor proteins were resolved on a 4-12% Nupage Bis-Tri gel and transferred to a polyvinylidene difluoride membrane. Phospho-mVEGFR2 and phospho-mPDGFRa were detected in the blot using an anti-phospho-tyrosine HRP antibody conjugate. Gel-loaded total receptor proteins were tested with antibodies on mPDGFRa or mVEGFR2.

Figure 15 shows the amino acid sequences of domains Vh and Vl of various identified Fab proteins. Fabs IE10, 1A11,3B2, 1C10, 3G7, 1F9 and IF2 were identified in a screening for phageFab that binds to murine PDGFRα. Fab 2B4 has been identified in a screening for Fab phage that binds to murine VEGFR2; mutation with displacement of the frame; * stop codon resulting from mutation with displacement of information; frame deletion.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel antibodies comprising single variable domain antigen (sVD) binding sites. The invention also provides antigen-binding proteins comprising both single domain antigen-binding sites and two-domain Fv-type antigen binding sites.

Single domain antigen binding sites are similar to variable immunoglobulin domains (e.g., Vh or Vl), but are antigen-specific binding capable in the absence of a second antigen-binding domain (e.g., a Vl or Vh counterpart). Single domains may in fact be incapable of stable association with the counterpart domains of binding. However, single domain antibodies can bind antigen with similar affinities and avidity to antibodies that include both V1 and VH domains. In addition, as natural antibodies, single domain antibodies can block receptor-ligand interactions. Consequently, antibodies of the invention comprising single domain sites are used for the same purposes as antibodies obtained, for example, from hybridomas, transgenic mice expressing antibodies. human, and Fab or scFv deletion domain phage display libraries.

Antibodies of the invention are immunoglobulin-like in various aspects. 1) IgG-like antibodies of the invention are heterotetramers, consisting of two of each of two dissimilar polypeptide chains. 2) The dissimilar polypeptide chains associate and may be covalently linked, for example, by disulfide bonds in the same manner as standard naturally occurring immunoglobulin light and heavy chain constant regions. 3) One of the polypeptide chains is capable of stable self-association in the naturally occurring immunoglobulin heavy chain mode. For example, one of the polypeptide chains may include one or more domains corresponding to CH2, CH3 or Ch4 of a naturally occurring immunoglobulin. In a preferred embodiment, an antibody of the invention comprises the constant domain structure of a naturally occurring IgG.

A huge advantage of an immunoglobulin-like structure is that antibody chains naturally pair when expressing. Unlike many prior art tetrameric antibodies, no further manipulation is required to obtain a homogeneous antibody preparation.

The antigen binding proteins of the invention comprise antigen binding sites that are unique variable domains (sVDs). In certain embodiments of the invention, an sVD binding site replaces a variable domain of an IgG-like antibody, and an scFv replaces the other variable domain. Examples of such embodiments are described in Figure 1A. In other embodiments of the invention, a RV binding site is grafted onto an IgG antibody at the N or C-terminus of the IgG light and heavy chain. Examples of these embodiments are shown in Figure 1B. SVDs may also be incorporated into an immunoglobulin type antibody at other positions. For example, sVDs can be fixed at the C terminus of the constant domains Ch and / or CL. In addition, by combining sVD binding site substitutions, as in Figure 1, sVD binding site constraints at the end of the Ch and / orCL constant domains, IgG-like antibodies that are multispecific and / or multivalent for a particular antigen can be produced. .

As you know, an Fv consists of two domains (for example, a Vh domain and a VL domain). In a standard antibody, Fv Vhe Vl domains, although associated to form an antigen binding site, are not directly linked, but are each joined to constant regions of bound antibodies. Fv domains Vh and Vl can also be joined with a synthetic linker to form a single chain Fv (scFv).

Antibody specificity refers to the selective recognition of the antibody by a particular epitope of an antigen. Natural antibodies, for example, are monospecific. Bispecific antibodies (BsAbs) are antibodies that have two different antigen binding sites or specificities. When an antigen binding protein has more than one specificity, recognized epitopes may be associated with a single antigen or with more than one antigen.

A natural antibody molecule is composed of two identical heavy chains and two identical light chains. Each light chain is covalently linked to a heavy chain by a disulfide interchain bond. The two heavy chains are further attached to each other multiple disulfide bonds in the hook region. Individual chains duplicate domains having similar sizes (about 110-125 amino acids) and structures, but different functions. The light chain comprises a variable domain (V1) and a constant domain (CL). The heavy chain comprises one variable domain (Vh) and, depending on the class or isotype of the antibody, three or four constant domains (CH1, CH2, Ch3 and Ch4). In mice and humans, the isotypes are IgA, IgD, IgE, IgG and IgM, with IgAe IgG further subdivided into subclasses or subtypes. The portion of an antibody consisting of V1 and Vh domains is designated "Fv" and constitutes the antigen binding site. A single chain Fv (scFV) is an engineered protein containing a V1 domain and a Vh domain in a polypeptide chain, wherein the N-terminus of one domain and the C-terminus of the other domain are joined by a flexible linker. "Fab" refers to the portion of the antibody consisting of the VL, VH, Cl and ChI domains.

The variable domains show considerable amino acid sequence variability from one antibody to the next, particularly in the location of the antigen binding site. Three regions, called "hypervariables" or "complementarity determining regions" (CDRs) are found in each of V1 and VH.

"Fc" is the designation for the portion of an antibody that comprises pair-formed heavy chain constant domains. In an IgGi antibody, for example, Fc comprises CH2 and CH3 domains. The Fcde of an IgA or IgM antibody further comprises a CH4 domain. Fc is associated with Fc receptor binding, complement-mediated cytotoxicity activation and antibody-dependent cellular cytotoxicity. Natural paraantibodies such as IgA and IgM, which are complexes of multiple IgG type proteins, complex formation requires constant Fc domains.

Finally, the "hook" region separates the Fab and Fc portions of the antibody, providing Fabs with respect to each other and with respect to Fc, as well as including multiple covalent disulfide bonds to heavy chains.

Antibodies of the invention have a combination of desirable aspects. Firstly, they are essentially homogeneous. By design, the wrong formation of antibody light and heavy chain pairs is greatly reduced or eliminated. For example, some bispecific antibodies make use of two different heavy chains to provide two specificities. Four combinations are possible when these heavy chains are arranged in an IgG-like molecule. Two of these consist of heavy decades with mismatch so that the product is monospecific. In the antibodies of the invention, mismatch is substantially eliminated.

A second aspect of the antibodies of the invention is that they are equivalent to each binding specificity. Many specific antibodies are monovalent for each of the antibody binding sites that are included. This is significant for antibody function because abivalence allows binding cooperativism and a significant increase in binding avidity with respect to a molecule comprising an antigen specific deletion site.

A third advantage of the antibodies of the invention is that one or more heavy chain constant domains that constitute the Fc region (e.g., CH2 and / or Ch3 for an IgGi molecule) of an anti-spontaneous and providing other antibody functions are present. In addition, multiple linking domains are separated from constant domains so that the functions provided by constant domains are unaffected. Constant domain functions include binding to certain accessory molecules (e.g., surface binding of soluble Fc cells and receptors, IgA J-chain association, IgM, IgA S protein), activation of the complement pathway (complement-dependent cytotoxicity, CDC), recognition of antibody binding to target cells by several different leukocyte populations (antibody-dependent cell-mediated cytotoxicity, ADCC) and opsonization (phagocytosis amelioration). Also, the Fc heavy chain constant domain (s) may confer increased half-life.

A fourth advantage of the proteins of the invention is that in vitro processing is not necessary to obtain the complete product. Bluntly arranged in an artificial way, each of the domains has a natural character that allows expression in a biological system. For example, bispecific antibodies may be expressed in prokaryotic and eukaryotic expression systems. The proteins that are produced are substantially bispecific.

SVD antigen binding sites and binding sites containing the Fc region for use in an antibody of the invention can be obtained by a variety of methods. The amino acid sequences of the Vh and / or V1 portions of a selected binding domain may be obtained from a naturally occurring antibody or may be chosen or modified, triad or selected for desired binding characteristics. For example, the Vh and / or Vl domains may be obtained directly from a monoclonal antibody having the desired binding characteristics. Alternatively, the Vh and / or Vl domains may be from libraries of V gene sequences from a mammal of choice. The elements of these libraries express random combinations of the Vh and / or V1 domains and are screened with any desired antigen to identify elements that have desired binding characteristics. Particularly preferred is a library of human V genes. The methods for screening are known by nature. Alternatively, the Vh and / or Vl domains from a selected non-human source may be incorporated into chimeric antibody comprising human constant domains. For example, for administration to a human, one may wish to use an antibody with one or more functional human constant domains wherein the Vh and Vl domains are selected from a non-human source. To maximize the domain associated function or reduce the immunogenicity of the antibody, human constant domains are preferred.

Alternatively, a domain V may be produced which is "humanized". Humanized variable domains are constructed, wherein amino acid sequences comprising one or more non-human complementarity determining regions (CDRs) are grafted onto human framework regions (FRs). For examples, see: Jones, P.T. et al., 1996, Nature 321, 522-25; Riechman, L. Et al., 1988, Nature 332,323-27; and US Patent 5,530,101 to Queen et al. A humanized construct is particularly valuable for the elimination of adverse immunogenic characteristics, for example, when an antigen-binding domain from a non-human source is desired to be used for treatment of a human. The variable domains have a high degree of structural homology, allowing for easy identification of amino acid residues in variable domains corresponding to CDRs and FRs. See, for example, Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD. Thus, amino acids that participate in antigen binding are easily identified. In addition, methods for conserving or improving affinity for antigen of humanized binding domains comprising grafted CDRs have been developed. One method is to include in the container variable domain foreign foreign residues that influence the conformation of the CDR regions. A second way is to graft foreign CDRs over human variable domains with the closest homology to the foreign variable region. Queen, C. et al., 1989, Proc. Natl. Acad. Know. USA 86, 10029-33. CDRs are more easily grafted onto different RFs by first amplifying the individual sequences using overlapping primers that include desired CDR sequences, and joining the resulting gene segments into subsequent amplification reactions. Grafting a CDR over a different variable domain may further involve replacing amino acid residues that are adjacent to the CDR in the amino acid sequence or allocated against the CDR in the duplicate variable domain structure that affects the CDR conformation. The humanized variable domains of the invention thus include human domains comprising one or more non-human CDRs as well as such domains in which other substitutions or changes have been made to conserve or improve binding characteristics.

An antibody of the invention may also employ variable domains that have been rendered less immunogenic by substitution of surface-exposed residues to make the antibody appear to belong to the immune system (Padlan, E.A., 1991, Mol. Immunol. 28, 489-98). Antibodies were modified by this process without loss of affinity (Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91, 969-973). Due to the inner envelope of amino acid residues in the vicinity of antigen binding site remain unchanged, affinity is conserved. Substitution of surface exposed residues according to the invention for reduced immunogenicity does not mean substitution of CDR residues or adjacent residues that influence binding characteristics.

It is often preferable to employ variable domains that are essentially human. Human binding domains can be obtained from phage display libraries, where combinations of human light and heavy chain variable domains are displayed on the filamentous phage surface (See, for example, McCafferty et al., 1990, Nature 348, 552-54; Aujame et al., 1997, Human Antibodies 8, 155-68). Combinations of variable domains are typically displayed in the phage as Fabs or scFvs. The library is screened for phage-containing variable domain combinations having desired antigen-deletion characteristics. Variable domain and single domain combinations exhibit high affinity for a selected antigen and small cross-reactivity for other related antigens. By screening very large repertoires of antibody fragments (see, for example, Griffiths et al., 1994, EMBO J. 13, 3245-60), a good diversity of high affinity binding domains is isolated, and many are expected to have sub-affinities. -nomolars for the desired antigen.

Alternatively, human binding domains may be obtained from transgenic animals in which unranked Ig gene segments have been introduced and in which mouse Ig endogenous genes have been inactivated (reviewed in Bruggemann and Taussig, 1997, Curr.Opin. Biotechnol. 8, 455 -58). Preferred transgenic animals contain very large contiguous Ig gene fragments that are above 1 Mb in size (Mendez et al., 1997, Nature Genet. 15, 146-56), but moderate affinity Mabshumans may be augmented by smaller gene Ioci-containing animaistransgenics. (See, for example, Wagner et al., Eur. J. Immunol. 42, 2672-81; Green et al., 1994, Nature Genet. 7, 13-21.) SVD binding sites can be obtained from Fvespecific regions. (which comprise both Vh and VL domains). It can often be shown that the binding affinity and specificity of an Fv region is given first by one of the variable domains. In addition, scFv can be obtained directly. Direct sources of sVDs include mammals (eg, camelids) that naturally express antibodies containing only the Vh domain and phage display libraries constructed to express only a single variable domain. For example, a library of human domain antibody phage display is commercially available from Domantis (Cambridge, UK). As exemplified herein, PDGFRα specific sVD binding sites were obtained from a phage library containing variants in which the V1 domain was spontaneously deleted.

In a physiological immune response, mutation and selection of expressed antibody degens lead to the production of antibodies having high affinity for its target antigen. The Vh and Vl domains incorporated into the antibodies of the invention may similarly be subjected to in vitro mutation and screening procedures to obtain high affinity variants. Thus, the binding domains of the invention include those for which deletion characteristics are enhanced by direct mutation or by affinity maturation methods. Affinity and specificity can be modified or improved by changing CDRs and screening antigen binding sites having desired characteristics (See, for example, Yang et al., 1995. J. Mol. Bio.254, 392-403). It should be understood that amino acid residues that are primary determinants of single domain antibody binding may be in Kabat-defined CDRs, but may include other residues as well, such as residues that may otherwise be buried at the Vh-V1 interface. of a Vh-V1 heterodimer. CDRs or other binding-determining residues are changed in a variety of ways. One way is to randomize individual residues or residue combinations such that identical antigen-binding sites, all of twenty amino acids, or a subset thereof, are found in particular positions. In addition, mutations are induced in a range of CDR residues by error-prone PCR methods (See, for example, Hawkins et al., 1992, J. Mol. Βίο. 226, 889-96). Containing gene phage display vectors encoding the binding domains (e.g., heavy and / or light chain variable region genes) can be propagated in E. coli mutator strains (See, for example, Low et al., 1996, J. Mol. Bio. 250, 359-68). These methods of mutagenesis are illustrative of many methods known to one skilled in the art.

Each antibody variable domain of the present invention may be a complete immunoglobulin light or heavy chain variable domain, or may be a functional equivalent or a mutant or derivative of a naturally occurring domain, or a synthetic domain constructed, for example, in vitro using a technique. as described in WO 93/11236 (Medical Research Council / Griffiths et al.). For example, it is possible to incorporate domains corresponding to antibody variable domains in which one or more amino acids are missing. Important feature is the ability of each variable domain to associate with a complementary variable domain to form an antigen binding site.

Antigen binding proteins of the invention have deletion sites for any epitope, antigenic site or protein. Of particular interest are antibodies that are usable for treating disease. Preferred antibodies neutralize receptor proteins such as receptors that are involved in angiogenesis and / or oncogenesis. Neutralizing a receptor means inactivating the intrinsic receptor kinase activity to transduce a signal. A reliable test for receptor neutralization is inhibition of receptor phosphorylation. The present invention is not limited to any particular receptor neutralization mechanism. Some possible mechanisms include preventing ligand binding to the extracellular receptor deletion domain, and preventing receptor dimerization or oligomerization. Other mechanisms cannot, however, be excluded.

Neutralization of receptor activation in a sample of endothelial or nonendothelial cells, such as tumor cells, can be performed in vitro or in vivo. Neutralizing activation of a receptor in a cell expressing receptor sample comprises contacting the cells with an antibody of the invention. In vitro, cells are contacted with the antibody before, simultaneously with, or after adding VEGF to the cell sample. In vivo, an antibody of the invention is contacted with a receptor upon administration to a mammal. Methods of administration to a mammal include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration.

Examples of these receptors include, but are not limited to, platelet-derived growth factor receptor (PDGF-R), VEGF receptors (e.g., VEGFR-2 / KDR / Flk-1, VEGFR1 / Flt-1, VEGFR3 / Flt-4) , epidermal growth factor receptor (EGFR), degrowth factor receptor as insulin (IGFR) and others. Other non-limiting examples of receptor tyrosine kinases include Flt-4, HER2 / neu, Tek and Tie2.

Other factors implicated as possible regulators of ingenesis and / or tumor growth in vivo include fibroblast growth factor (FGF), and nerve growth factor (NGF). Corresponding receptors are fibroblast growth factor (FGF-R). ), and nerve growth factor (NGFR). Another receptor implicated in cell migration, morphological changes and invasiveness is the macrophage stimulating protein receptor ("MSP-R" or "RON"). Receptors of interest include human proteins and homologues of other mammals. Antibodies of the invention may incorporate Ig antigen binding domains. from any source. For example, antibodies are known to the receptors listed above and are sources of Vh and Vl domains for the antibodies of the present invention. Examples of KDR-specific scFv variable region binding domains include, for example, the IMC-IC11 Vh and Vl domains (VH nucleotide and deamino acid sequences: SEQ ID NOs: 1 and 2; VL nucleotide and deamino acid sequences: SEQ ID Nos: 3 and 4) (see WO 00/44777), IMC-2C6 (VH nucleotide and amino acid sequences: SEQ ID Nos: 5 and 6; VL nucleotide and amino acid sequences: SEQ ID Nos. : 7 and 8) (see WO 03/075840), and IMC-1121 (VH nucleotide and amino acid sequences: SEQ ID Nos: 5 and 6; V1 nucleotide and amino acid sequences: SEQ ID Nos: 9 and 10) ( see, WO 03/075840). Examples of specific Flt-I deletion domains include 6.12 (VH nucleotide and deamino acid sequences: SEQ ID Nos: 11 and 12; VL nucleotide and deamino acid sequences: SEQ ID Nos: 13 and 14) and IMC-18F1 (sequences VH denucleotide and amino acid sequences: SEQ ID Nos: 27 and 28; VL denucleotide and amino acid sequences: SEQ ID Nos: 29 and 30).

EGFR-specific binding domains include, for example, ERBITUX® (Cetuximab; IMC-C225) (Vh nucleotide and amino acid sequences: SEQ ID Nos: 15 and 16; VL nucleotide and deamino acid sequences: SEQ ID Nos: 17 and 18) as described in WO 96/40210 eIMCl 1F8 (VH nucleotide and amino acid sequences: SEQ ID Nos: 19 and 20; VL nucleotide and amino acid sequences: SEQ ID Nos: 21 and 22). An example of an IGFR-specific binding domain is IMC-A12 (VH nucleotide and amino acid sequences: SEQ ID Nos: 23 and 24; VL nucleotide and amino acid sequences: SEQ ID Nos: 25 and 26). Antibodies that bind to FGT receptors (see, WO 2005/037235) (see, WO 2005/037235) include, for example, FR1-H7 (VH denucleotide and amino acid sequences: SEQ ID Nos: 31 and 32); V1 denucleotide and amino acid sequences: SEQ ID Nos: 33 and 34), FRl-Al (VH nucleotide and amino acid sequences: SEQ ID Nos: 35 and 36; VL nucleotide and amino acid sequences: SEQ ID Nos : 37 and 38), eFR1-4H (VH nucleotide and amino acid sequences: SEQ ED Nos: 39 and 40; VL nucleotide and amino acid sequences: SEQ ID Nos 41e 42). RON or MSP-R binding antibodies (see WO 2005/120557) include IMC-41A10 (VH nucleotide and amino acid sequences: SEQ ID Nos: 43 and 44; V1: SEQ nucleotide and amino acid sequences Nos. 45 and 46) and IMC-41B12 (VH nucleotide and deamino acid sequences: SEQ ID Nos: 47 and 48; VL nucleotide and deamino acid sequences: SEQ ID Nos: 49 and 50). Antibodies that bind to PDGFRα include, for example, 3G3 and 7G11 (Loizos et al., 2005, Mol.Cancer Ther. 4: 369).

In addition, portions of the binding domains listed above, such as the CDR regions, may be incorporated into the binding domains used to produce the binding proteins described herein.

Certain preferred antibodies bind to two of the receptors listed above. In one embodiment of the invention, a bispecific antigen deletion protein binds to and blocks the activation of two different tyrosine kinase receptors involved in angiogenesis. In this embodiment, the antibody binds to PDGFR and a VEGF receptor such as VEGFR2 / Flk-1 / KDR. In another embodiment, the antibody binds to KDR and FLT-I.

In another embodiment, an antibody of the invention binds to HER2 and EGFR. In yet another embodiment, an antibody to the invention binds to EGFR and IGFR.

In another embodiment, an antigen binding protein of the invention binds to EGFR and VEGFR. In a preferred embodiment, VEGFR is VEGFR2. The antibody is useful for blocking stimulation of vascular epithelial cells by blocking signal transduction through both EGFR and VEGFR. This is particularly usable where angiogenesis occurs in response to EGFR ligands, particularly TGFÎ ±, secreted by tumor cells.

Antibodies of the invention may be used to reticular antigens on target cells with antigens on immune system effector cells. This may be useful, for example, by promoting targeted immune responses against cells having particular antigens of interest on the cell surface. According to the invention, immune system effector cells include antigen-specific cells such as T cells that activate cellular immune responses and non-specific cells such as macrophages, neutrophils and natural killer (NK) cells that mediate cellular immune responses.

Antibodies of the invention may have a binding site for any cell surface antigen of an immune system effector cell. Such cell surface antigens include, for example, cytokine and lymphokine receptors, Fc, CD3, CD16, CD28, CD32, and CD64 receptors. In addition to the antigen binding sites provided by sVDs, bispecific antibodies may include Fvs provided binding sites. Fvs can be obtained from antibodies to the above antigens from combinatorial libraries, as well as by other methods known in the art. Bispecific antibodies that are specific for cytokine and lymphokine receptors may also include binding sites comprising amino acid sequences that correspond to all or part of the natural ligand for the receptor. For example, when the cell surface antigen is an IL-2 receptor, an invention bispecific antibody may have an antigen binding site that comprises a corresponding amino acid sequence or IL-2. Other elymphokine cytokines include, for example, interleukins such as interleukin-4 (IL-4) and interleukin-5 (IL-5), and colony stimulating factors (CSFs) such as degranulocyte macrophage CSF (GM-CSF), and CSF of granulocyte (G-CSF).

Antibodies of the invention are produced by expressing two strands of polypeptide which taken together comprise at least one single domain antigen binding site. The two polypeptide chains each comprise at least one heavy chain constant domain that is capable of dimerization (e.g., CH2 and / or Ch3). Antibodies are conveniently produced in E. coli using DNA constructs that comprise bacterial secretion signal sequences at the start of each polypeptide chain. A variety of bacterial signal sequences are known in the art. A preferred signal sequence is from the erBrin carotovora pelB gene. DNA fragments encoding the antibodies will be cloned, for example, into vectors employing human cytomegalovirus (HCMV) enhancers and enhancers for expression in mammalian cells such as CHO, NSO, COS-7and PER.C6 cells, and cell lines of lymphoid origin such as lymphoma, myeloma and hybridoma cells. (See, for example, Bendig et al., US Patent 5,840,299; Maeda et al. (1991) Hum. Antibod. Hybridomas 2, 124-34).

A selectable marker is a gene encoding a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Typical selectable markers encode proteins that (a) confer resistance to antibiotics or other toxins, for example ampicillin, neomycin, methotrexate or tetracycline, (b) complement sauxotrophic deficiencies, or (c) provide critical nutrients not available from complex media, for example, the gene. encoding D-alanine racemase parabacilli. A particularly usable selectable marker provides methotrexate resistance. For example, cells transformed with the DHFR deletion gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a DHFR-antagonist. An appropriate host cell when wild type DHFR is employed is the DHFR activity-deficient hamsterchinese ovary (CHO) cell line prepared and propagated as described by Urlaub and Chasin (1980) Proc. Natl. Acad. Know. USA 77, 4216. Transformed cell cells are then exposed to increased levels of methotrexate. This leads to the synthesis of multiple copies of the DHFR gene, and concomitantly multiple copies of other DNA comprising expression vectors, such as DNA encoding the antibody or fragment. of antibody.

When it is desired to express a yeast gene construct, an example of a selection gene suitable for yeast use is the trpl gene present in yeast plasmid YRp7. Stinchcombet al. (1979) Nature, 282, 39; Kingsman et al. (1979) Gene 7, 141. Genetrpl provides a selection marker for a mutant strain of yeast that is deficient in tryptophan, for example, ATCC No. 4,476 or PEP4-1. Jones (1977) Genetics 85, 12. The presence of triplet lesion in the yeast host cell genome then provides an effective environment for detecting culture transformation in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids containing the Leu2 gene. .

Transformed host cells are cultured by methods known in the art in a liquid medium containing carbon-assimilable sources, for example carbohydrates such as glucose or lactose, nitrogen, for example, amino acids, peptides, proteins or their degradation products such as peptides, ammonium salts or others, and inorganic salts, for example sodium, potassium, magnesium and calcium sulfates, phosphates and / or carbonates. The medium furthermore contains, for example, growth promoting substances such as trace elements, for example iron, zinc, manganese and others.

Antibodies that bind to growth factor receptors are preferably capable of blocking the inactivation of receptortirosin kinase (RTK) activity. Inhibition of tyrosine kinase may be determined using well known methods, for example, by measuring the level of recombinant receptor kinase receptor phosphorylation, and / or phosphorylation of natural or synthetic substrates. Thus, phosphorylation assays are useful in determining the RTK antagonists of the present invention. Phosphorylation can be detected, for example, by using a phosphotyrosine-specific antibody in an ELISA or western blot. Some tests for tyrosine kinase activity are described in Panek et al., J. Pharmacol.Exp. Thera. (1997) 283: 1433-44 and Batley et al., Life Sci. (1998) 62: 143-50.

In addition, methods for protein expression detection can be used to determine RTK antagonists, where protein expression being measured is mediated by RTK. These methods include immunohistochemistry (IHC) for protein expression detection, fluorescence, in situ hybridization (FISH) for detection of dogene amplification, competitive radioligand binding assays, solid matrix blotting techniques such as Northern and Southern blot, reverse transcriptase polymerase chain (RT-PCR) and ELISA. See, for example, Grandis et al., Cancer, (1996) 78: 1284-92; Shimizu et al., Japan J. Cancer Res., (1994) 85: 567-71; Sauter et al., Am. J. Path., (1996) 148: 1047-53; Collins, Glia (1995) 15: 289-96; Radinsky et al., Clin. Cancer Res. (1995) 1: 19-31; Petrides et al., Cancer Res., (1990) 50: 3934-39; Hoffmann et al., Anticancer Res., (1997) 17: 4419-26; Wikstrand et al., Cancer Res. (1995) 55: 3140-48.

The ability of an antibody to block ligand binding can be measured, for example, by a competitive in vitro assay. In this test, an RTK ligand (e.g. EGF to EGFR) is immobilized, and a binding test is performed to determine the effectiveness of the antibody to competitively inhibit RTK binding to the immobilized ligand.

In vivo tests may also be used to determine RTK antagonists. For example, receptor tyrosine kinase inhibition can be observed by mitogenic assays using receptor ligand-stimulated cell lines in the presence or absence of inhibitor. For example, EGF-stimulated A431 (American Type Culture Collection (ATCC), Rockville, MD) cells can be used to test for EGFR inhibition. Another method involves testing the inhibition of growth of EGFR-expressing tumor cells, using, for example, human tumor cells injected into a mouse. See US patent no. 6,365,157 (Rockwell et al.).

Preferred antibodies of the present invention have specificity and are capable of binding to two different antigens simultaneously. Different antigens may be located in different cells or cell names. Antigen cross-linking can be shown in vitro, for example by providing a solid surface to which a first antigen is attached, adding bispecific antibodies specific for the first antigen and a second antigen for which the binding protein is also specific, and detecting the presence of the second bound antigen. .

Preferred antibodies of the invention are capable of blocking the interaction between two receptors and their respective ligands. For example, a KDR and Flt-I specific antibody inhibits VEGF-induced cell migration as well as PIGF-induced cell migration. Combination of two receptor binding specificities into specific antibodies may be more effective in inhibiting the migration of cells from individual parent antibodies (see, for example, Zhu, Z., WO2004 / 003211).

Compared to antibodies that are monospecific, bispecific antibodies may be more potent inhibitors of cell function. For example, VEGF-stimulated cell functions such as endothelial cell proliferation and VEGF and PlGF-induced migration from human leukemia cells may be more potent. efficiently inhibited by bispecific antibodies even when the affinity for one or both of the two target antigens is reduced. A specific (monovalent) antibody to both KDR and Flt-I may be more effective in inhibiting VEGF or PlGF-induced cell migration than a monospecific scFv directed to either target antigen (WO 2004/003211).

In another example, an antibody having dual specificity for both EGFR (or Her2 / neu) and IGFR that is capable of binding to both receptors and blocking interaction with their specific ligands is used to neutralize both EFG and IGF stimulated receptor activation. downstream signal transduction. Stimulation of either deEGFR or IGFR is observed to result in activation (e.g. phosphorylation) of common downstream signal transduction molecules, including Akt and p44 / 42, although in different extensions. In certain tumor cells, inhibition of deEGFR function may be offset by up-regulation of other growth factor receptor de-signaling pathways, and particularly by IGFR stimulation. In contrast to treatment with an antibody that selects a receptor, and does not completely block both Aktcomo p44 / 42 phosphorylation, incubating tumor cells with an EGFR-binding antibody like IGFR blocks both Akt and p44 phosphorylation. Consequently, inhibition of IGFR signaling results in inhibition of tumor growth and increased sensitivity to tumor cells in certain therapeutic agents.

Inhibition of phosphorylation of such common signal transduction cascade components is also observed with antibodies that bind other RTKs, such as RON. Consequently, antigen-deletion proteins are generally useful for treating neoplastic diseases characterized by cell transformation or growth resulting from activation of multiple signal transduction pathways.

Antibodies of the invention are usable for treating a variety of proliferative disorders. For example, the present invention provides for treatment of tumors that express and are stimulated by more than one receptor tyrosine kinase. Stimulation through more than one receptor can result in uncontrolled growth that is insensitive to blocking each receptor alone. In addition, stimulus from a second receptor may increase the activation observed in response to stimulation through a first receptor. Alternatively, contributions from individual recipients may be multiplicative. In each of the above cases, significantly improved inhibition of tumor growth is observed in the presence of an antigen binding protein that blocks both receptors.

Antibodies of the invention are useful for treating diseases in which receptor stimulation is via EGFR paracrine and / or autocrine activation. For example, tumors expressing EGFR are characteristically sensitive to EGF present in their environment, and may still be stimulated by EGF or TGF-α produced in the tumor. Although not intended to be limited by any particular mechanism, diseases and conditions that can be treated or prevented by the present methods include, for example, those in which tumor growth is stimulated. The method is thus effective for treating a solid tumor that is not vascularized or not yet substantially vascularized.

Some antibodies of the invention are useful for inhibiting the angiogenesis associated with a hyperproliferative disease. For example, by blocking tumor-associated angiogenesis, tumor growth may be inhibited. In one embodiment, the antibody binds to a tumor-associated RTK and inhibits the production of angiogenic ligands (ie, VEGF) by the tumor, and also binds to a VEGF receptor associated with vasculature cells to inhibit their proliferation. cells In a different embodiment, the antibody binds to multiple VEGF receptors, such that the VEGF ligand or other VEGFR ligand (e.g. PIGF) is prevented from binding to more than one type of VEGF receptor.

Tumors that can be treated include primary tumors and metastatic tumors as well as refractory tumors. Refractory tumors include tumors that fail to respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone, or combinations thereof. Refractory tumors also include tumors that appear to be inhibited by treatment with such agents, but recur within five years, sometimes up to ten years or more after treatment is discontinued. Tumors may express EGFR or other RTK at normal levels or may overexpress RTK at levels, for example at least 10, 100, or 1000 times normal levels.

Examples of EGFR-expressing tumors that are stimulated by an EGFR ligand include carcinomas, gliomas, sarcomas, adenocarcinomas, adenosarcomas, and adenomas. These tumors can occur virtually all over the body, including, for example, breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow. , blood, thymus, uterus, testes, cervix or liver. Some tumors that have been observed as EGFR overexpressing that can be treated according to the present invention include, but are not limited to, colorectal and head and neck tumors, especially head and neck squamous cell carcinoma, brain tumors such as glioblastomas, and tumors of the lung, breast, pancreas, esophagus, bladder, kidney, ovary, cervix and prostate. Non-limiting examples of tumors that have, as noted, constitutively active (i.e. non-regulated) receptor tyrosine kinase activity include gliomas, non-small cell lung carcinomas, ovarian carcinomas and prostate carcinomas. Other examples of tumors include Kaposi's sarcoma, central nervous system neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and cerebrospinal metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multifomiosomal. Overexpression of other RTKs may produce similar decreasing defects. For example, more metastatic bone cancers emerge from primary prostate, breast, or lung tumors. Prostate tumors may initially be hormone dependent, but the loss of this dependence coincides with IGFR-mediated stimulation of bone-migrating cells.

Antibodies are also usable for treating hyperproliferative diseases other than tumors comprising administering to the mammal an effective amount of the antibody of the present invention. As described herein, "hyperproliferative disease" is defined as a condition caused by overgrowth of non-cancerous cells expressing an EGFR family member or other receptor cytosine kinase. Excess cells generated by a hyperproliferative disease express RTK at normal levels or may overexpress RTK.

Examples of hyperproliferative disease include psoriasis, actinic keratoses and seborrheic keratoses, warts, keloid scars, and eczema. Also included are hyperproliferative diseases caused by viral infections such as papillomavirus infection. For example, psoriasis occurs in very different variations and degrees of severity. Different types of psoriasis exhibit features such as blisters with pus (pustular psoriasis), severe skin eschar (erythrodermic psoriasis), drop-like spots (guttate psoriasis), and smooth inflamed lesions (reverse psoriasis). Treatment of all types of psoriasis (e.g., psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis, arthropathic psoriasis, parapsoriasis, pustulosepalmoplantar) is contemplated by the invention.

In accordance with the invention, antibodies may be chemically or biosynthetically conjugated to other agents such as antineoplastic or antiangiogenic agents for treating disease. Antitumor-linked antitumor agents include any agents that destroy or damage a tumor to which the antibody has bound or in the cell medium in that the antibody bound. For example, an antitumor agent is toxic as a chemotherapeutic agent or a radioisotope. Chemotherapeutic agents are conjugated to the antibody using conventional methods (See, for example, Hermentin and Seiler (1988) Behring Inst.Mitt. 82, 197-215), including peptide and non-peptide linkers.

Antibodies of the invention may also be linked to detectable signal producing agents usable in vivo and in vitro for diagnostic purposes. The signal producing agent produces a measurable signal that is detectable by external means, usually electromagnetic radiation measurement. For the most part, the signal producing agent is an enzyme or chromophore, or emits light by fluorescence, phosphorescence or chemiluminescence. Chromophores include dyes that absorb ultraviolet or visible light in the region, and may be substrates or degradation products of enzyme-catalyzed reactions.

The invention further contemplates the use of antibodies with diagnostic agents or treatment incorporated in secondary reagents. For example, a member of a binding pair is bound to the antibody of the invention. Antineoplastic agents, for example, are conjugated to second members of these pairs and are thus directed to the site where the antibody is bound. In a preferred embodiment, biotin is conjugated to an invention antibody, and thus provides a target for an antineoplastic or other evaporation agent that is conjugated to avidin or streptavidin. In addition, biotin or another such moiety is bound to an antibody of the invention and used as a reporter, for example in a diagnostic system where a detectable disinfectant producing agent is conjugated to avidin or streptavidin.

Antibodies may be administered in combination with one or more appropriate adjuvants, such as cytokines (IL-10 and IL-13, for example) or other immune stimulants such as, but not limited to, aquimokine, tumor-associated antigens, and peptides. It should be noted, however, that administration of an antibody alone is sufficient to prevent, inhibit or reduce tumor progression from a therapeutically effective modulator.

In some embodiments, it may be desirable to administer an antibody of the invention that binds to an RTK and blocks ligand binding in combination with another ligand-binding antigen binding protein. Ligand binding antibodies are well known in the art and include, for example, anti-VEGF (Avastin®; bevacizumab).

Antibodies of the invention should also be used in combination treatment methods by administration with an antineoplastic agent such as a chemotherapeutic agent or a radioisotope. Suitable chemotherapeutic agents are known to those skilled in the art and include irinotecan (CPT-11), anthracyclines (eg, daunomycin and doxorubicin). ), methotrexate, vindesine, neocarzinostatin, cisplatin, chlorambucil, cytosine arabinoside, 5-fluorouridine, melfalan, ricin and calicheamicin. An antibody and an antiangiogenic or antineoplastic agent are administered to a patient in amounts effective to inhibit angiogenesis and / or reduce tumor growth. Antibodies should also be administered in combination with other treatment regimens, for example with treatments such as radiation therapy. For examples of combination therapies, see, for example, US 6,217,866 (Schlessinger et al.) (Anti-EGFR antibodies in combination with antineoplastic agents); WO 99/60023 (Waksal et al.) (Anti-EGFR antibodies in combination with radiation).

Any suitable antineoplastic agent may be used, such as a chemotherapeutic agent, radiation or a combination thereof. Antineoplastic agents known in the art or being evaluated may be grouped into classes based on their target or mode of action. For example, alkylating agents include, but are not limited to cisplatin, cyclophosphamide, melphalan and dacarbazine. Examples of antimetabolites include, but are not limited to, doxorubicin, epaclitaxel daunorubicin, gemcitabine, and irinotecan topoisomerase inhibitors (CPT-11), aminocamptothecin, camptothecin, DX-8951f, and topotecan (topoisomeride I-10) and topoisomeride I (10) VM-26) (topoisomerase II). For radiation, the source can be either external (external beam radiation therapy - EBRT) or internal (brachytherapy - BT) to the patient being treated. These classifications may be usable for choosing an antineoplastic agent use. For example, it is noted that IGFR-binding antibodies may be particularly effective when administered with a detopoisomerase inhibitor.

The dose of the antioneoplastic agent administered depends on a number of factors, including, for example, the type of agent, the type and severity of the tumor being treated, and the route of administration of the agent. It should be emphasized, however, that the present invention is not limited to any particular dose.

In a combination therapy, the antibody is administered before, during or after initiation of therapy with another agent as well as any combination thereof, that is, before and during, before and after, after, or before, during and after Initiate therapy with an anioneoplastic agent. For example, for treatment of a tumor or neoplastic disease, the antibody may be administered from 1 to 30 days, preferably 3 to 20 days, more preferably from 5 to 12 days before starting radiation therapy. In a preferred embodiment of the invention, chemotherapy is administered concurrently with, prior to or subsequent to antibody therapy.

In the present invention, any suitable route or method may be used to administer the antibodies of the invention and optionally co-administer the antineoplastic agents, receptor antagonists, or other pharmaceutical composition. For example, antineoplastic agent regimens used in accordance with the invention include any regimen believed to be optimally suited for the treatment of a patient neoplastic condition. Different malignancies may require the use of specific antitumor antibodies and specific antineoplastic agents that will be determined on a patient to patient basis. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration. The dose of the antineoplastic agent administered depends on a number of factors, including, for example, the type of neoplastic agent, the type and severity of the tumor being treated, and the route of antineoplastic agent administration. It should be emphasized, however, that the present invention is not limited to any particular method or route of administration.

It is understood that the antibodies of the invention, when used in a mammal for purposes of prophylaxis or treatment, will be administered in the form of a composition further comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the invention may, as known in the art, be formulated to provide sustained or delayed, rapid, active ingredient release following administration to the mammal.

The present invention also includes kits for inhibiting tumor growth and / or angiogenesis, or treating other diseases, comprising a therapeutically effective amount of an invention antibody. Human or humanized antibodies are preferred. The kits may further contain any appropriate antagonist of, for example, another growth factor receptor involved in tumorigenesis or angiogenesis (e.g. EGFR, VEGFR, VEGFR-I / Flt-1, VEGFR-2 / Flk-1 / KDR, IGFR , PDGFR, NGFR, FGFR, etc., as described above). Alternatively, or in addition, the kits of the present invention may further comprise an antineoplastic agent. Examples of suitable anti-neoplastic agents in the context of the present invention are described herein. The kits of the present invention may further comprise an adjuvant; Examples are also described above.

Also included within the scope of the present invention is the use of the antibodies of the present invention in vivo and in vitro for research or diagnostic methods which are well known in the art. Diagnostic methods include kits containing the antibodies of the present invention.

Therefore, the present receptor binding antibodies can thus be used in vivo and in vitro for research, diagnostic, prophylaxis or treatment methods which are well known in the art. Of course, it should be understood and expected that variations of the principles of the invention described herein may be made by a person skilled in the art and it is intended that such modifications should be included within the scope of the present invention. All references mentioned herein are incorporated by reference in their entirety.

EXAMPLES

The following examples further illustrate the invention, but should not be construed as limiting the scope of the invention in any way. Detailed descriptions of conventional methods such as those employed in vector and plasmid construction, insertion of genes encoding polypeptides into such vectors and plasmids, introduction of plasmids in host cells, and the expression and determination thereof of genes and gene products can be obtained from numerous publications, including Sambrook, J. et al., (1989) Molecular Cloning: A LaboratoryManual, 2a. Edition, Cold Spring Harbor Laboratory Press; and Coligan, J. et al. (1994) Current Protocols in Immunology, Wiley & Sons, Incorporated.

Selection of human anti-mPDGFRα antibodies from the phage display Fab library. An extensive native Dyax Fabhuman phage display library (containing 3.7 χ 10 10 clones) was used to select antibodies directed against mPDGFRa-Fc protein (R&D Systems (Minneapolis, MN). The library lot (100 μΐ) was grown in Phase Iog in 20 ml 2YTAG medium, recovered with M13K07 helper phage, and amplified overnight in 2YTAK medium (2YT containing 100 μg / ml ampicillin and 50 μg / ml kanamycin) at 30 ° C. Phage preparations were precipitated at 4% of PEG, 0.5 M NaCl. Phage preparations were resuspended in 1 ml 3% skimmed milk / PBS containing 240 µg of unrelated IgG and incubated at 37 ° C for 1 h to block non-specific binding and binding to Fc label of mPDGFRa protein.

Three rounds of selection were performed on mPDGFRa-Fc with decreasing amounts of protein (50, 10 and 2 μg, respectively) coated in selection tubes. For each round of selection, mPDGFRa-Fc coated MaxisorpStar tubes (Nunc, Rosklide, DenMark) were first blocked with 3% milk / PBS at 37 ° C for 1 h, and then incubated with the phage preparation at room temperature for 1 h. Ostubes were washed 15 times with PBST (PBS containing 0.1% Tween 20) followed by 15 washes with PBS. Bound phage were eluted at room temperature for 10 min with 1 ml of a freshly prepared 100 mM triethylamine solution (Sigma). The eluted phage was incubated with 10 ml half-log phase TG1 cells at 37 ° C for 30 min stationary and 30 min with shaking. The infected TG1 cells were granulated, deposited in three 2YTAG plates, and incubated overnight at 30 ° C. All colonies cultured on the plates were scraped in 3-5 ml 2YTA medium, mixed with glycerol (final concentration: 10%), aliquoted and stored at -80 ° C. For subsequent selection cycles, the phage lot (100 μ) from the previous selection cycle was amplified and used for selection after the procedure described above with decreased amount of mPDGFRa / Fc to coat the Maxisorp Star tubes.

To estimate incoming phages, 10 μΐ of phages prior to selection were used to infect TG1 cells, and then titrated onto 2YTAG plates. To estimate phage recovery from selection, 10 μΐ of eluted phage TG1-infected cells from the above selection procedure were titrated in 2YTAG plates. Recovery yields for each selection cycle were calculated and increased in successive cycles (first cycle, 1x10%; second cycle, 4x10 "6%; third cycle, 2 χ 10" 4%) although mPDGFRa / Fc coating was decreased.

ELISA screening for antibodies with binding and blocking activities. After the 2nd and 3rd rounds of selection, 190 clones from each cyclophores were randomly selected and tested for binding and deblocking by both phage ELISA and Fab ELISA. Briefly, TG1 clones recovered after the 2nd and 3rd rounds of selection were randomly selected and cultured at 37 ° C in 96-well plates. To produce phages, cells were rescued with the M13K07 helper phage as described above. To produce soluble Fab, cells were incubated in 2YTA medium containing 1 mM IPTG. For the ELISA deletion, phage preparations and soluble Fab-containing cell culture supernatants were blocked with 1/6 volume of 18% milk / PBS at room temperature for 1 h. The preparation of blocked or cell culture supernatant phages were then added to 96-well mPDGFRa / Fc coated (Nunc) demicrotiter plates (1μg / ml, 50 μΐ at 4 ° C overnight) and incubated at room temperature 1 H. After incubation at room temperature for 1 h, the plates were washed 3 times with PB ST.

For phage ELISA, the plates were incubated with an anti-M13 phage antibody-HRP conjugate (Amersham Biosciences). For Fab ELISA, plates were incubated with an anti-human Fab antibody-HRP conjugate (Jackson ImmunoResearch Laboratory). After three washings, color was revealed by the addition of TMB peroxidase substrate (KPL, Gaithersburg, MD), and absorbance was measured at 450 nM using a microplate reader (Molecular Device, Sunnyvale, CA). IgG IC11 binding ELISAs were also performed to eliminate Fc-specific antibodies identified as binding agents in library screening. More than 77% of clones selected after 2nd selection, and 99% of clones recovered after 3rd edition were positive in mPDGFRα binding test, suggesting a high efficiency of the selection process.

For the blocking ELISA, 50 μ prepar phage preparations or Fabs culture supernatants were mixed with a fixed amount of mPDGFRa-Fc (0.5 μg / ml) and incubated at room temperature durantemin. The mixture was then transferred to rhPDGF-AA pre-coated 96-well plates (0.5 μg / ml; R&D Systems, Minneapolis, MN) and incubated at room temperature for 1 h. To quantify the bound DemPDGFRa-Fc protein, the plates were then incubated at room temperature for 1h with an antibody-HRP anti-human-Fc horseradish conjugate, followed by three washes and addition of the TMS peroxidase substrate. The bound mPDGFRa-Fc protein was quantified by reading the absorbance at 450 nM. Blocking activity was indicated by decreased ELISA signal detected by antibody-HRP anti-human-Fc conjugate. About 4.2% of the clones that bound mPDGFRa showed PDGF-AA blocking activity.

Based on blocking test results, 14 clones, including a non-blocking binder as a control clone (3F3), were initially selected for another study. Phagemid DNAs were isolated and DNA sequences were determined by dideoxynucleotide sequencing. VH and VL gene sorting and alignments were performed using the Andrew C.R. Martin's Bioinformatics Group (www.bioinf.org.uk) and MagAlign DNAstar's Bioinformatics website.

Analysis of selected anti-mPDGFRα antibodies. Among 14 clones, 9 distinct antibodies were revealed (Table 1). In addition to IC10 and IF2, no identical VHs or V | Ls were found. Interestingly, four of the antibodies with stronger blocking activity had incomplete light chains. Clones IF2 and IF9 had deletions of V1 in the frame (leader peptide sequence followed by Cl sequence). Clones IC10 and 1F2 shared the same Vh gene. However, since the IC10 coding sequence V1 included a stop codon, ThisCl was not expressed. Clone 3G7 including a stop codon appears at the 5 'end of the VL gene. Both IC10 and 3G7 demonstrated a strong lock. Fab expression and binding activity appeared to be very low, but can be accounted for by poor binding of anti-human Fab secondary reagents. Table 1 - Anti-mPDGFRa Antibodies from a Fabs Phage Display Library

<table> table see original document page 41 </column> </row> <table>

Nucleotide and amino acid sequences for Fabs are as follows: 1E10 Vh domain: SEQ ID Nos: 51 and 52; E10 V1 domain: SEQ ID Nos: 53 and 54; 1A12 Vh domain: SEQ ID Nos: 55 and 56; 1A12 V1 domain: SEQ ID Nos: 57 and 58; 3B2 Vh domain: SEQ IDNs: 59 and 60; 3B2 V1 domain: SEQ ID Nos: 61 and 62; 1C10 Vh domain: SEQ ID Nos: 63 and 64; 1C10 V1 domain: SEQ ID Nos: 65 and 66 3G7 Vh domain: SEQ ID Nos: 67 and 68; 3G7 dodomain V1 nucleotide sequence: SEQ ID No: 69; 3G7 V1 domain: QAW; Vhde domain 1F9: SEQ ID Nos: 70 and 71; 1F9 V1 domain: SEQ ID Nos: 72 and 73; 1F2 Vh domain: SEQ ID Nos: 74 and 75; 1F2 V1 domain: SEQ ID Nos: 76 and 77. Figure 15 shows each of the domains (including the V1 truncations or 3G7, 1F9 and 1F2 deletions) and the locations of the CDR regions.

Fab fragments of six clones were expressed in E. coli HB2151 host cells, and purified by the G-protein column by affinity chromatography. Phagemids from the individual selected cloners were used to transform a non-suppressing E. coli host HB2151. Expression of Fabs fragments in HB2151 was induced by culturing the cells in 2YTA medium containing 1 mM deIPTG at 30 ° C. A periplasmic extract of the cells was prepared as described by Lu (χ). Soluble Fab protein was purified using a protein G column following the manufacturer's protocol (AmershamBiosciences). To examine the purity and molecular weight of the preparation, purified antibodies were electrophoresed on 4-12% Bis-Tris NuPAGE ™ gel (Invitrogen) and visualized by staining with the SimplyBlue ™ SafeStain solution (Invitrogen).

Purified binding proteins were analyzed by SDS-PAGE (Figure 2). Full light chain Fabs appear in Figure 2A with molecular weights of about 50,000. In contrast, unreduced fragments of 1F2 and 1F9 gave small bands (-DTT: MW ~ 37,500) as compared to a standard control Fab (Figure 2B). Under deduction conditions (+ DTT), two bands were evident, the upper band consistent with the normal size of VH = CH1 fragments, and the lower band consistent with light chains having CL fragments (MW ~ 12,500) masking the variable domains.

Binding of mPDGFRa and blocking mPDGFRa / PDGF-AA by anti-mPDGFRa antibodies. Soluble Fab fragments of six anti-mPDGFRa clones were quantitatively compared in their antigen binding efficiency and potency to block the demPDGFRa / PDGF-AA interaction. In the binding assay, Fab was first incubated in a 96-well plate coated with PDGFRa / Fc fusion protein (1 μg / ml) at room temperature for 1 h, followed by incubation with an anti-anti-HRP Fab antibody conjugate. human coelhodurant for 1 h more. The plate-bound HRP antibody was then quantified by the addition of peroxidase substrate. Consistent with the triageminitial result (Table 1), Fab 1F2 binds much more efficiently to mPDGFRa than all other Fab proteins (Figure 3A).

Binding kinetics of various anti-mPDGFRα antibodies were determined by surface plasmon resonance using a BIAcore 3000 biosensor and evaluated using the BIA Evaluation 2.0 program (Biacore, Inc., Uppsala, Sweden). The affinity constant, Kd, was calculated from the dissociation rate (Δ off) / association rate (Migrate) correlation. The values described represent the mean ± standard deviation of at least two determinations for Fabs and three determinations for IgG. (Table 2). Consistent with the binding assays, the sVD 1F2 antibody has a much higher affinity (Kd) of about 0.5 nM, more than 23 times higher than the selected normal Fab, IE10.

In Table 2, Fab 1F2-2H is an engineered divalent Fab containing a second identical VH domain expressed as a paraCL fusion. The divalent Fab was expressed in E. coli and purified by protein G chromatography. SDS-PAGE analysis of purified 1F2-2H Fab demonstrated a single protein band of approximately 50 kD, similar to standard Fab defragment (Figure 5). The affinity of the divalent Fab is 79.5 pM, compared to 418 pM for the monovalent 1F2 Fab, indicating a 5.2-fold improvement (Table 2).

<table> table see original document page 43 </column> </row> <table>

In the blockade test, various amounts of antibodies were incubated with a fixed amount of mPDGFRa / Fc fusion in solution for 30 min, and the mixtures were then transferred to rhPDGF-AA coated 96-well plates and incubated for 1 h. Linked mPDGFR was then quantified by quantification of bound anti-human Fc-Ab. Fb 1F2 strongly blocked the interaction of mPDGFRa / PDGF-AA (figure-3B) with an IC50 value of 12 nM compared with 57, 140 and 220 nM for 1F9, IE10 and IA1, respectively. On an equal molar basis, divalent Fab showed moderately improved activity in both amPDGFRα binding and blocking receptor / ligand interaction (Figure 6).

Cloning, expression and purification of complete IgG. Fabs from selected clones were converted to full length IgGs by cloning the VH and VL domains into the heavy chain expression vector, pDFc, and light chain expression vector, pLCk, respectively. PDFc have Hind III and Nhe I cloning sites between the leader peptide and CH1 sequence encoding genes. PLCk has Hind III eBsiW I cloning sites between a leader peptide sequence and CL. Briefly, the VH and VL decoding genes were amplified by reverse transcription PCR phagemid DNAs and cloned into their respective expression vectors. The heavy and light chain expression vectors (VH-pDFc and VL-pLCk) were then co-coded. transfected into COS-7 cells for transient expression as previously described. Cell culture media were collected at 48 and 96 hours after transfection, and pooled. Antibodies were purified from cell culture supernatant by affinity chromatography using protein A columns following the manufacturer's protocol (Amersham Biosciences). After IgG function was confirmed, the expressed pairs of VH and VL genes were cloned into single expression vectors and transfected into COS-7 cells. Purification of antibodies has been described above.

Three IgG-like constructs were produced from Ig1F2 containing only one VH (see Figure 7), including four VHs tetravalent IF2-2H IgG (A), and 1F2 Vh divalent IF2 expressed as a fusion to both Ch and Cl (B and C). Plasmids to express light and heavy chains were used to co-transfect COS-7 cells for transient expression. Antibody expression levels were monitored by ELISA using cell culture supernatants. 1F2-2H expression was consistently lower than both divalent 1F2 antibodies, about one quarter of divalent 1F2 antibodies. Cell culture supernatant antibodies were purified by affinity chromatography using a protein A column.

Clone IE10, a blocker with abnormal antibody components, was also converted to a full length IgG. The IE10 Vh and V1 genes were first cloned into pDFc and pLCk, respectively. As above, after DNA sequencing and confirmation of IE10 IgG binding activity, the expressed VH / VL pair was cloned into a single expression vector which was then used to transfect COS-7 cells followed by antibody purification.

Purified antibodies were analyzed by SDS-PAGE to determine the purity and molecular weight of the preparation. Different antibodies 1F2-CH / CL and 1F2-CL / CH, with MW ~ 125,000, migrated faster than normal IgG (1E10 and 2B4; left panel of Figure 8). When antibodies were treated with DTT before electrophoresis ( right panel of figure 8), the heavy and light chain components have been resolved. The 1F2-CH / CL antibody had a higher band correlated with a heavy chain similar to that of IE10 and 2B4, and a fast migrating lower band with near-anticipated mobility (MW ~ 12,500, CL only). Antibody 1F2-CL / CH, on the other hand, showed that the lower band represents a light chain with mobility similar to that of IE10 and 2B4, and the upper band correlates with the ChI-Fc polypeptide only (MW ~ 37,500).

Binding and blocking activities of full-length anti-mPDGFRa antibodies. All three purified 1F2 variant antibodies, 1F2-2H, 1F2-CH / CL and 1F2-CL / CH and the IE10 antibody were compared for their binding affinity to mPDGFRa by ELISA. Various amounts of antibodies were incubated in the ELIS plate coated with mPDGFRa. / Fc, antibodies bound to mPDGFRα were then detected by an anti-human HRP-κ antibody conjugate. The ELISA result indicates that although 1F2-2H IgG has four binding sites, the IF2-2H binding activity was ~ 8x lower than both divalent 1F2 IgGs (1F2-CH / CL and 1F2-CL / CH (Figure 1). On the other hand, both divalent 1F2 (1F2-CH / CL) and 1F2-CL / CH) had very similar binding affinity shown by ELISA (Figure 9A) and BIAcore analysis (Table 2). increased by about one magnitude from parental 1F2 Fab and IE10 IgG increased 20-fold from its Fab (Table 2). Overall, the binding affinities of both divalent 1F2 IgGs in mPDGFRa were about a greater magnitude than IE10 IgG (Figure 9A and Table 2).

Quantitative blocking tests were also performed to evaluate anti-mPDGFRa antibodies. As described above, various amounts of antibodies were incubated with a fixed amount of mPDGFRa / Fc fusion in solution for 30 min, and the mixtures were then transferred to rhPDGF-AA coated 96-well plates and incubated for 1 h. Bound mPDGFRα was then quantified by quantification of bound anti-human Fc-Ab. Consistent with the deletion data, the divalent 1F2-CH / CL and 1F2-CL / CH were better blockers than 1F2-2H IgG and 1E10 IgG: IC50 values were 3.2, 2.7, 17, and 9.6 nM for 1F2-CH / CL, 1F2-CL / CH, 1F2-2H IgG and IE10 IgG, respectively (Figure 9B).

Bispecific antibodies. An anti-Flk-1 antibody, 2B4, was also isolated from the Fab Dyax phage library using the same procedure described above except that Maxisorp Star tubes were coated with mVEGFR2-Fc fusion protein. The 2B4 Fab was Flk-I specific and blocked the Flk-IATEGFi65 interaction. The 2B4 DeFab Vh and Vl domains are provided by SEQ ID Nos: 79 and 81, respectively. Figure 15 shows each of the domains and regions of the CDR regions. The 2B4 Fab was converted to complete IgG1 as described above. 2B4 and IgG Fab binding kinetics were determined by BIA core analysis. The 2B4 and IgG Fab binding affinities for mVEGFR2 are 6.7 ± 3.0nM and 0.39 ± 0.1 nM, respectively. 2B4 IgG blocks the VEGFR2 / VEGFi65 interaction with an IC50 value of approximately 3.5 nM (Figure 13B).

Construction of Domain-Based Bispecific Antibodies (Anti-FlP-1 mPDGFRα χ) - Domain-specific bispecific antibodies were constructed in two formats, the scFv format and the Fab format (Figure 10, A and B). In scFv format, PCR fragments encoding the 2B4 VH and VL genes were first assembled using overlapping PCR. The 2B4 VL COOH terminus was linked to the VH 2B4 NH2 terminus via a 5 amino acid linker (Ala-Ser-Thr-Lys-Gly, obtained from the N-terminus of a CL domain) (Figure 10A). The resulting gene encoding 2B4 VL-VH was then cloned into the pDFc vector viaHindIII / Nhe I binding for expression of the scFv2B4-CH fusion (consisting of 2B4 VL-2B4 VH - CH1 - CH2 - CH3). The scFv2B4-CH expression vector was then paired with the 1F2VH-CL expression vector as described above by co-transfecting COS-7 cells for transient expression.

In Fab format, the 1F2 Vh gene and 2B4 Vl were first pooled using overlap PCR. In this fusion, the Vh 1F2 COOH terminus was linked to the 2B4 L amino terminus via a 5 amino acid linker of the 5 'end of Ch. The gene encoding VH-2B4 VL 1F2 was then cloned into the pLCk vector via the HindIII / BsiW sites for protein expression. Melting Point 1F2 VH-2B4 VL-CL. The 2B4 Vh gene was cloned into pDFc primer as described above for VH expression. Plasmids for expressing light and heavy chains, paired as described in Figure 10B, were used to co-transfect COS-7 cells for transient expression.

After the chlorins were tested by ELISA for dual deletion activities, the Ig expression constructs were combined into a single expression vector which was then used to transfect COS-7 cells for transient expression of engineered IgG. Antibodies were purified from cell culture supernatant as described above.

Expression and purification of bispecific antibodies combase in the domains. Antibodies 1F2 / scFv2B4 and 1F2-2B4 were produced by transfection and transient expression in COS-7 cells (100-200 ml cell cultures). Antibodies were purified from cell culture supernatants by affinity chromatography using protein A columns.

Purified bispecific antibodies (1F2 / scFv and 1F2-2B4) and their parental antibodies 1F2 and 2B4 were analyzed by SDS-PAGE (Figure 8). The lower mobility of untreated bispecific antibodies (-DTT) correlated with their high molecular hair (~ 175,000), as compared to dB-2B4 and divalent 1F2-CH / CL IgG that migrated at the molecular weight position ~ 125,000. The two components of each of four antibodies were resolved separately after DTT treatment. Comparing to a standard IgG (2B4), the lower band of the 1F2 / scFv2B4 antibody (the VH-CL fusion) is comparable to a standard IgG yeast chain. The upper band correlates with a scFv heavy chain fusion protein (MW ~ 62,500). On the other hand, the upper band of 1F2-2B4 corresponds to the heavy chain of a standard IgG (2B4) and the lower band is observed at an expected position for an HCV-light chain fusion (MW ~ 37,500).

Dual specificity of bispecific domain based antibodies. In a cross-linking test, bispecific 1F2-2B4 antibody and monospecific 1F2-CH / CL and 2B4 antibodies were first incubated with mPDGFRa or mVEGFR2 in solution and then transferred to a second receptor-coated microtiter plate (mVEGFR2 or mPDGFRa, respectively), followed, by incubation with a biotin-labeled anti-polyclonal to the first receptor. Only BsAb, but not parental monospecific 2B4 IgG and 1F2-CH / CL, was able to derectulate both target receptors.

The antigen binding efficiency of domain-based bispecific antibodies was determined in immobilized mPDGFRa and Flk-1.ELISA showed that bispecific antibody, 1F2 / scFv2B4 and 1F2-2B4, bound to mPDGFRa and Flk-1, but not as well. efficiently as the 1F2 parental and 2B4 antibody (Figure 12). However, in both cases, binding of the 1F2-2B4 antibody was slightly more efficient compared to 1F2 / scFv2B4. As expected, FlB-I-specific antibody 2B4 does not bind to mPDGFRa (Fig. 12A), nor is 1F2 of demPDGFRa-specific antibody binding to Flk-I (Fig. 12B). Binding kinetics of specific antibodies for mPDGFRa and Flk-I were determined by surface deplasmon resonance using a BIAcore instrument (Table 3). Consistent with ELISA observations, 1F2-2B4 antibody has higher affinity for mPDGFRa as Flk-I compared to 1F2 / scFv2B4 antibody.

<table> table see original document page 49 </column> </row> <table> <table> table see original document page 50 </column> </row> <table>

Figure 13 shows that both specific antibodies inhibit mPDGFRα from binding to immobilized PDGF-AA (Figure 13A) with an estimated IC50 of 9.9 nM and 25.3 nM for 1F2 / scFv2B4 and 1F2-2B4, respectively. Antibodies also block Flk-I from binding to immobilized aVEGF165 (Figure 13B) with an IC50 value of 9.5 nM and 19.5 nM, respectively. As expected, 2B4 had no effect on mPDGFRa / PDGF-AA interaction, while 1F2 had no effect on Flk-I / VEGFi65 interaction.

Although the bispecific antibody binding affinity is minor in mPDGFRα binding and VEGFR2 binding compared to respective monospecific divalent molecules, 1F2-CH / CL and 2B4 IgG (table 3) when examined for binding to cell-expressed receptors on eEnd cells . 1 (the cells expressing both mPDGFRα and mVEGFR2), BsAb demonstrated higher efficiency than either parent monospecific bivalent antibodies. The mean defluorescence intensity (MFI) in cells was 15.7, 31 and 36.9 for 1F2-CH / CL (mPDGFRa binding), 2B4 IgG (mVEGFR2 binding) and 1F2-2B4IgG (mPDGFRa binding and nVEGFR2), respectively.

Inhibition of ligand-induced activation of mV? GFR2 inPDGFR. Expression of mVEGFR2 and mPDGFRa was demonstrated in eEnd.l cells via FACS analysis. Cells were incubated with 2B4 IgG, 1F2-CH / CL or 1F2-2B4 IgG (10 μ ^ πιΐ) at 4 ° C for 1 h, followed by incubation with a PE labeled goat anti-human Fc antibody (Jackson ImmunoResearch Lab.) For an additional 1 h, and analysis on a Guava Easycyte system (Guava Technologies, Inc ,,. Hayward, CA).

To examine receptor phosphorylation, eEnd cells. 1 were deposited on 6 cm plates and cultured at 70-80% confluence, after which the cells were washed twice in PBS and cultured overnight in serum free medium. Cells were first incubated with various antibodies at 37 ° C for 30 min, followed by stimulation with VEGF or PDGF-AA at 37 ° C for 15 min. Cells were lysed in delysis buffer (50 mM Tris-HCl, pH 7.4, 150 nM NaCl, 1% Triton X-100, 1mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 nM Na3VO4, 1 µg / ml delaupeptin, 1 μg / ml pepstatin and 1 μg / ml aprotinin) for 1 h, followed by centrifugation of the lysate at 12,000 rpm for 10 min at 4 ° C. Receptors were immunoprecipitated from cell lysate supernatant using either anti-mPDGFRa (eBioscience, San Diego, CA) or anti-mVEGFR2 (Santa Cruz, Biotech, Santa Cruz, CA) antibody, followed by the addition of 20μl of Pro-A / G-Sepharose (Santa Cruz Biotech). Precipitated receptor proteins were resolved on a 4-12% Nupage Bis-Tris gel (Invitrogen) and transferred to a depolyvinylidene difluoride membrane. Phospho-mVEGFR2 and phospho-mPDGFRa were detected in the noblot using an anti-phospho-tyrosine HRP antibody conjugate (Santa CruzBiotech). Gel-loaded total receptor proteins were tested with antibodies in either mPDGFRa or mVEGFR2 (both from Santa Cruz Biotech).

As shown in Figure 14, BsAb inhibited both PDGF and VEGF stimulated phosphorylation of mPDGFRa receptors in VEGFR2, while monospecific parental antibodies only blocked activation of a single receptor stimulated by its ligandocognate. As a control, the anti-EGFR antibody, C225, had no effect on ligand-stimulated activation of any receptor. SEQUENCE LISTING

<110> ImClone Systems, Inc.Zhenping, Zhu

<120> Functional Antibodies

<130> 11245/54376

<140> PCT / US07 / 04 051

<141> 2007-02-15

<150> 60 / 773.994

<151> 2006-02-15

<160> 81

<170> PatentIn version 3.3

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Tyr Asp Wing Ser Asn Read Asp Thr Gly Val Pro Ser Arg Phe Ser Gly50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Read Thr Ile Be Ser Read Gln Ala65 70 75 80

Glu Asp Phe Wing Val Tyr Phe Cys Gln Gln Wing Lys Wing Phe Pro Pro 90 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys100 105

<210> 11

<211> 348

<212> DNA

<213> Mus musculus

<220>

<221> CDS <222> (1) .. (348)

<400> 11

cag gtc aaa ctg cag cag tct ggg gcaGln Val Lys Leu Gln Gln Ser Gly Ala1 5

gag ctt gtc aag cca ggg gcc 48

Glu Leu Val Lys Pro Gly Ala10 15

tca gtc aag ttg tcc tgc aca gct tct ggc ttc aac att aaa gac acc 96

Be Val Lys Read Be Cys Thr Wing Be Gly Phe Asn Ile Lys Asp Thr20 25 30

tat ata cac tgg gtg aag cag age cct gaa cag ggc ctg gag tgg att 144

Tyr Ile His Trp Val Lys Gln Ser Pro Glu Gln Gly Leu Glu Trp Ile35 40 45

gga agg till gat cct ag gat aat act aaa tat gac ccg aag ttc 192

Gly Arg Ile Asp Pro Pro Asn Asp Asn Thr Lys Tyr Asp Pro Lys Phe50 55 60

cag ggc aag gcc act ata aca gca gac aca tcc tcc aat aca gcc tac 240

Gln Gly Lys Wing Thr Ile Thr Wing Asp Thr Be Ser Asn Thr Wing Tyr65 70 75 80atg cag ctc cgc age ctg aca

Met Gln Read Arg Be Read Thr Be Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

gcc ctc cca ccg ttc tac tt gac tac tgg ggc cat ggc acc acg gtc 336

Wing Pro Le Pro Phe Tyr Phe Asp Tyr Trp Gly His Gly Thr Thr Val100 105 110

acc gtc tcc tca 348

Thr Val Ser Ser115

<210> 12

<211> 116

<212> PRT

<213> Mus musculus

<4 00> 12

Gln Val Lys Leu Gln Gln Ser Gly Wing Glu Leu Val Lys Pro Gly Wing 10 15 15

Be Val Lys Read Be Cys Thr Wing Be Gly Phe Asn Ile Lys Asp Thr20 25 30

Tyr Ile His Trp Val Lys Gln Ser Pro Glu Gln Gly Leu Glu Trp Ile35 40 45

Gly Arg Ile Asp Pro Pro Asn Asp Asn Thr Lys Tyr Asp Pro Lys Phe50 55 60

Gln Gly Lys Wing Thr Ile Thr Wing Asp Thr Be Ser Asn Thr Wing Tyr65 70 75 80

Met Gln Read Arg Be Read Thr Be Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

Wing Pro Le Pro Phe Tyr Phe Asp Tyr Trp Gly His Gly Thr Thr Val100 105 110

Thr Val Ser Ser115

<210> 13

<211> 327

<212> DNA

<213> Mus musculus

<220>

<221> CDS <222> (1) .. (327) <4 00> 13

gac till gag ctc act cag tet cca aaa ttc atg tcc aca tca gta gga 48

Asp Ile Glu Read Thr Gln Be Pro Lys Phe Met Be Thr Be Val Gly1 5 10 15

gac agg gtc age gtc acc tgc aag gcc agt cag aat gtg gat act aat 96

Asp Arg Val Be Val Thr Cys Lys Wing Be Gln Asn Val Asp Thr Asn20 25 30

gta gcc tgg tat caa cag aaa cca ggg caa tet cct aaa gea ctg att 144

Val Wing Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Wing Leu Ile35 40 45

tac tcg gea tcc tac cgg tac agt gga gtc cct gat cgc ttc aca ggc 192

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly50 55 60

agt gga tet ggg aca gat ttc act ctc acc until age aat gtg cag tet 240

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65, ■ 70 75 80

gaa gac ttg gea gag tat ttc tgt cag caa tat aac acts ttt cct tac 288

Glu Asp Read Wing Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Phe Pro Tyr85 90 95

acg ttc gga ggg ggg acc aag ctg gaa ata aaa cgg gcg 327

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala100 105

<210> 14

<211> 109

<212> PRT

<213> Mus musculus

<400> 14

Asp Ile Glu Read Thr Gln Be Pro Lys Phe Met Be Thr Be Val Gly15 10 15

Asp Arg Val Be Val Thr Cys Lys Wing Be Gln Asn Val Asp Thr Asn20 25 30

Val Wing Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Wing Leu Ile35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser65 70 75 80

Glu Asp Read Wing Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Phe Pro Tyr85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala100 105 <210> 15

<211> 357

<212> DNA

<213> Mus musculus

<220>

<221> CDS <222> (1) .. (357)

<400> 15

cag gtg cag ctg aag cag tca gga cct ggc cta

Gln Val Gln Leu Lys Gln Be Gly Pro Gly Leu Val Gln Pro Be Gln1 5 10 15

age ctg tcc until acc tgc aca gtc tet ggt ttc tca tta act aac tat 96

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr20 25 30

ggt gta cac tgg gtt cgc cag tet cca gga aag ggt ctg gag tgg ctg 144

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu35 40 45

gga gtg ata tgg agt ggt gga aac aca gac tat aat aca cct ttc aca 192

Gly Val Ile Trp Being Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr50 55 60

tcc aga ctg age till aac aag gac aat tcc aag age caa gtt ttc ttt 240

Ser Arg Read Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80

aaa atg aac agt ctg caa tet aat gac aca gcc ata tat tac tgt gcc 288

Lys Met Asn Be Read Gln Be Asn Asp Thr Wing Ile Tyr Tyr Cys Ala85 90 95

ag gcc ctc acc tac tat gat tac gag ttt gct tac tgg ggc caa ggg 336

Arg Wing Read Thr Tyr Tyr Asp Tyr Glu Phe Wing Tyr Trp Gly Gln Gly100 105 110

act ctg gtc act gtc tet gea 357

Thr Leu Val Thr Val Ser Ala115

<210> 16

<211> 119

<212> PRT

<213> Mus musculus

<400> 16

Gln Val Gln Leu Lys Gln Be Gly Pro Gly Leu Val Gln Pro Be Gln1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr20 25 30

Gly Val His Trp Val Arg Gln Be Pro Gly Lys Gly Leu Glu Trp Leu35 40 45Gly Val Ile Trp Be Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr50 55 60

Ser Arg Read Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80

Lys Met Asn Be Read Gln Be Asn Asp Thr Wing Ile Tyr Tyr Cys Ala85 90 95

Arg Wing Read Thr Tyr Tyr Asp Tyr Glu Phe Wing Tyr Trp Gly Gln Gly100 105 110

Thr Leu Val Thr Val Ser Ala115

<210> 17

<211> 321

<212> DNA

<213> Mus musculus

<220>

<221> CDS <222> (1) .. (321)

<4 00> 17

gac by ttg ctg act by cag tet cca gtc by ctg tet gtg by agt cca gga 48

Asp Ile Leu Read Le Gln Ser Pro Val Ile Leu Read Val Ser Pro Gly15 10 15

gaa aga gtc agt ttc tcc tgc agg gcc agt cag agt att ggc aca aac 96

Glu Arg Val Be Phe Be Cys Arg Wing Be Gln Be Ile Gly Thr Asn20 25 30

aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 144

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Read Leu Ile35 40 45

aag tat gct tet gag tet till tet ggg till cct tcc agg ttt agt ggc 192

Lys Tyr Wing Be Glu Be Ile Be Gly Ile Pro Be Arg Phe Be Gly50 55 60

agt gga tca ggg aca gat ttt act ctt age till aac agt gtg gag tet 240

Ser Gly Ser Gly Thr Asp Phe Thr Read Ser Ile Asn Ser Val Glu Ser65 70 75 80

gaa gat att gea gat tat tac tgt caa caa aat aat aac tgg cca acc 288

Glu Asp Ile Asp Wing Tyr Tyr Cys Gln Asn Asn Asn Asp Trp Pro Thr85 90 95

acg ttc ggt ggt ggg acc aag ctg gag ctg aaa 321

Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys100 105

<210> 18 <211> 107 <212> PRT <213> Mus musculus

<400> 18

Asp Ile Leu Read Thr Gln Be Pro Val Ile Leu Be Val Ser Pro Gly1 5 10 15

Glu Arg Val Be Phe Be Cys Arg Wing Be Gln Be Ile Gly Thr Asn20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Read Leu Ile35 40 45

Lys Tyr Wing Be Glu Be Ile Be Gly Ile Pro Be Arg Phe Be Gly50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Read Ser Ile Asn Ser Val Glu Ser65 70 75 80

Glu Asp Ile Asp Wing Tyr Tyr Cys Gln Asn Asn Asn Asp Trp Pro Thr85 90 95

Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys100 105

<210> 19 <211> 363

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (363)

<4 00> 19

cag gtg cag ctg

Gln Val Gln Leu Gln Glu Ser Gly Pro1 5

acc ctg tcc ctc acc tgc act gtc tct

Thr Leu Be Leu Thr Cys Thr Val Ser

20 25

gga ctg gtg aag cct tca cag 48

Gly Leu Val Lys Pro Ser Gln10 15

ggt ggc tcc till age agt ggt 96

Gly Gly Ser Ile Ser Ser Gly30

gat tac tac tgg agt tggAsp Tyr Tyr Trp Ser Trp35

tgg att ggg tac till tatTrp Ile Gly Tyr Ile Tyr50

ctc aag agt cga gtc accLeu Lys Ser Arg Val Thr

till cgc cag ccc cca gggIle Arg Gln Pro Pro Gly40

tac agt ggg age acc gacTyr Ser Gly Ser Thr Asp55 60

atg tcc gta gac acg tccMet Ser Val Asp Thr Ser

aag ggc ctg gag 144

Lys Gly Leu Glu45

tac aac ccg tcc 192

Tyr Asn Pro Ser

aag aat cag ttt 240

Lys Asn Gln Phe65 70 75 80

tcc ctg aag gtc aac tct gtg acc gcc gca gac acg gct gtg tat tac 288

Ser Leu Lys Val Asn Ser Val Thr Wing Asp Wing Thr Val Wing Tyr Tyr85 90 95

tgt gcg aga gtg tcg att ttt gga gtg ggg aca ttt gac tac tgg ggc 336

Cys Wing Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr Trp Gly100 105 110

cg ggc acc cg gtc acc gtc tca age 363

Gln Gly Thr Read Val Val Val Ser Ser 120 120

<210> 20

<211> 121

<212> PRT

<213> Homo sapiens

<4 00> 20

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Gln15 10 15

Thr Read Be Read Thr Cys Thr Val Be Gly Gly Be Ile Be Gly20 25 30

Asp Tyr Tyr Trp Being Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser50 55 60

Leu Lys Be Arg Val Thr Met Be Val Asp Thr Be Lys Asn Gln Phe65 70 75 80

Ser Leu Lys Val Asn Ser Will. Thr Wing Asp Wing Thr Wing Val Tyr Tyr85 90 95

Cys Wing Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr Trp Gly100 105 110

Gln Gly Thr Read Val Val Val Ser Ser 120 120

<210> 21

<211> 321

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (321) <400> 21

gaa att gtg atg aca cag tct cca gcc acc ctg tct ttg tct cca ggg 4 8

Glu Ile Val Met Thr Gln Be Pro Wing Thr Read Be Read Pro Gly1 5 10 15

gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt age age tac 96

Glu Arg Wing Thr Read Le Be Cys Arg Wing Be Gln Be Val Ser Be Tyr20 25 30

tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc up to 144

Leu Trp Wing Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu Ile35 40 45

tat gat gea tcc aac agg gcc act ggc till cca gcc agg ttc agt ggc 192

Tyr Asp Wing Ser Asn Arg Wing Thr Gly Ile Pro Wing Arg Phe Ser Gly50 55 60

agt ggg tct ggg aca gac ttc act ctc acc until age age cta gag cct 240

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Glu Pro65 70 75 80

gaa gat ttt gea gtg tat tac tgt cac cag tat ggt age aca cct ctc 288

Glu Asp Phe Wing Val Tyr Cyr His Gln Tyr Gly Ser Thr Thr Leu85 90 95

act ttc ggc gga ggg acc

Thr Phe Gly Gly Gly Thr Lys Wing Glu Ile Lys100 105

<210> 22

<211> 107

<212> PRT

<213> Homo sapiens

<4 00> 22

Glu Ile Val Met Thr Gln Be Pro Wing Thr Read Be Read Pro Gly1 5 10 15

Glu Arg Wing Thr Read Le Be Cys Arg Wing Be Gln Be Val Ser Be Tyr20 25 30

Leu Trp Wing Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu Ile35 40 45

Tyr Asp Wing Ser Asn Arg Wing Thr Gly Ile Pro Wing Arg Phe Ser Gly50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Glu Pro65 70 75 80

Glu Asp Phe Wing Val Tyr Cyr His Gln Tyr Gly Ser Thr Thr Leu85 90 95

Thr Phe Gly Gly Gly Thr Lys Wing Glu Ile Lys100 105

<210> 23

<211> 390

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (390)

<4 00> 23

gag gtg cag ctg gtg cag tct ggg gct gag gtg

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Ser15 10 15

tcg gtg aag gtc tcc tgc aag gct tct gga ggc acc ttc age act tatSer Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr20 25 30

gct till age tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atgAla Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly

gga ggg up to cct till ttt ggt aca gea aac tac gea cag aag ttcGly Gly Ile Ile Pro Ile Phe Gly Thr Wing Asn Tyr Wing Gln Lys Phe50 55 60

cag ggc aga gtc acg att acc gcg gac aaa tcc acg age aca gcc tacGln Gly Arg Val Ile Thr Thr Wing Asp Lys Ser Thr Be Thr Wing Tyr65 70 75 80

atg gag ctg age age ctg aga tct gag gac acg gcc gtg tat tac tgtMet Glu Leu Be Ser Leu Arg Ser Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

gcg aga gcg cca tta cga ttt ttg gag tgg tcc acc caa gac cac tacAla Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr100 105 110

tac tac tac tac atg gac tgg tgg ggc aaa ggg acc acg gtc acc gtcTyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Thr Val115 120 125

tca ageSer Ser130

<210> 24

<211> 130

<212> PRT

<213> Homo sapiens

<4 00> 24

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Ser15 10 15

Be Val Lys Val Be Cys Lys Wing Be Gly Gly Thr Phe Be Be TyrAla Ile Be Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Wing Asn Tyr Wing Gln Lys Phe50 55 60

Gln Gly Arg Val Thr Ile Thr Asp Wing Lys Be Thr Be Thr Wing Tyr65 70 75 80

Met Glu Read Be Ser Read Ar Be Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

Arg Wing Pro Wing Read Arg Phe Read Glu Trp Be Thr Gln Asp His Tyr100 105 110

Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val115 120 125

Ser Ser30

<210> 25

<211> 327

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (327)

<4 00> 25

tct tct gag ctg act cag gac cct gct gtg

Be Ser Glu Leu Thr Gln Asp Pro Val Wing Be Val Wing Leu Gly Gln15 10 15

aca gtc agg till aca tgc caa gga gac age ctc aga age tat tat gea 96

Thr Val Arg Ile Thr Cys Gln Gly Asp Be Read Arg Be Tyr Tyr Ala20 25 30

age tgg tac cag cag aag cca gga cag gcc cct gta ctt gtc until tat 144

Ser Trp Tyr Gln Lys Pro Gly Gln Pro Wing Val Leu Val Ile Tyr35 40 45

ggt aaa aac aac cgg ccc tca ggg to cca gac cga ttc tct ggc tcc 192

Gly Lys Asn Asn Arg Pro Be Gly Ile Asn Arg Pro Phe Be Gly Ser50 55 60

age tca gga aac aca gct tcc ttg acc till act ggg gct cag gcg gaa 240

Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Wing Gln Wing Glu65 70 75 80

gat gag gct gac tat tac tgt aac tcc cgg gac aac agt gat aac cgt 288Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Asn Ser Asp Asn Arg85 90 95

ctg ata ttt ggc ggc ggg acc aag ctg acc gtc ctc agt 327

Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser100 105

<210> 26

<211> 109

<212> PRT

<213> Homo sapiens

<400> 26

Be Ser Glu Leu Thr Gln Asp Pro Val Wing Be Val Wing Leu Gly Gln1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Be Read Arg Be Tyr Tyr Ala20 25 30

Ser Trp Tyr Gln Lys Pro Gly Gln Pro Wing Val Leu Val Ile Tyr35 40 45

Gly Lys Asn Asn Arg Pro Be Gly Ile Asn Arg Pro Phe Be Gly Ser50 55 60

Be Ser Gly Asn Thr Wing Be Read Thr Ile Thr Gly Wing Gln Wing Glu65 70 75 80

Asp Glu Wing Asp Tyr Tyr Cys Asn Be Arg Asp Asn Be Asp Asn Arg85 90 95

Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser100 105

<210> 27

<211> 378

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (378)

<400> 27

cag gcg cag gtg gtg gag tct ggg gga ggc gtg gtc cag tct ggg agg 48

Gln Wing Gln Val Val Glu Be Gly Gly Gly Val Val Gln Be Gly Arg1 5 10 15

tcc ctg aga ctc tcc tgt gca gcg tct gga ttc gcc ttc agt age tac 96

Be Read Arg Read Be Cys Wing Be Gly Phe Wing Be Phe Be Tyr20 25 30

ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45

gca gtt ata tgg tat gat gga agt aat aaa tac tat gca gac tcc gtg 192

Wing Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Wing Asp Ser Val

50 55 60

agg ggc cga ttc acc till tcc aga gac aat tcc gag aac acg ctg tat 240

Arg Gly Arg Phe Thr Ile Be Arg Asp Asn Be Glu Asn Thr Read Tyr65 70 75 80

ctg caa atg aac age ctg aga gcc gag gac acc gct gtg tat tac tgt 288

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

gcc aga gat cac tat ggt tcg ggg gtg cac cac tat ttc tac tac ggt 336

Arg Asp Wing His Tyr Gly Ser Gly Val His His Tyr Phe Tyr Tyr Gly100 105 110

ctg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca 378

Read Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser115 120 125

<210> 28

<211> 126

<212> PRT

<213> Homo sapiens

<4 00> 28

Gln Wing Gln Val Val Glu Be Gly Gly Gly Val Val Gln Be Gly Arg15 10 15

Be Read Arg Read Be Cys Wing Be Gly Phe Wing Be Phe Be Tyr20 25 30

Gly Met His Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val35 40 45

Val Ile Wing Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Wing Asp Ser Val50 55 60

Arg Gly Arg Phe Thr Ile Be Arg Asp Asn Be Glu Asn Thr Read Tyr65 70 75 80

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

Arg Asp Wing His Tyr Gly Ser Gly Val His His Tyr Phe Tyr Tyr Gly100 105 110

Read Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser115 120 125 <210> 29

<211> 324

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (324)

<4 00> 29

gaa att gtg ttg acg cag tct cca ggc acc ctg tct ttt tct cca gggGlu Ile Val Leu Thr Pro Gly Thr Leu Ser Leu Pro Pro Gly15 10 15

gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt age age ageGlu Arg Wing Thr Read Le Ser Cys Arg Wing Ser Gln Ser Val Ser Ser 25 25 30

tac tta gcc tgg tac cag aaa cct ggc'cag gct ccc agg ctc ctcTyr Leu Wing Trp Tyr Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu35 40 45

till ggt gea tcc age agg gcc act ggc till cca gac agg ttc agtIle Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser50 55 60

ggc agt ggg tct ggg aca gac ttc act ctc acc till age aga ctg gagGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80

cct gaa gat ttt gea gtg tat tac tgt cag cag tat ggt age tca ccgPro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Tyr Gly Ser Ser Pro85 90 95

ctc act ttc ggg ggg ggg acc aag gtg gag until aaaLeu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys100 105

<210> 30

<211> 108

<212> PRT

<213> Homo sapiens

<4 00> 30

Glu Ile Val Leu Thr Gln Be Pro Gly Thr Read Leu Be Read Pro Gly15 10 15

Glu Arg Wing Thr Read Le Be Cys Arg Wing Be Gln Be Val Be Ser20 25 30

Tyr Leu Wing Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu35 40 45

Ile Tyr Gly Ala Be Being Arg Wing Thr Gly Ile Pro Asp Arg Phe Ser50 55 60Gly Be Gly Be Gly Thr Asp Phe Thr

Pro Glu Asp Phe Wing Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro85 90 95

Read Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys100 105

<210> 31 <211> 363

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (363)

<4 00> 31

gag gtg cag ctg gtg cag tct ggg gct gag gtg

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Wing 10 15 15

tca gtg aag gtt tcc tgc aag gtt tct gga tac acc ttc acc gac tac 96

Be Val Lys Val Be Cys Lys Val Be Gly Tyr Thr Phe Thr Asp Tyr20 25 30

tac atg cac tgg gtg caa cag gcc cct gga aaa ggg ctt gag tgg atg 144

Tyr Met His Trp Val Gln Gln Pro Wing Gly Lys Gly Leu Glu Trp Met35 40 45

gga ctt gtt gat cct gaa gat ggt gaa aca until tac gea gag aag ttc 192

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Wing Glu Lys Phe50 55 60

cag ggc aga gtc acc ata acc gcg gac acg tct aca gac aca gcc tac 240

Gln Gly Arg Val Thr Ile Thr Wing Asp Thr Be Asp Thr Wing Tyr65 70 75 80

atg gag ctg age age ctg aga tct gag gac acg gcc gtg tat tac tgt 288

Met Glu Read Be Ser Read Ar Be Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

gcg aga gat gac tac atg gac gtc tgg ggc aaa ggc acc ctg gtc acc 336

Wing Asp Asp Asp Tyr Met Asp Val Trp Gly Lys Gly Thr Read Val Thr100 105 110

gtc tca age gcc tcc acc

Val Ser Ser Ala Ser Thr Lys Gly Pro115 120

<210> 32

<211> 121

<212> PRT

<213> Homo sapiens

<4 00> 32Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Wing 5 10 15

Be Val Lys Val Be Cys Lys Val Be Gly Tyr Thr Phe Thr Asp Tyr20 25 30

Tyr Met His Trp Val Gln Gln Pro Wing Gly Lys Gly Leu Glu Trp Met35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Wing Glu Lys Phe50 55 60

Gln Gly Arg Val Thr Ile Thr Wing Asp Thr Be Asp Thr Wing Tyr65 70 75 80

Met Glu Read Be Ser Read Ar Be Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

Wing Asp Asp Asp Tyr Met Asp Val Trp Gly Lys Gly Thr Read Val Thr100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro115 120

<210> 33 <211> 330 <212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (330)

<4 00> 33

gaa acg aca ctc acg cag tct cca gac acc ctg tct ttg tct cca

Glu Thr Thr Read Thr Gln Be Pro Asp Thr Read Be Read Pro Gly15 10 15

gga gcc gcc acc ctc tcc tgt agg gcc agt cag agt gtt age ggc agtGlu Gly Wing Thr Leu Ser Cys Arg Wing Ser Gln Ser Val Ser Gly Ser20 25 30

gcg ttg gcc tgg tac cag aaa cct ggc cag gct ccc aga ctc ctcAla Leu Wing Trp Tyr Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu35 40 45

until tat gat gea tcc agt agg gcc act ggc gtc cca gac agg ttc agtIle Tyr Asp Ala Ser Ser Arg Ala Thr Gly Val Pro Asp Arg Phe Ser50 55 60

ggc agt ggg tcg ggg gea gac ttc agt ctc acc up aga ctg gagGly Ser Gly Ser Gly Ala Asp Phe Ser Leu Thr Ile Ser Arg Leu Glu65 70 75 80cct gaa gat ttt gca gtg tat tcc tgt cag caa tat ggt age tca cct 28

Pro Glu Asp Phe Wing Val Tyr Ser Cys Gln Gln Tyr Gly Ser Ser

85 90 95

ctc act ttc ggc cct ggg acc aaa gtg gat

Read Thr Phe Gly Pro Gly Thr Lys Val Asp

100 105 110

<210> 34

<211> 110

<212> PRT

<213> Homo sapiens

<400> 34

Glu Thr Thr Read Thr Gln Be Pro Asp Thr Read Be Read Pro Gly1 5 10 15

Glu Gly Wing Thr Read Be Cys Arg Wing Be Gln Be Val Ser Gly Ser20 25 30

Wing Leu Wing Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu35 40 45

Ile Tyr Asp Wing Ser Be Arg Wing Thr Gly Val Pro Asp Arg Phe Ser50 55 60

Gly Ser Gly Ser Gly Wing Asp Phe Be Read Thr Ile Be Arg Read Glu65 70 75 80

Pro Glu Asp Phe Wing Val Tyr Ser Cys Gln Tyr Gly Ser Ser Pro85 90 95

Read Thr Phe Gly Pro Gly Thr Lys Val Asp Val Lys Arg Thr100 105 110

<210> 35

<211> 334

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (333)

<400> 35

cag gtc cag ctg gtg cag tet ggg gct gag gtg aag aag cct ggg tcc 48

Gln Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Ser

1 5 10 15

tcg gtg aag gtc tcc tgc aag gct tet gga tcg acc ttc acc ggc tac 96

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ser Thr Phe Thr Gly Tyr20 25 30tat atg cac tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144

Tyr Met His Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

gga agg till cct till ctt ggt ata gea aac tac gea cag aag ttc 192

Gly Arg Ile Ile Pro Ile Read Gly Ile Wing Asn Tyr Wing Gln Lys Phe50 55 60

cag ggc aga gtc acg att acc gcg gac aaa tcc acg age aca gcc tac 240

Gln Gly Arg Val Thr Ile Thr Wing Asp Lys Be Thr Be Thr Wing

65 70 75 80

atg gag ctg age age ctg aga tet gag gac acg gcc gtg tac tac tgt 288

Met Glu Read Be Ser Read Ar Be Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

ggg aga gga gga gat ctg ggc ggt atg gac gtc tgg ggc caa ggg a 334

Wing Arg Gly Gly Asp Read Gly Gly Met Met Asp Val Trp Gly Gln Gly100 105 110

<210> 36

<211> 111

<212> PRT

<213> Homo sapiens

<4 00> 36

Gln Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Ser15 10 15

Be Val Lys Val Be Cys Lys Wing Be Gly Be Thr Phe Thr Gly Tyr20 25 30

Tyr Met His Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Arg Ile Ile Pro Ile Read Gly Ile Wing Asn Tyr Wing Gln Lys Phe50 55 60

Gln Gly Arg Val Thr Ile Thr Asp Wing Lys Be Thr Be Thr Wing Tyr65 70 75 80

Met Glu Read Be Ser Read Ar Be Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

Wing Arg Gly Gly Asp Read Gly Gly Met Met Asp Val Trp Gly Gln Gly100 105 110

<210> 37

<211> 342

<212> DNA

<213> Homo sapiens

<220> <221> CDS <222> (1) .. (342)

<400> 37

gaa att gtg ctg act cag tct cca ctc tcc ctg cct gtc acc cct gga 48

Glu Ile Val Leu Thr Gln Ile Pro Leu Be Valu Pro Gly1 5 10 15

gag ccg gcc tcc to tcc tgc agg tct agt cag age ctc cgg cat agt 96

Glu Pro Wing Be Ile Be Cys Arg Be Be Gln Be Read Arg His Ser20 25 30

aat gga tac aac tat ttg cat tgg tac cg cag aag cca ggg cag tct 14 4

Asn Gly Tyr Asn Tyr Read Asp Trp Tyr Read Gln Lys Pro Gly Gln Ser35 40 45

cca cag ctc ctg until tat ttg gct tct aat cgg gcc

Pro Gln Leu Leu Ile Tyr Leu Wing Be Asn Arg Wing Be Gly Val Pro50 55 60

gac agg ttc agt ggc agt gga tca ggc aca gat ttt aca ctg aaa until 240

Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read Lys Ile65 70 75 80

age aga gtg gag gct gag gat gtt ggg gtt tat tac tgc atg caa gct 288

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala85 90 95

cta caa att cct ccg act ttc ggc cct ggg acc aaa gtg gat by aaa 336

Read Gln Ile Pro Pro Thr Phe Gly Gly Thr Lys Val Asp Ile Lys100 105 110

cga act 342

Arg thr

<210> 38

<211> 114

<212> PRT

<213> Homo sapiens

<400> 38

Glu Ile Val Leu Thr Gln Ile Pro Leu Be Valu Pro Gly15 10 15

Glu Pro Wing Be Ile Be Cys Arg Be Be Gln Be Read Arg His Ser20 25 30

Asn Gly Tyr Asn Tyr Read Asp Trp Tyr Read Gln Lys Pro Gly Gln Ser35 40 45

Pro Gln Leu Leu Ile Tyr Leu Wing Be Asn Arg Wing Be Gly Val Pro50 55 60

Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Thr Read Lys Ile65 70 75 80Ser Arg Val Glu Wing Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala85 90 95

Read Gln Ile Pro Pro Thr Phe Gly Gly Thr Lys Val Asp Ile Lys100 105 110

Arg thr

<210> 39

<211> 372

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (372)

<400> 39

cag gtg cag ctg gtg gag tt ggc cca gga ctg gtg aag cct tcg gag 48

Gln Val Gln Leu Val Glu Phe Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15

acc ctg tcc ctc acc tgc act gtc tct ggt ggc tcc to agt agt tac 96

Thr Read Be Read Thr Cys Thr Val Be Gly Gly Be Ile Be Ser Tyr20 25 30

tac tgg age tgg till cgg cag ccc cca ggg aag gga ctg gag tgg att 144

Tyr Trp Being Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile35 40 45

ggg tat till tat tac agt ggg age acc aac tac aac ccc tcc ctc aag 192

Gly Tyr Ile Tyr Tyr Being Gly Being Thr Asn Tyr Asn Pro Being Read Lys50 55 60

agt cga gtc gcc ata tca gta gac acg tcc aag aac cag ttc tcc ctg 240

Ser Arg Val Wing Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80

aag ctg age tct gtg acc gcc gcg gac acg gcc gtg tat tac tgt gcg 288

Lys Leu Ser Ser Val Thr Wing Asp Thr Wing Val Tyr Tyr Cys Ala85 90 95

aga gag tat tat tat gat agt agt ggt tat tac ttt tat gct ttt gat 336

Arg Glu Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Phe Tyr Ala Phe Asp100 105 110

till tgg ggc caa ggg acc acg gtc acc gtc tca age 372

Ile Trp Gly Gln Gly Thr Thr Thr Thr Ser Ser 120

<210> 40

<211> 124

<212> PRT

<213> Homo sapiens

<400> 40Gln Val Gln Leu Val Glu Phe Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15

Thr Read Be Read Thr Cys Thr Val Be Gly Gly Be Ile Be Ser Tyr20 25 30

Tyr Trp Being Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile35 40 45

Gly Tyr Ile 'Tyr Tyr Be Gly Be Thr Asn Tyr Asn Pro Be Read Lys50 55 60

Ser Arg Val Wing Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80

Lys Leu Ser Ser Val Thr Wing Asp Thr Wing Val Tyr Tyr Cys Ala85 90 95

Arg Glu Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Phe Tyr Ala Phe Asp100 105 110

Ile Trp Gly Gln Gly Thr Thr Thr Thr Ser Ser 120

<210> 41

<211> 333

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (333)

<4 00> 41

ctg cct gtg ctg act cag ccc tcLeu Pro Val Leu Thr Pro Gln Pro Ser1 5

gcg tct ggg acc ccc ggg cag 4 8

Wing Gly Thr Pro Gly Gln10 15

agg gtc tcc to tct tgt tct gga age age tcc aac to gga agt aat 96

Arg Val Be Ile Be Cys Be Gly Be Be Asn Ile Gly Be Asn

20 25 30

tat gta tac tgg tac cag cag ctc cca gga acg gcc ccc aaa ctc ctc 144

Tyr Val Tyr Trp Tyr Gln Glu Leu Pro Gly Thr Wing Pro Lys Leu Leu

35 40 45

until ttt agg aat aat cag cgg ccc tca ggg gtc cct gac cga ttc tct 192

Ile Phe Arg Asn Asn Gln Arg Pro Be Gly Val Pro Asp Arg Phe Be

50 55 60

ggc tcc aag tct ggc act tca gcc tcc ctg gcc until agt ggg ctc cggGly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ile Ser Gly Leu Arg65 70 75 80

240tcc gag gat gag gct gat tat tac tgt gca gca tgg gat gac age ctg 288

Be Glu Asp Glu Wing Asp Tyr Tyr Cys Wing Wing Trp Asp Asp Ser Leu85 90 95

agt ggt tgg gtg ttc ggc gga ggg acc aag ctg acc gtc cta ggt 333

Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly100 105 110

<210> 42

<211> 111

<212> PRT

<213> Homo sapiens

<400> 42

Leu Pro Val Leu Thr Pro Gln Pro Pro Wing Be Gly Thr Pro Gly Gln1 5 10 15

Arg Val Be Ile Be Cys Be Gly Be Be Asn Ile Gly Be Asn20 25 30

Tyr Val Tyr Trp Tyr Gln Glu Leu Pro Gly Thr Wing Pro Lys Leu Leu35 40 45

Ile Phe Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser50 55 60

Gly Be Lys Be Gly Thr Be Wing Be Read Wing Wing Ile Be Gly Read Arg65 70 75 80

Be Glu Asp Glu Wing Asp Tyr Tyr Cys Wing Wing Trp Asp Asp Ser Leu85 90 95

Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly100 105 110

<210> 43

<211> 381

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (381)

<400> 43

cag gtg cag ctg cag gag tcc ggc cca gga ctg gtg aag cct tcg gag 48

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu1 5 10 15

by ctg tcc ctc acc tgc act gtc tet ggt ggc tcc by agt agt cac 96

Ile Leu Be Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Be Ser His His20 25 30tac tgg agt tgg gtc cgg cag ccc ggg aag gga ctg gag tgg att 144

Tyr Trp Being Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile35 40 45

ggg tac till tat tac agt ggg age acc aac tac aac ccc tcc ctc aag 192

Gly Tyr Ile Tyr Tyr Being Gly Being Thr Asn Tyr Asn Pro Being Read Lys50 55 60

agt cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg 240

Be Arg Val Thr Ile Be Val Asp Thr Be Lys Asn Gln Phe Be Leu

65 70 75 80

aac ctg age tet gtg acc gct gcg gac acg gcc gtg tat tat tgt gcg

Asn Leu Be Ser Val Thr Wing Asp Thr Wing Val Tyr Tyr Cys Wing

85 90 95

aga att ccc aac tac tat gat agt ggt tat tat ccc ggt tac tgg 336

Arg Ile Pro Asn Tyr Tyr Asp Arg Be Gly Tyr Tyr Pro Gly Tyr Trp

100 105 110

tac ttc gat cg tgg ggc cgt ggc acc ctg gtc acc gtc tca age 381

Tyr Phe Asp Read Trp Gly Arg Gly Thr Read Val Val Val Ser Ser 120 120 125

<210> 44

<211> 127

<212> PRT

<213> Homo sapiens

<4 00> 44

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu15 10 15

Ile Read Be Read Thr Cys Thr Val Be Gly Gly Be Ile Be Be His20 25 30

Tyr Trp Being Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile35 40 45

Gly Tyr Ile Tyr Tyr Being Gly Being Thr Asn Tyr Asn Pro Being Read Lys50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80

Asn Leu Be Ser Val Thr Wing Asp Thr Wing Val Tyr Tyr Cys Ala85 90 95

Arg Ile Pro Asn Tyr Tyr Asp Arg Be Gly Tyr Tyr Pro Gly Tyr Trp100 105 110

Tyr Phe Asp Read Trp Gly Arg Gly Thr Read Val Val Val Ser Ser115 120 125 <210> 45

<211> 345

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (345)

<400> 45

cag gct gtg ctg act cag ccg tct tcc ctc tct gca cct cct gga gca 48

Gln Wing Val Leu Thr Gln Pro Be Being Read Be Wing Pro Pro Gly Wing 10 15 15

tca gcc agt ctc acc tgc acc ttg cgc agt gtc ttc aat gtt gat tcc 96

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Phe Asn Val Asp Ser20 25 30

tac agg ata tcc tgg tac cag cag aag cca ggg agt cct ccc cag tat 144

Tyr Arg Ile Ser Trp Tyr Gln Gln Lys Pro

35 40 45

ctc ctg agg tac aaa tca gac tca gat aag cag cag ggc tct gga gtc 192

Leu Leu Arg Tyr Lys Be Asp Be Asp Lys Gln Gln Gly Be Gly Val50 55 60

ccc age cgc ttc tct gga tcc aaa gat gct tcg gcc aat gca ggg att 240

Pro Be Arg Phe Be Gly Be Lys Asp Wing Be Wing Asn Wing Gly Ile65 70 75 80

tta ctc up to tct ggg ctc cag tct gag gat gag gct gac tat tac tgt 288

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Wing Asp Tyr Tyr Cys

85 90 95

atg att tgg cac age age gct tgg gtg ttc ggc gga ggg acc aag ctg 336

Met Ile Trp His Ser Be Trp Wing Val Phe Gly Gly Gly Thr Lys Leu100 105 110

acc gtc cta 345

Thr Val Leu115

<210> 46

<211> 115

<212> PRT

<213> Homo sapiens

<4 00> 46

Gln Wing Val Leu Thr Gln Pro Be Being Read Be Wing Pro Pro Gly Wing 10 15 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Phe Asn Val Asp Ser20 25 30

Tyr Arg Ile Be Trp Tyr Gln Lys Pro Gly Be Pro Pro Gln Tyr35 40 45Leu Read Arg Tyr Lys Be Asp Ser Asp Lys Gln Gly Ser Gly Val50 55 60

Pro Be Arg Phe Be Gly Be Lys Asp Wing Be Wing Asn Wing Gly Ile65 70 75 80

Leu Leu Ile Be Gly Leu Gln Be Glu Asp Glu Wing Asp Tyr Tyr Cys85 90 95

Met Ile Trp His Ser Be Trp Wing Val Phe Gly Gly Gly Thr Lys Leu100 105 110

Thr Val Leu115

<210> 47

<211> 372

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (372)

<400> 47

gag gtc cag ctg gtg cag tct ggg ggaGlu Val Gln Leu Val Gln Ser Gly Gly1 5

ggc ttg gtc aag cct gga ggg 48

Gly Leu Val Lys Pro Gly Gly10 15

tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt age tat 96

Be Read Arg Read Be Cys Wing Wing Be Gly Phe Thr Phe Be Tyr20 25 30

ggt atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg

Wing Met His Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val35 40 45

gca gtt ata tca tat gat gga agt aat aaa tac tac gca gac tcc gtg 192

Wing Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Wing Asp Ser Val

50 55 60

aag ggc cga ttc acc up to tcc aga gac aat tcc aag aac acg ctg tat 240

Lys Gly Arg Phe Thr Ile Be Arg Asp Asn Be Lys Asn Thr Read Tyr65 70 75 80

ctg caa atg aac age ctg aga gct gag gac acg gct gtg tat tac tgt 288

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys

85 90 95

gcg agg ttc agt ggc tgg cc aac aac tac tac tac tac ggt atg gac 336

Wing Arg Phe Ser Gly Trp Pro Asn Asn Tyr Tyr Tyr Tyr Gly Met Asp100 105 110

gtc tgg ggc caa ggg acc acg gtc acc gtc tca age 372

Val Trp Gly Gln Gly Thr Thr

120

<210> 48

<211> 124

<212> PRT

<213> Homo sapiens

<400> 48

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly15 10 15

Be Read Arg Read Be Cys Wing Wing Be Gly Phe Thr Phe Be Tyr20 25 30

Wing Met His Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val35 40 45

Wing Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Wing Asp Ser Val50 55 60

Lys Gly Arg Phe Thr Ile Be Arg Asp Asn Be Lys Asn Thr Read Tyr65 70 75 80

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

Wing Arg Phe Ser Gly Trp Pro Asn Asn Tyr Tyr Tyr Tyr Gly Met Asp100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser115 120

<210> 49

<211> 339

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (339)

<4 00> 49

gat gtt gtg atg act cag tct cca ctc tcc ctg ccc gtc acc cct gga 48

Asp Val Val Met Thr Gln Be Pro Read Be Read Pro Val Thr Pro Gly15 10 15

gag ccg gcc tcc to tcc tgc agg tctGlu Pro Ala Ser Ile Ser Cys Arg Ser20 25

aat gga ttc aac gtg gat tgg tacAsn Gly Phe Asn Tyr Val Asp Trp Tyr

agt cag age ctc ctg cat agt 96

Ser Gln Ser Read Leu His Ser30

ctg cag aag cca ggg cag tct 144

Read Gln Lys Pro Gly Gln Sercca cac ctc ttg up to ttc ggt tet tat cgg gcc tcc ggg gtc cct 192

Pro His Leu Read Ile Tyr Phe Gly Be Tyr Arg Wing Be Gly Val Pro50 55 60

gac agg ttc agt ggc agt gga tca ggc aca gat ttt aca ctg aaa until 240

Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read Lys Ile65 70 75 80

age aga gtg gag gct gag gat gtt ggg gtt tat tac tgc atg caa gct 288

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala85 90 95

ctg caa act cct ccc tgg acg ttc ggc caa ggg acc aag gtg

Read Gln Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile100 105 110

now 339

Arg

<210> 50

<211> 113

<212> PRT

<213> Homo sapiens

<4 00> 50

Asp Val Val Met Thr Gln Be Pro Read Be Read Pro Val Thr Pro Gly15 10 15

Glu Pro Wing Be Ile Be Cys Arg Be Be Gln Be Read His Read20 25 30

Asn Gly Phe Asn Tyr Val Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser35 40 45

Pro His Leu Read Ile Tyr Phe Gly Be Tyr Arg Wing Be Gly Val Pro50 55 60

Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read Lys Ile65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala85 90 95

Read Gln Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile100 105 110

Arg

<210> 51 <211> 369 <212> DNA <213> Homo sapiens

<220>

<221> CDS <222> (1) .. (369)

<4 00> 51

cag gta cag ctg cag cag tca ggt cca gga ctg

Gln Val Gln Leu Gln Gln Be Gly Pro Gly Leu Val Lys Pro Be Gln15 10 15

acc ctc tca ctc acc tgt gcc until tcc ggg gac aat gtc tet age aac 96

Thr Read Be Read Thr Cys Wing Ile Be Gly Asp Asn Val Be Ser Asn20 25 30

agt gct act tgg aat tgg until agg cag tcc cca tcg aga ggc ctt gag 144

Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Be Arg Gly Leu Glu35 40 45

tgg ctg gga agg tac tac agg tcc aag tgg tat aat gag tat gea 192

Trp Read Gly Arg Thr Tyr Tyr Arg Be Lys Trp Tyr Asn Glu Tyr Ala50 55 60

gta tet gtg aaa agt cga ata acc till aac gga gac aca tcc aag aac 240

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Gly Asp Thr Be Lys Asn65 70 75 80

cag ttc tc ctg cag ttg aac tet gtg act ccc gac gac acg gct gta 288

Gln Phe Be Read Gln Read Asn Be Val Thr Pro Asp Asp Thr Wing Val85 90 95

tat tat tgt gea aga gga tat aga gag age cac tgg acc tat gac tac 336

Tyr Tyr Cys Arg Wing Gly Tyr Arg Glu Be His Trp Thr Tyr Asp Tyr100 105 110

tgg ggc ctg gg acc ctg gtc acc gtc tca age 369

Trp Gly Leu Gly Thr Leu Val Thr Val Ser Ser115 120

<210> 52

<211> 123

<212> PRT

<213> Homo sapiens

<4 00> 52

Gln Val Gln Leu Gln Gln Be Gly Pro Gly Leu Val Lys Pro Be Gln15 10 15

Thr Read Be Read Thr Cys Wing Ile Be Gly Asp Asn Val Be Ser Asn20 25 30

Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Be Arg Gly Leu Glu35 40 45

Trp Read Gly Arg Thr Tyr Tyr Arg Be Lys Trp Tyr Asn Glu Tyr Ala50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Gly Asp Thr Be Lys Asn65 70 75 80

Gln Phe Be Read Gln Read Asn Be Val Thr Pro Asp Asp Thr Wing Val85 90 95

Tyr Tyr Cys Arg Wing Gly Tyr Arg Glu Be His Trp Thr Tyr Asp Tyr100 105 110

Trp Gly Leu Gly Thr Leu Val Thr Val Ser Ser115 120

<210> 53

<211> 327

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1). . (327)

<4 00> 53

gaa acg aca ctc acg cag tct cca ggg acc ctg tca ttg tct cca ggg 48

Glu Thr Thr Read Thr Gln Be Pro Gly Thr Read Le Be Pro Gly15 10 15

gaa agg ggc acc ctc tcc tgc agg tcc agt cag agt gtt acc aga aac 96

Glu Arg Gly Thr Read Be Cys Arg Be Be Gln Be Val Thr Arg Asn20 25 30

tac ttg gcc tgg tac cag cg aaa gct ggc cag gct ccc agg ctc ctc 144

Tyr Leu Wing Trp Tyr Gln Gln Lys Wing Gly Gln Wing Pro Arg Leu Leu35 40 45

till ggt gea tcc act agg gcc act ggc till cca ctc agg ttc agt 192

Ile Tyr Gly Wing Ser Be Arg Wing Thr Gly Ile Pro Read Arg Phe Ser50 55 60

ggc agt ggg tct ggg aca gac ttc act ctc acc till age gga ctg gag 240

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Glu65 70 75 80

cct gaa gat tt gea gtg tat ttc tgt cag cag tat gag till tca ccg 288

Pro Glu Asp Phe Wing Val Tyr Phe Cys Gln Gln Tyr Glu Ile Ser Pro85 90 95

ccc act ttc ggc gga ggg acc aag gtc gag until aaa cga 327

Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg100 105

<210> 54

<211> 109

<212> PRT

<213> Homo sapiens <400> 54

Glu Thr Thr Read Thr Gln Be Pro Gly Thr Read Le Be Pro Gly1 5 10 15

Glu Arg Gly Thr Read Be Cys Arg Be Be Gln Be Val Thr Arg Asn20 25 30

Tyr Leu Wing Trp Tyr Gln Gln Lys Wing Gly Gln Wing Pro Arg Leu Leu35 40 45

Ile Tyr Gly Wing Ser Be Arg Wing Thr Gly Ile Pro Read Arg Phe Ser50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Glu65 70 75 80

Pro Glu Asp Phe Wing Val Tyr Phe Cys Gln Gln Tyr Glu Ile Ser Pro85 90 95

Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg100 105

<210> 55

<211> 360

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (360)

<400> 55

cag gtg cag ctg cag gag tcc ggc cca gga ctg gtg aag cct tcg gag 48

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu1 5 10 15

acc ctg tcc ctc acc tgc act gtc tct ggt ggc tcc until age age agt 96

Thr Read Be Read Thr Cys Thr Val Be Gly Gly Be Ile Be Ser20 25 30

tat tac tac tgg ggc tgg till cga cag ccc cca ggg aag ggg ctg gag 144

Tyr Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu35 40 45

tgg att ggg agt till tat tat act ggg age age tac tac aac ccg tca 192

Trp Ile Gly Ser Ile Tyr Tyr Thr Gly Ser Tyr Tyr Asn Pro Ser50 55 60

ctc aag agt cga gtc acc ata tcc tta gac acg tcc aag aat gag ttc 240

Read Lys Be Arg Val Thr Ile Be Read Asp Thr Be Lys Asn Glu Phe

65 70 75 80

tcc ctc aag ctg age tct gtg acc gcc gea gac acg gcc gtg tat tac 288Ser Leu Lys Leu Ser Ser Val Thr Ala Asp Thr Ala Val Tyr Tyr85 90 95

tgt gcg aga gcg ggt ggc agt tcg acc tgg ttc gac ccc tgg ggc cag 336

Cys Wing Arg Wing Gly Gly Be Being Thr Trp Phe Asp Pro Trp Gly Gln100 105 110

ggc acc ctg gtc acc

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 56 <211> 120 <212> PRT <213> Homo

<400> 56

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu1 5 10 15

Thr Read Be Read Thr Cys Thr Val Be Gly Gly Be Ile Be Ser20 25 30

Tyr Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Thr Gly Ser Tyr Tyr Asn Pro Ser50 55 60

Read Lys Be Arg Val Thr Ile Be Read Asp Thr Be Lys Asn Glu Phe65 70 75 80

Be Leu Lys Leu Be Ser Val Thr Wing Asp Thr Wing Val Tyr Tyr85 90 95

Cys Wing Arg Wing Gly Gly Be Being Thr Trp Phe Asp Pro Trp Gly Gln100 105 110

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 57 <211> 339 <212> DNA <213> Homo sapiens

<220>

<221> CDS <222> (1) .. (339)

<400> 57

gat gtt gtg atg act cag tet cca ctc tcc ctg ccc gtc acc cet gga 48Asp Val Val Met Thr Gln Ser Pro Leu Pro Le Val Pro Pro Gly

1 5 10 15

gag ccg gcc tcc to tcc tgc agg tet agt cgg age ctc ctg cat agt 96

Glu Pro Wing Be Ile Be Cys Arg Be Be Arg Be Read Leu His Ser20 25 30

aat gga tac aac tat ttg gat tgg tac ctg cag

Asn Gly Tyr Asn Tyr Read Asp Trp Tyr Read Gln Lys Pro Gly Gln Ser

35 40 45

cca cag ctc ctg until tat ttg ggt tet aat cgg gcc tcc ggg gtc cct 192

Pro Gln Leu Leu Ile Tyr Leu Gly Be Asn Arg Wing Be Gly Val Pro50 55 60

gac agg ttc agt ggc agt gga tca ggc aca gat ttt aca ctg aaa until 240

Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Gly Read Lys Ile

65 70 75 80

age aga gtg gag gct gag gat gtt gga gtt tat tac tgc atg caa gct 288

Ser Arg Val Glu Wing Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Wing

85 90 95

cta cga ctg act ctg act ttc ggc cct ggg acc aaa gtg gat by aaa 336

Read Arg Thr Pro Read Le Phe Gly Pro Gly Thr Lys Val Asp Ile Lys100 105 110

cga 339

Arg

<210> 58

<211> 113

<212> PRT

<213> Homo sapiens

<400> 58

Asp Val Val Met Thr Gln Be Pro Read Be Read Pro Val Thr Pro Gly15 10 15

Glu Pro Wing Be Ile Be Cys Arg Be Be Arg Be Read Leu His Ser20 25 30

Asn Gly Tyr Asn Tyr Read Asp Trp Tyr Read Gln Lys Pro Gly Gln Ser35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Be Asn Arg Wing Be Gly Val Pro50 55 60

Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read Lys Ile65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala85 90 95Leu Arg Thr Pro Read Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys100 105 110

Arg

<210> 59

<211> 351

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (351)

<400> 59

gag gtc cag ctg gtg cag tct ggg gga ggc ttg

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly15 10 15

tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc gtc

Be Read Arg Read Be Cys Wing Wing Be Gly Phe Thr Val Be Asn20 25 30

tac gtg age tgg gtc cgc cag gct cca ggg aag ggg ctg

Tyr Val Ser Trp Val Arg, Gln Pro Wing Gly Lys Gly Leu Glu Trp Val35 40 45

tca gtt att tat age ggt ggt age aca tac tac gca gac tcc gtg aag 192

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Wing Asp Ser Val Lys50 55 60

ggc aga ttc acc until tcc aga gac aat tcc aag aac acg ctg tat ctt 240

Gly Arg Phe Thr Ile Be Arg Asp Asn Be Lys Asn Thr Leu Tyr Leu65 70 75 80

caa atg aac age ctg aga gcc gag gac acg gct gtg tat tac tgt gcg 288

Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys Ala85. 90. 95

ggc ca <? tgg ctg ggg ggg ttt gac tac tgg ggc cag gga acc ctg 336

Arg Gly Gln Trp Read Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110

gtc acc gtc tca age 351

Val Thr Val Ser Ser115

<210> 60

<211> 117

<212> PRT

<213> Homo sapiens

<4 00> 60

Pro Gly Gly15

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln1 5 10Ser Leu Arg Leu Ser Cys Wing Ser Gly Phe Thr Val Ser Asn20 25 30

Tyr Val Ser Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Wing Asp Ser Val Lys50 55 60

Gly Arg Phe Thr Ile Be Arg Asp Asn Be Lys Asn Thr Leu Tyr Leu65 70 75 80

Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys Ala85 90 95

Arg Gly Gln Trp Read Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110

Val Thr Val Ser Ser115

<210> 61

<211> 327

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (327)

<4 00> 61

gaa acg aca ctc acg cg tct cca ggg acc ctg tct ttg tct cca

Glu Thr Thr Read Thr Gln Be Pro Gly Thr Read Le Be Pro Gly1 5 .10 15

gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt age age age 96

Glu Arg Wing Thr Read Le Be Cys Arg Wing Be Gln Be Val Be Ser20 25 30

aac tta gcc tgg tac cag c aaa cct ggc cag gct ccc agg ctc ctc 144

Asn Leu Wing Trp Tyr Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu35 40 45

till ggt gea tcc act agg gcc act ggc till cca gac agg ttc agt 192

Ile Tyr Gly Ala Ser Be Arg Wing Thr Gly Ile Pro Asp Arg Phe Ser50 55 60

ggc agt ggg tct ggg aca gac ttc act ctc acc until age aga ctg gag 240

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Be Arg Leu Glu65 70 75 80

cct gaa gat ttt gea gtg tac tac tgt cag cag tat ggt age tca ctt 288

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu85 90 95tgg acg ttc ggc caa ggg acc aag gtg gaa aaa cga 327

Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg100 105

<210> 62

<211> 109

<212> PRT

<213> Homo sapiens

<400> 62

Glu Thr Thr Read Thr Gln Be Pro Gly Thr Read Le Be Pro Gly15 10 15

Glu Arg Wing Thr Read Le Be Cys Arg Wing Be Gln Be Val Be Ser20 25 30

Asn Leu Wing Trp Tyr Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu35 40 45

Ile Tyr Gly Ala Ser Be Arg Wing Thr Gly Ile Pro Asp Arg Phe Ser50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Be Arg Leu Glu65 70 75 80

Pro Glu Asp Phe Wing Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu85 90 95

Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg100 105

<210> 63

<211> 360

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (360)

<4 00> 63

gag gtc cag ctg gta cag tet gga gctGlu Val Gln Leu Val Gln Ser Gly Ala1 5

gag gtg aag aag cct ggg gcc 48

Glu Val Lys Pro Lys Gly Ala10 15

tca gtg aag gtc tcc tgc aag gct tet ggt tac acc ttt acc age tat 96

Be Val Lys Val Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Be Tyr20 25 30

ggt till age tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144

Gly Ile Ser Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45gga tgg till age gct tac ag ggt aac aac tat gea cag aag ctc 192

Gly Trp Ile Be Wing Tyr Asn Gly Wing Asn Tyr Wing Gln Lys Leu50 55 60

cag ggc aga gtc acc atg acc aca gac aca tcc acg age aca gcc tac 240

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Be Thr Be Wing Tyr65 70 75 80

atg gag ctg agg age ctg aga tet gac gac acg gcc gtg tat tac tgt 288

Met Glu Read Arg Be Read Arg Be Asp Asp Thr Wing Val Tyr Tyr Cys85 90 95

ggg aga gga tac ggg age tac caa aaa cgt ttt gac tac tgg ggc cag 336

Wing Arg Gly Tyr Gly Ser Tyr Gln Lys Arg Phe Asp Tyr Trp Gly Gln100 105 110

ggc acc ctg gtc acc

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 64

<211> 120

<212> PRT

<213> Homo sapiens

<4 00> 64

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Wing 10 15 15

Be Val Lys Val Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Be Tyr20 25 30

Gly Ile Ser Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Trp Ile Be Wing Tyr Asn Gly Wing Asn Tyr Wing Gln Lys Leu50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Be Thr Be Wing Tyr65 70 75 80

Met Glu Read Arg Be Read Arg Be Asp Asp Thr Wing Val Tyr Tyr Cys85 90 95

Wing Arg Gly Tyr Gly Ser Tyr Gln Lys Arg Phe Asp Tyr Trp Gly Gln100 105 110

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 65 <211> 384 <212> DNA <213> Homo sapiens

<220>

<221> CDS <222> (1) .. (384)

<400> 65

gat att gtg atg acc cac act cca ctc tct ctg tcc gtc acc cct gga 48

Asp Ile Val Met Thr His Thr Pro Read Be Read Be Val Thr Pro Gly1 5 10 15

cag ccg gcc tcc to tcc tgc aag tct agt cag age ctc ctg cat agt 96

Gln Pro Wing Be Ile Be Cys Lys Be Be Gln Be Read His Read Ser20 25 30

gat gga aag acc ttg tat tgg tac ctg cag aag cca ggc cag cct 144

Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro35 40 45

cca cag ctc ctg until tat gaa gtt tcc aac cgg ttc tct gga gtg cca 192

Pro Gln Read Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro50 55 60

gag agg ttc agt ggc age ggg tca ggg aca gct ttc aca ctg aaa to 240

Glu Arg Phe Be Gly Be Gly Be Gly Thr Wing Phe Thr Read Lys Ile65 70 75 80

age agg gtg gag gct gag gat gtt gga gtt tat tac tgc atg caa cgt 288

Ser Arg Val Glu Wing Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Arg85 90 95

ata gag ttt ccg ccc cct cac ttt cgg cgg agg gac caa ggt gga gat 336

Ile Glu Phe Pro Pro Pro His Phe Arg Arg Arg Asp Gln Gly Gly Asp100 105 110

caa acg aac tgt ggc tgc acc up to tgt ctt cat ctt ccc gcc up to tga 384

Gln Thr Asn Cys Gly Cys Thr Ile Cys Read His Read Pro Ala Ile115 120 125

<210> 66

<211> 127

<212> PRT

<213> Homo sapiens

<4 00> 66

Asp Ile Val Met Thr His Thr Pro Read Be Read Be Val Thr Pro Gly1 5 10 15

Gln Pro Wing Be Ile Be Cys Lys Be Be Gln Be Read His Read Ser20 25 30

Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro35 40 45

Pro Gln Read Leu Ile Tyr Glu Val Ser Asn Arg Phe Be Gly Val Pro50 55 60Glu Arg Phe Ser Gly Be Gly Ser Gly Thr Wing Phe Thr Leu Lys Ile65 70 75 80

Ser Arg Val Glu Wing Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Arg85 90 95

Ile Glu Phe Pro Pro Pro His Phe Arg Arg Arg Asp Gln Gly Gly Asp100 105 110

Gln Thr Asn Cys Gly Cys Thr Ile Cys Read His Read Pro Ala Ile115 120 125

<210> 67

<211> 360

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (360)

<400> 67

gag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg gcc 4 8

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Wing 5 10 15

tca gtg aag gtc ttc tgc aag gct ttc gga tac acc ttc acc ggc tac 96

Be Val Lys Val Phe Cys Lys Wing Be Gly Tyr Thr Phe Thr Gly Tyr20 25 30

tat atg cac tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144

Tyr Met His Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

gga tgg until aac cct aac agt ggt ggc aca aac tat gea cag aag ttt 192

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Wing Gln Lys Phe50 55 60

cag ggc agg gtc acc atg acc agg gac acg tcc until age aca gcc tac 240

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Be Ile Be Thr Wing Tyr65 70 75 80

atg gag ctg age agg ctg aga tct gac gac acg gcc gtg tat tac tgt 288

Met Glu Read Be Arg Read Be Arg Asp Asp Thr Wing Val Tyr Tyr Cys85 90 95

gcg aga gct cct tgg agt ggt cgc aga cgc ttt gac tac tgg ggc cag 336

Wing Arg Wing Pro Trp Be Gly Arg Arg Arg Phe Asp Tyr Trp Gly Gln100 105 110

gga acc ctg gtc acc gtc tca age 360

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 68 <211> 120 <212> PRT

<213> Homo sapiens

<400> 68

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Wing 5 10 15

Be Val Lys Val Phe Cys Lys Wing Be Gly Tyr Thr Phe Thr Gly Tyr20 25 30

Tyr Met His Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Wing Gln Lys Phe50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Be Ile Be Thr Wing Tyr65 70 75 80

Met Glu Read Be Arg Read Be Arg Asp Asp Thr Wing Val Tyr Tyr Cys85 90 95

Wing Arg Wing Pro Trp Be Gly Arg Arg Arg Phe Asp Tyr Trp Gly Gln100 105 110

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 69

<211> 12

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1) .. (12)

<4 00> 69cag gcg tgg tgaGln Wing Trp1

<210> 70

<211> 360

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (360) <4 00> 70

gag gtg cag ctg gtg cag tct gag gct gag gtg

Glu Val Gln Leu Val Gln Ser Glu Wing Glu Val Lys Lys Pro Gly Ala15 10 15

tca gtg aag gtt tcc tgc aag gct tct gga tac acc ttc act age tatSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr20 25 30

gct atg cat tgg gtg cgc cag gcc ccc gga caa agg ctt gag tgg atgAla Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met35 40 45

gga tgg till aac gct ggc aat ggt aac aca aaa tat tca cag aag ttcGly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe50 55 60

cag ggc aga gtc acc att acc agg gac acg tcc till age aca gcc tacGln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80

atg gag ctg age agg ctg aga tct gac gac acg gcc gtg tat tac tgtMet Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys85 90 95

ggg aga gga tac ggg age tac caa aaa cgt ttt gac tac tgg ggc cagAla Arg Gly Tyr Gly Ser Tyr Gln Lys Arg Phe Asp Tyr Trp Gly Gln100 105 110

ggc acc ctg gtc acc gtc tca ageGly Thr

<210> 71

<211> 120

<212> PRT

<213> Homo sapiens

<4 00> 71

Glu Val Gln Leu Val Gln Ser Glu Wing Glu Val Lys Lys Pro Gly Ala15 10 15

Be Val Lys Val Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Be Tyr20 25 30

Wing Met His Trp Val Arg Gln Pro Wing Gly Gln Arg Leu Glu Trp Met35 40 45

Gly Trp Ile Asn Wing Gly Asn Gly Asn Thr Lys Tyr Be Gln Lys Phe50 55 60

Gln Gly Arg Val Thr Thr Ile Thr Thr Asp Thr Be Ile Be Thr Wing Tyr65 70 75 80

Met Glu Leu Be Arg Leu Arg Be Asp Asp Thr Wing Val Tyr Tyr Cys85 90 95Ala Arg Gly Tyr Gly Be Tyr Gln Lys Arg Phe Asp Tyr Trp Gly Gln100 105 110

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 72

<211> 33

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (33)

<4 00> 72

cag gtc caa ctg cag gag ctc gag until aaa cgt 33

Gln Val Gln Leu Gln Glu Leu Glu Ile Lys Arg1 5 10

<210> 73

<211> 11

<212> PRT

<213> Homo sapiens

<4 00> 73

Gln Val Gln Leu Gln Glu Leu Glu Ile Lys Arg15 10

<210> 74

<211> 360

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (360)

<4 00> 74

gag gtc cag ctg gta cag tet gga gct gag gtg aag aag cct ggg gcc 48

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Wing 10 15 15

tca gtg aag gtc tcc tgc aag gct tet ggt tac acc ttt acc age tat 96

Be Val Lys Val Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Be Tyr

20 25 30

ggt till age tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg

Gly Ile Ser Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

gga tgg till age gct tac ag ggt aac aca aac tat gea cag aag ctc 192

Gly Trp Ile Ser Wing Tyr Asn Gly Asn Thr Asn Tyr Wing Gln Lys Leu50 55 60cag ggc aga gtc acc atg acc aca gac aca tcc acg age aca gcc tac 240

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Be Thr Be Wing Tyr65 70 75 80

atg gag ctg agg age ctg aga tet gac gac acg gcc gtg tat tac tgt 288

Met Glu Read Arg Be Read Arg Be Asp Asp Thr Wing Val Tyr Tyr Cys85 90 95

ggg aga gga tac ggg age tac caa aaa cgt ttt gac tac tgg ggc cag 336

Wing Arg Gly Tyr Gly Ser Tyr Gln Lys Arg Phe Asp Tyr Trp Gly Gln100 105 110

ggc acc ctg gtc acc

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 75

<211> 120

<212> PRT

<213> Homo sapiens

<4 00> 75

Glu Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys Pro Gly Wing 10 15 15

Be Val Lys Val Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Be Tyr20 25 30

Gly Ile Ser Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Met35 40 45

Gly Trp Ile Be Wing Tyr Asn Gly Wing Asn Tyr Wing Gln Lys Leu50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Be Thr Be Wing Tyr65 70 75 80

Met Glu Read Arg Be Read Arg Be Asp Asp Thr Wing Val Tyr Tyr Cys85 90 95

Wing Arg Gly Tyr Gly Ser Tyr Gln Lys Arg Phe Asp Tyr Trp Gly Gln100 105 110

Gly Thr Read Val Val Val Ser Ser 120 120

<210> 76

<211> 33

<212> DNA

<213> Homo sapiens <220>

<221> CDS <222> (1) .. (33)

<400> 76

cag gtc caa ctg cag gag ctc gag until aaa cgt 33

Gln Val Gln Leu Gln Glu Leu Glu Ile Lys Arg1 5 10

<210> 77

<211> 11

<212> PRT

<213> Homo sapiens

<4 00> 77

Gln Val Gln Leu Gln Glu Leu Glu Ile Lys Arg15 10

<210> 78

<211> 351

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1) .. (351)

<4 00> 78

cag ctg cag ctg cag gag tcg ggg gga ggc ttg gtc cag cct ggg ggg 48

Gln Leu Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

15 10 15

tcc ctg aaa ctc tcc tgt gea gcg tet gga ttc acc ttc agt age tat 96

Be Leu Lys Leu Be Cys Wing Ala Be Gly Phe Thr Phe Be Tyr20 25 30

ggc atg cac tgg gtc cg cg gct cca ggc aag ggg ctg gag tgg gtg 14 4

Gly Met His Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val35 40 45

gea gtt ata tgg tat gat gga agt aat aaa tac tat gea gac tcc gtg 192

Wing Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Wing Asp Ser Val

50 55 60

aag ggc cga ttc acc up to tcc aga gac aat tcc agg aac acg ctg tat 240

Lys Gly Arg Phe Thr Ile Be Arg Asp Asn Be Arg Asn Thr Read Tyr65 70 75 80

ctg caa atg aac age ctg aga gcc gag gac acg gct gtg tat tac tgt 288

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys

85 90 95

gcg act tat cct tt gga gtg gtt cac aac tgg ggc cag ggc acc ctg 336

Tyr Pro Wing Phe Gly Val Val His Asn Trp Gly Gln Gly Thr Leu100 105 110

gtc acc gtc tca age 351

Val Thr Val Ser Ser115 <210> 79

<211> 117

<212> PRT

<213> Homo sapiens

<4 00> 79

Gln Leu Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly15 10 15

Be Leu Lys Leu Be Cys Wing Ala Be Gly Phe Thr Phe Be Tyr20 25 30

Gly Met His Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val35 40 45

Val Ile Wing Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Wing Asp Ser Val50 55 60

Lys Gly Arg Phe Thr Ile Be Arg Asp Asn Be Arg Asn Thr Read Tyr65 70 75 80

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys85 90 95

Tyr Pro Wing Phe Gly Val Val His Asn Trp Gly Gln Gly Thr Leu100 105 110

Val Thr Val Ser Ser115

<210> 80

<211> 339

<212> DNA

<213> Homo sapiens

<220>

<221> CDS <222> (1) .. (339)

<400> 80

gaa att gtg ctg act cag tct cca ctc tcc ctg cct gtc acc cct gga 48

Glu Ile Val Leu Thr Gln Ile Pro Leu Be Valu Pro Gly15 10 15

gag ccg gcc tcc to tcc tgc agg tct agt cag age ctc ctg cat agt 96

Glu Pro Wing Be Ile Be Cys Arg Be Be Gln Be Read His Read20 25 30

aat gga ttc aac ttg gat tgg tac ctg cag aag cca ggg cag tct 144

Asn Gly Phe Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser35 40 45cca cac ctc ttg till tat ttc ggt tet tat cgg gcc tcc ggg gtc cct 192

Pro His Leu Read Ile Tyr Phe Gly Be Tyr Arg Wing Be Gly Val Pro50 55 60

gac agg ttc agt ggc agt gga tca ggc aca gat ttt aca ctg aaa until 240

Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read Lys Ile65 70 75 80

age aga gtg gag gct gag gat gtt ggg att tat tac tgc atg caa aat 288

Ser Arg Val Glu Wing Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Asn85 90 95

ctt caa act ccg tgg acg ttc ggc caa ggg acc aag gtg gat by aaa 336

Read Gln Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys100 105 110

cga 339

Arg

<210> 81

<211> 113

<212> PRT

<213> Homo sapiens

<400> 81

Glu Ile Val Leu Thr Gln Ile Pro Leu Be Valu Pro Gly15 10 15

Glu Pro Wing Be Ile Be Cys Arg Be Be Gln Be Read His Read20 25 30

Asn Gly Phe Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser35 40 45

Pro His Leu Read Ile Tyr Phe Gly Be Tyr Arg Wing Be Gly Val Pro50 55 60

Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read Lys Ile65 70 75 80

Ser Arg Val Glu Wing Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Asn85 90 95

Read Gln Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys100 105 110

Arg- 45 -NYOl 1314242 vl

- 1 -NY01 1314242 ν1

Claims (37)

Antigen-binding protein, characterized in that it comprises a complex of two first polypeptides and two second polypeptides, each first polypeptide having a first antigen-binding site located at the N-terminus of an immunoglobulin yeast chain constant domain (CL domain) referred to in Cl domain capable of stable association with a first immunoglobulin heavy chain constant domain (CrI domain), each second polypeptide having a second antigen binding site located at the N-terminus of a Ch 1 domain, said Ch domain Followed by one or more chain constant domains heavy cells capable of stable self-association, wherein at least one said first antigen binding site and said second antigen binding site is a single variable domain (sVD). Antigen binding protein according to claim 1, characterized in that both said first and second antigen binding sites are unique variable domains (sVDs). Antigen-binding protein according to claim 1, characterized in that one of said first and second antigen-binding sites is a single variable domain (sVD) and the other of said first and second antigen-binding sites is a chain. single Fv (scFv). Antigen-binding protein, characterized in that it comprises a complex of two first polypeptides and two second polypeptides, each first polypeptide comprising an immunoglobulin heavy chain composed of a variable domain and constant domains, each second polypeptide comprising a deimmunoglobulin light chain composed of one. variable domain and constant domain, wherein the first two polypeptides and the second second polypeptides stably associate to form a deimmunoglobulin-like molecule, wherein the variable domains of the immunoglobulin heavy chains stably associate with the light chain variable domains of immunoglobulin to form first antigen binding sites; and wherein one or both of said first and second polypeptides further comprises a second single chain variable domain (sVD) antigen binding site at the N-terminus or C-terminus. Antigen binding protein according to any one of claims 1 to 4, characterized in that said first antigen binding sites and said second antigen binding sites have different specificities. Antigen-binding protein according to claim 5, characterized in that said different specificities are for epitopes residing on different antigens. Antigen-binding protein according to claim 5, characterized in that said different specificities are for epitopes residing on the same antigen. Antigen binding protein according to either of Claims 1 and 2, characterized in that said first antigen binding sites of said second antigen binding sites have the same specificity. Antigen binding protein according to any one of claims 1 to 4, characterized in that said constant domains are human constant domains. Antigen binding protein according to any one of claims 1 to 4, characterized in that said single variable domain is human. Antigen binding protein according to any one of claims 1 to 4, characterized in that it binds to an Fc receptor. Antigen-binding protein according to any one of claims 1 to 4, characterized in that it influences complement-dependent cytotoxicity (CDC). Antigen binding protein according to any one of claims 1 to 4, characterized in that it influences antibody-dependent cell-mediated acitotoxicity (ADCC). Antigen binding protein according to any one of claims 1 to 4, characterized in that it is bound to an anti-tumor agent. An antigen binding protein according to any one of claims 1 to 4, characterized in that it is bound to a detectable signal production agent. Antigen binding protein according to any one of claims 1 to 4, characterized in that at least one of said first and second antigen binding sites is specific for a tyrosine kinase receptor (RTK). Antigen binding protein according to claim 16, characterized in that it blocks the binding of tyrosine kinase receptor binding. Antigen-binding protein according to claim 16, characterized in that it neutralizes doreceptor tyrosine kinase activation. Antigen binding protein according to claim 16, characterized in that it inhibits tyrosine kinase receptor signal transduction. Antigen binding protein according to claim 16, characterized in that the tyrosine kinase receptor is human. Antigen binding protein according to claim 16, characterized in that the tyrosine kinase receptor is selected from the group consisting of PDGFRα, VEGFR1, VEGFR2, VEGFR3, EGFR, HER2, IGFR, FGFR, NGFR, RON, Tek. and Tie2. Antigen binding protein according to claim 16, characterized in that one of said first and second antigen binding sites is specific for PDGFRα and the other of said first and second antigen binding sites is specific for VEGFR2. Antigen binding protein according to claim 16, characterized in that one of said first and second antigen binding sites is specific for VEGFR1 and the other of said first and second antigen binding sites is specific for VEGFR2. Antigen binding protein according to claim 16, characterized in that one of said first and second antigen binding sites is IGFR specific and the other said first and second antigen binding sites are specific for EGFR or HER2. A method of neutralizing the activation of a receptor tyrosine kinase comprising treating a cell with an antigen binding protein as defined in any one of claims 1 to 4 specific for said tyrosine kinase receptor in an amount sufficient to neutralize it. the activation of the receiver. Angiogenesis inhibition method, characterized in that it comprises treating a mammal with an antigen-binding protein as defined in any one of claims 1 to 4, in an amount sufficient to neutralize receptor activation. Tumor growth reduction method, characterized in that it comprises treating a mammal with an antigen deletion protein as defined in any one of claims 1 to 4, in an amount sufficient to reduce tumor growth. A method according to any one of claims 26 and 27, characterized in that the antigen binding protein binds PDGFRα and VEGFR2 and inhibits ligand-induced activation of PDGFRae ligand-induced activation of VEGFR2. A method according to any one of claims 26 and 27, characterized in that the antigen binding protein binds VEGFR1 and VEGFR2 and inhibits VEGFR2 ligand-induced activation and VEGFR2 ligand-induced activation. The method according to claim 27, characterized in that the antigen binding protein binds to EGFR and IGFR inhibits both EGFR ligand-induced activation and IGFR ligand-induced activation. The method of claim 27, characterized in that the antigen binding protein binds HER2 and IGFR inhibits HER2 ligand-induced activation and IGFR ligand-induced activation. The method of claim 27, further comprising administering an effective amount of an antineoplastic agent. 33. An antigen-binding protein production method comprising: a) co-expressing in a host cell a recombinant DNA construct encoding a first polypeptide having a first N-terminal antigen-binding site of a C-constant. immunoglobulin light chain (CL domain), said Cl domain capable of stable association with a first immunoglobulin heavy chain constant domain (ChI domain), a recombinant DNA construct encoding a second polypeptide having a second antigen binding site located at the N-terminus of a domain Ch 1, said ChI domain followed by one or more self-stable heavy chain constant domains, wherein at least one of said first antigen binding site and said second antigen binding site is a single variable domain (sVD); a while and in a mode sufficient for pe allow polypeptide expression and antibody formation; and b) recovering the antigen binding protein. The method of claim 33, characterized in that the constructs are in the same DNA expression vector. A method according to claim 33, characterized in that the constructs are in different DNA expression vectors. The method according to claim 33, characterized in that the host cell is a bacterial cell, a yeast cell or a mammalian cell. A method according to claim 33, characterized in that the antibody is secreted from the host cell.