KR20050021001A - Anti-igf-i receptor antibody - Google Patents

Abstract

An object of the present invention is to specifically bind to the insulin-like growth factor-I receptor, to inhibit the cellular activity of the receptor by antagonizing the receptor, and also to antibody, antibody fragments and antibodies substantially free of potent activity on the receptor. To provide derivatives.

Thus, in the first embodiment, the amino acid sequence of both the light and heavy chain variable regions, the cDNA sequence of the gene for the light and heavy chain variable regions, the identity of the CDRs (regions determining complementarity), the identity of the surface amino acids, and In view of the means of expression in the recombinant form, a fully characterized rodent antibody EM164 is provided herein.

In a second embodiment, a resurfacing or antibody adapted for human body of antibody EM164 is provided. Antibody residues or antibody fragments whose surface is exposed in antibody EM164 are replaced in both the light and heavy chains to become more similar to known human antibody surfaces. Such antibodies adapted to the human body may have increased utility as therapeutic or diagnostic agents, as compared to rodent EM164. Human-adapted variants of the antibody EM164 also include here their respective amino acid sequences of both the light and heavy chain variable regions, the DNA sequence of the gene for the light and heavy chain variable regions, the identity of the CDRs, the identity of the CDR surface amino acids, And fully characterized in view of the description of their means of expression in the recombinant.

In a third embodiment, an antibody is provided that can inhibit the growth of at least about 80% cancer cells in the presence of growth stimulants such as, for example, serum, insulin-like growth factor-I and insulin-like growth factor-II.

In a fourth embodiment, there is provided an antibody or antibody fragment having a heavy chain comprising a CDR having an amino acid sequence expressed by each of SEQ ID NOS: 1-3:

SYWMH (SEQ ID NO: 1),

EINPSNGRTNYNEKFKR (SEQ ID NO: 2),

GRPDYYGSSKWYFDV (SEQ ID NO: 3);

There is provided an antibody or antibody fragment having a light chain comprising a CDR having an amino acid sequence expressed by SEQ ID NOS: 4-6:

 RSSQSIVHSNVNTYLE (SEQ ID NO: 4);

 KVSNRFT (SEQ ID NO: 5);

 FQGSHVPPT (SEQ ID NO: 6).

In a fifth embodiment, an antibody is provided that has a heavy chain having an amino acid sequence that shares at least 90% sequence homology with the amino acid sequence expressed by SEQ ID NO: 7:

QVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSSKWYFDVWGAGTTVTVSS (SEQ ID NO: 7).

Similarly, antibodies are provided having a light chain having an amino acid sequence that shares at least 90% sequence homology with the amino acid sequence expressed by SEQ ID NO: 8:

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 8).

In a sixth embodiment, an antibody having a light chain variable region adapted or resurfaced to a human body having an amino acid sequence corresponding to one of SEQ ID NOS: -9-12 is provided.

DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 9);

DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 10);

DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 11); or

DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 12).

Similarly, antibodies having a heavy chain variable region adapted or resurfaced to a human body having an amino acid sequence corresponding to SEQ ID NO: 13 are provided:

QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSSKWYFDVWGQGTTVTVSS (SEQ ID NO: 13).

In a seventh embodiment, antibodies or antibody fragments having improved properties are provided by the present invention. For example, antibodies or antibody fragments with improved affinity for the IGF-I receptor are provided by the affinity maturation of the antibody or antibody fragments according to the invention.

In addition, the present invention provides a conjugate of the above antibodies, wherein the cytotoxic factor is covalently linked via an link that can be cleaved directly or through cleavage, to the antibody fragment of the invention or to an antibody fragment bound to an epitope. More preferably, the cytotoxic factor is taxol, maytansinoids, CC-1065 or CC-1065 analogs.

The invention also provides labeled antibodies and fragments thereof for use in research or diagnostic related applications. More preferably, the label is a radiolabel, a fluorescent label, a fluorophore, an imaging factor or a metal ion.

Diagnostic methods are provided in which the labeled antibodies or fragments are administered to a subject that appears to have cancer and the distribution of the label in the subject is measured or observed.

In an eighth embodiment, the present invention provides a method for treating a subject having cancer by administering the antibody, antibody fragment or antibody conjugate of the present invention in combination with or alone with other cytotoxic factors or therapeutic factors. The cancer may be one or many of breast cancer, colon cancer, ovarian cancer, malignant bone tumor, cervical cancer, prostate cancer, lung cancer, lubricous carcinoma, pancreatic cancer, or other cancers in which IGF-I receptor markers are still highly measured.

Description

Human Insulin-Like Growth Factor-I Receptor Antibody {ANTI-IGF-I RECEPTOR ANTIBODY}

The present invention relates to antibodies coupled with human insulin-like growth factor-I receptor (IGF-I receptor). In particular, the present invention relates to anti-IGF-I-receptor antibodies that inhibit the cellular function of the IGF-I-receptor. The present invention also anti-IGF-I-receptor antibodies that antagonize the effects of IGF-I, IGF-II and serum on the growth and survival of tumor cells and substantially lack the activity of agonists. It is about. The invention also relates to fragments of the antibody, humanized and resurfaced versions of the antibody, and to conjugates, antibody derivatives, diagnostics, investigations and therapeutic uses of the antibody. The present invention relates to an improved antibody or fragments thereof made from the antibodies and fragments thereof. In another aspect, the invention relates to a vector comprising a polynucleotide and a polynucleotide encoding an antibody or fragments thereof.

Insulin-like growth factor-I receptor (IGF-I receptor) is a transmembrane with two extracellular alpha chains and two membrane-spanning beta chains of disulfide-linked β-α-α-β arrays. Heterotetrameric protein.

By the extracellular domain of the IGF-I receptor, the binding of insulin-like growth factor-I and insulin-like growth factor-II ligands activates intracellular tyrosine kinases, resulting in autophosphorylation and substrate phosphorylation of the receptor. The IGF-I receptor has 84% high sequence similarity in the beta chain tyrosine kinase domain and is homologous to the insulin receptor with 48% low sequence similarity in the alpha chain extracellular cysteine high content domain (Ulrich, A.et al., 1986, EMBO, 5,2503-2512; Fujita-Yamaguchi, Y. et al., 1986, J. Biol . Chem ., 261, 16727-16731; Le Royth, D. et al., 1995, Endocrine Reviews , 16, 143-163). IGF-I receptors and their ligands (IGF-I, IGF-II) play an important role in adult physiological processes, including growth and development during development, metabolism, cell division, and cell differentiation (LeRoith , D., 2000, Endocrinology , 141, 1287-1288; Le Royth, D., 1997, New England J. Med. , 336,633-640).

IGF-I and IGF-II function as endocrine hormones in the blood, which are remarkably present in complexes of IGF-binding proteins, and as locally produced paracrine and self-secreting growth factors (Humbel, RE, 1990, Eur . J. Biochem ., 190, 445-462; Cohick, WS and Clemmons, DR, 1993, Annu . Rev. Physiol. 55,131-153).

IGF-I receptors have been involved in promoting growth, transformation and survival of tumor cells (Baserga, R. et al., 1997 , Biochem. Biophys. Acta , 1332, F105-F126; Blakesly, VA et al., 1997 , Journal of Endocrinology , 152,339-344; Kaleko, M., Rutter, WJ, and Miller, AD 1990, Mol. Cell. Biol., 10,464-473). As such, tumors that are known to be expressed higher than normal levels of the IGF-I receptor are several types of tumors such as breast cancer, colon cancer, ovarian cancer, synovial sarcoma, and biliary tract cancer (Khandwala, HM et al., 2000, Endocrine Reviews , Wrener, H. and Le Royth, D., 1996, Adv. Cancer Res ., 68, 183-223; Happerfield, LC et al., 1997, J. Pathol ., 183,412-417; Frire, S.et al., 1999, Gut , 44,704-708; van Dam, PA et al., 1994, J. Clin . Pathol ., 47,914-919; Xie, Y. et al., 1999, Cancer Res ., 59,3588-3591 Bergmann, U. et al., 1995, Cancer Res ., 55, 2007-2011).

In vitro, IGF-I and IGF-II have been shown to be effective in inducing mitogens against several human tumor cell lines, such as lung cancer, breast cancer, colon cancer, osteosarcoma, and cervical cancer (Ankrapp, DPand). Bevan, DR, 1993, Cancer Res ., 53,3399-3404; Cullen, KJ, 1990, Cancer Res ., 50, 48-53; Hermanto, U. et al., 2000, Cell Growth & Differentiation , 11,655-664; Guo , Y Set al., 1995, J. Am . Coll . Surg ., 181, 145-154; Kappel, CC et al., 1994, Cancer Res ., 54,2803-2807; Steller, MA et al., 1996, Cancer Res ., 56,1761-1765). These tumors and tumor cell lines also express high levels of IGF-I or IGF-II that can stimulate their secretion in auto- or collateral secretion (Qiunn, KA et al., 1996, J. Biol . Chem. ., 271,11477-11483).

Epidermal studies show a correlation between increased risk of prostate cancer, colon cancer, lung cancer and breast cancer and improved plasma concentrations of IGF-I (and low levels of IGF-binding protein-3) (Chan, JM et al., 1999, Science , 279,563-566; Wolf, A. et al., 1998, J. Natl. Cancer Inst. , 90,911-915; Ma, J. et al., 1999, J. Natl . Cancer Inst ., 91,620-625; Yu, H. et al., 1999, J. Natl . Cancer Inst ., 91,151-156; Hankinson, SE et al., 1998, Lancet , 351, 1393-1396). Strategies to lower the concentration of IGF-I in the plasma or to block the role of IGF-I receptors are necessary for cancer prevention (Wu, Y. et al., 2002, Cancer Res ., 62,1030- 1035; Grimmberg, A and Cohen P., 2000, J. Cell . Physiol ., 183, 1-9)

IGF-I receptors protect tumor cells from apoptosis caused by removal of growth factors, anchorage independent or cytotoxic drug prescription (Navarro, M. and Baserga, R., 2001). , Endocrinologly , 142, 1073-1081; Baserga, R. et al., 1997, Biochem . Biophys. Acta, 1332, F105-F126). Domains of IFG-I receptors important for cleavage induction, transformation and anti-apoptotic activity have been identified by mutation analysis.

For example, tyrosine 1251 residues of the IGF-I receptor have been shown to play an important role in anti-cell death and transformation activity, not by mitogenic activity (O'Connor, R. et al., 1997, Mol . Cell . Biol ., 17,427-435; Miura, M. et al., 1995, J. Biol. Chem. , 270,22639-22644). Intracellular signaling pathways of ligand-activated IGF-I receptors are associated with the phosphorylation of tyrosine residues on insulin receptor substrates (IRS-1 and IRS-2) that replenish the membrane with phosphatidylinositol-3-kinase. Related. Membrane-bound phospholipid products of PI-3-kinase activate the serine / threonine kinase Akt, and their substrates include the pro-apoptotic protein BAD, phosphorylated in an inactivated state (Datta, SR, Brunet, A. and Greenberg, ME, 1999, Genes & Development , 13,2905-2927; Kulik, G., Klippel, A. and Weber, MJ, 1997, Mol . Cell. Biol. 17,1595-1606). Cleavage-induced signaling of the IGF-I receptor in MCF-7 human breast cancer cells requires PI-3 kinase and is independent of cleavage-activated protein kinase. In contrast, survival signals in mutated mouse pheochromocytoma PC12 cells require both PI-3-kinase and cleavage-induced protein kinase pathways (Dufourny, B. et al., 1997, J. Biol. Chem. , 272,31163-31171; Parrizas, M., Saltiel, ARand LeRoith, D., 1997, J. Biol . Chem ., 272,154-161).

Downregulation of IGF-I receptors by anti-sense strategies has been shown to reduce tumorigenicity in vivo and in vitro of various tumor cell lines (Resnicoff, M). et al., 1994, Cancer Res ., 54,4848-4850; Lee, C.-T. et al., 1996, Cancer Res. , 56,3038-3041; Muller, M. et al., 1998, Int . J. Cancer , 77,567-571; Trojan, J. et al., 1993, Science, 259 , 94-97; Liu, X. et al., 1998, Cancer Res ., 58,5432-5438; Shapiro, DNet al., 1994, J. Clin . Invest ., 94, 1235-1242). In addition, negative mutants of dominant IGF-I receptors show reduced tumorigenicity in vivo and reduced in vitro growth of transformed murine-1 cells overexpressing IGF-I receptors (Prager, D. et al., 1994, Proc . Natl . Acad . Sci . USA, 91, 2181-2185).

Tumor cells that express antisense in the IGF-I receptor mRNA undergo large amounts of apoptosis when injected into the animal in a chamber of live diffusion. Given that tumor cells are more sensitive to apoptosis by inhibition of IGF-I receptors than normal cells, these results make IGF-I receptors an attractive therapeutic target ( Resnicoff, M. et al., 1995, Cancer Res ., 55, 2463-2469; Baserga, R., 1995, Cancer Res ., 55, 249-252).

Another method for inhibiting the function of the IGF-I receptor in tumor cells is to use an anti-IGF-I receptor antibody that is involved in the extracellular domain of the IGF-I receptor and inhibits its activity. Several attempts have been reported to express murine monoclonal antibodies against the IGF-I receptor, two inhibitory antibodies IR3 and 1H7 are possible and their use has been reported in the research papers of several IGF-I receptors.

Two antibodies, IR2, that show immunoprecipitation friendly to the insulin receptor selective to the binding insulin receptor, IR1 and IGF-I receptors (somatomedin-C receptor) and weak immunoprecipitation to the insulin receptor. IR3 antibodies that produced IR3 have been identified using partially purified placental preparat of insulin receptors to immunize mice (Kull, FC et al., 1983, J. Biol. Chem ., 258, 6561-6566). .

1H7 antibody was expressed by immunized rats with purified placental preparat of IGF-I receptor that produced inhibitory antibody 1H7 in addition to three stimulating antibodies (Li, S.-L. et al., 1993, Biochem Biophys.Res.Commun., 196, 92-98; Xiong, L. et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 5356-5360).

According to another report, a panel of mouse monoclonal antibodies specific for human IGF-I receptors divided into seven groups by binding competition studies and stimulation of IGF-I in combination with their immunity or 3T3 cell infection. Was obtained by immunization of mice infected with 3T3 cells expressing high levels of IGF-I receptor (Soos, MA et al., 1992, J. Biol. Chem., 267, 12955-12963).

As such, although IR3 antibodies are most commonly used as inhibitory antibodies to IGF-I receptor studies in vitro, they exhibit agonistic activity in infected 3T3 and CHO cells expressing human IGF-I receptors. (Kato, H. et al., 1993, J. Biol. Chem., 268, 2655-2661; Steele-Perkins, G. and Roth, RA, 1990, Biochem. Biophys. Res. Commun., 171 , 1244-1251). Similarly, among the panel of antibodies found by Soos et al., Most inhibitory antibodies 24-57 and 24-60 also show agonist activity in infected 3T3 cells (Soos, MA et al., 1992, J. Biol. Chem., 267, 12955-12963). Although IR3 antibodies have been reported to inhibit the binding of IGF-I (but not IGF-II) to express intact intracellular receptors after solubilization, they function both IGF-I and IGF-II. Inhibits and stimulates DNA synthesis in cells in vivo (Steele-Perkins, G. and Roth, RA, 1990, Biochem. Biophys. Res. Commun., 171, 1244-1251). Binding epitopes of IR3 antibodies are estimated from the hypothetical insulin-IGF-I receptor construct ranging from 223-274 of the IGF-I receptor (Gustafson, TA and Rutter, WJ, 1990, J. Biol. Chem., 265, 18663-18667; Soos, MA et al., 1992, J. Biol. Chem., 267, 12955-12963).

The MCF-7 breast cancer cell line has been used as a model cell line to formally describe the growth responses of IGF-I and IGF-II in vitro (Dufourny, B. et al., 1997, J. Biol. Chem). , 272, 31163-31171). In MCF-7 cells, IR3 antibodies incompletely block approximately 80% of the stimulatory effects of exogenous addition of IGF-I and IGF-II in the absence of serum. In addition, IR3 antibodies do not strongly inhibit the growth (less than 25%) of MCF-7 cells in 10% serum (Cullen, K. J. et al., 1990, Cancer Res., 50, 48-53). The weak inhibition of thermo-stimulated growth of MCF-7 cells by these in vitro IR3 antibodies is related to in vivo studies that IR3 antibodies do not inhibit the growth of MCF-7 xenografts in very nude mice (Arteaga, CL et al., 1989, J. Clin. Invest., 84, 1418-1423.

The weak agonist activity of IR3 and other antibodies and the ability to significantly inhibit the growth of tumor cells, such as MCF-7 cells, within the physiological state of plasma stimulation, resulted in serum-stimulated growth of tumor cells. There is a need for new anti-IGF-I receptor antibodies that are strongly inhibited and do not exhibit agonistic activity by themselves.

1 shows fluorescence activated cell sorting (FACS) analysis for purified EM164 antibodies that specifically bind to cells overexpressing the human Y1251F IGF-I receptor or human insulin receptor.

2 shows the binding titration curves of EM164 antibody that binds to the biotin labeled human IGF-I receptor.

3 shows that the binding of biotin-labeled IGF-I to human breast cancer MCF-7 cells is inhibited by EM164 antibody.

4 shows that autophosphorylation of IGF-I receptor stimulated by IGF-I in MCF-7 cells is inhibited by EM164 antibody.

5 shows that IRS-1-phosphorylation stimulated by IGF-I in MCF-7 cells is inhibited by EM164 antibody.

Figure 6 shows that signal transduction stimulated by IGF-I in SaOS-2 cells is inhibited by EM164 antibody.

7 shows the effect of EM164 antibody on the growth of MCF-7 cells under different growth conditions as assessed by MTT assay.

8 shows the effect of EM164 antibody on the growth and survival of MCF-7 cells under various serum concentrations.

9 shows that growth and survival of NCL-H838 cells stimulated by IGF-I and serum are inhibited by EM164 antibody.

Figure 10 shows the effect of treatment with EM164 antibody, Taxol, or a combination of EM164 antibody and Taxol in the growth of Calu-6 lung cancer transplanted into rodents.

FIG. 11 shows competition between binding of EM164 antibody (v.1.0) and human rodent EM164 antibodies adapted for human body.

12 shows the cDNA (SEQ ID NO: 49) and amino acid sequence (SEQ ID NO: 50) for the variable region of the light chain foremost and rodent anti-IGF-I receptor antibody EM164. The arrow marks the start of structure 1. The three CDR sequences according to Kabat are underlined.

FIG. 13 shows the cDNA (SEQ ID NO: 51) and amino acid sequence (SEQ ID NO: 52) for the variable region of the light chain foremost and rodent anti-IGF-I receptor antibody EM164. The arrow marks the start of structure 1. The three CDR sequences according to Kabat are underlined.

14 shows the light and heavy chain CDR amino acid sequences of antibody EM164 as determined from Chothia standard definition. Also shown are AbM modeling software definitions for the light chain CDRs. Light chain: CDR1 is SEQ ID NO: 4, CDR2 is SEQ ID NO: 5, and CDR3 is SEQ ID NO: 6. Heavy chain: CDR1 is SEQ ID NO: 1, CDR2 is SEQ ID NO: 2, CDR3 is SEQ ID NO: 3. AbM heavy chain: CDR1 is SEQ ID NO: 53, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 55.

FIG. 15 shows the light and heavy chain amino acid sequences for the anti-IGF-I-receptor antibody EM164 linked to the bacterial line sequence for Cr1 (SEQ ID NO: 56) and J558.c (SEQ ID NO: 57) genes. The dash (-) indicates sequence identity.

16 shows the plasmids used to generate and express chimeric EM164 antibodies adapted for human body of genetic recombination. A) light chain cloning plasmid, B) heavy chain cloning plasmid, C) mammalian antibody expression plasmid.

FIG. 17 is the amino acid sequence of the light chain screened from 127 antibodies in the set of structural files used to predict the surface residues of EM164, showing that most of the ten amino acid sequences are homologous to each other. em164LC (SEQ ID NO: 58), 2jel (SEQ ID NO: 59), 2pcp (SEQ ID NO: 60), 1nqb (SEQ ID NO: 61), 1kel (SEQ ID NO: 62), 1hyx (SEQ ID NO : 63), 1igf (SEQ ID NO: 64), 1tet (SEQ ID NO: 65), 1clz (SEQ ID NO: 66), 1bln (SEQ ID NO: 67), 1cly (SEQ ID NO: 68), Consensus (SEQ ID NO: 69).

FIG. 18 is the amino acid sequence of the heavy chain screened from 127 antibodies in the set of structural files used to predict the surface residues of EM164, showing that most of the ten amino acid sequences are homologous to each other. em164HC (SEQ ID NO: 70), 1 nqb (SEQ ID NO: 71), 1 ngp (SEQ ID NO: 72), 1 fbi (SEQ ID NO: 73), 1afv (SEQ ID NO: 74), 1yuh (SEQ ID NO) : 75), 1plg (SEQ ID NO: 76), 1d5b (SEQ ID NO: 77), 1ae6 (SEQ ID NO: 78), 1axs (SEQ ID NO: 79), 3hfl (SEQ ID NO: 80), Consensus (SEQ ID NO: 81).

FIG. 19 shows the average accessibility of each of the (A) light chain and (B) heavy chain variable region residues from the ten most homologous structures. Numbers indicate the Kabat antibody sequence position number.

20 shows light chain variable region amino acid sequences for rodent EM164 (muEM164) and human adapted EM164 (huEM164) antibodies. muEM164 (SEQ ID NO: 82), huEM164 V1.0 (SEQ ID NO: 83), huEM164 V1.1 (SEQ ID NO: 84), huEM164 V1.2 (SEQ ID NO: 85), huEM164 V1.3 ( SEQ ID NO: 86).

Figure 21 shows heavy chain variable region amino acid sequences of rodent EM164 (muEM164, SEQ ID NO: 87) and human adapted EM164 antibody (huEM164, SEQ ID NO: 88).

22 shows the amino acid sequence of the light chain (DNA, SEQ ID NO: 89, amino acid SEQ ID NO: 90) and heavy chain (DNA, SEQ ID NO: 91, amino acid SEQ ID NO: 92) and the huEM164 v1.0 variable region Show the DNA.

23 shows EM164 v1.1 (DNA, SEQ ID NO: 93; amino acid SEQ ID NO: 94), v1.2 (DNA, SEQ ID NO: 95; amino acid SEQ ID NO: 96) and v1 adapted for human body .3 shows amino acid sequence and light chain variable region DNA for (DNA, SEQ ID NO: 97; amino acid SEQ ID NO: 98).

24 shows that growth and survival of MCF-7 cells stimulated by IGF-I is inhibited by EM164 v1.0 antibody and rodent EM164 antibody adapted to human body.

25 shows that EM164 inhibits the MCF-7 cell cycle stimulated by IGF-I.

26 shows that EM164 inhibits the anti-apoptotic effect of IGF-I and serum. Treatment with EM164 kills cells as evidenced by the increased concentration of cleaved CK18 protein.

FIG. 27 shows the effect of treatment with EM164 antibody, gemcitabine, or a combination of EM164 antibody and gemcitabine in the growth of human BxPC-3 pancreatic cancer xenografted to immunodeficient rodents.

We have discovered and developed new antibodies that specifically bind to human insulin-like growth factor-I receptor (IGF-IR) at the cell surface. Antibodies and fragments have a special ability to inhibit the cellular function of the receptor without the ability to activate the receptor itself. Thus, while known antibodies that specifically bind and inhibit IGF-IR activate the receptor without the IGF-IR ligand, the antibodies or fragments of the invention substantially antagonize the activity of the agonist while antagonizing IGF-IR. Few. In addition, the antibodies and antibody fragments of the present invention inhibit the growth of human tumor cells, such as MCF-7 cells, by 80% or more in the presence of serum, which is greater than the inhibition obtained with known anti-IGF-IR antibodies. .

The present invention proceeds from a rodent anti-IGF-IR antibody, here EM164, and has been fully characterized in terms of amino acid sequences on both the light and heavy chains, the identification of CDRs, the identification of surface amino acids, and the means of expression in the recombination form.

In FIG. 15, the bacterial sequence associated with the sequence of EM164 is shown. The comparison identifies certain human mutations in EM164, including one in each of CDR1 in the light chain and CDR2 in the heavy chain.

The first amino acid and DNA sequences of the antibody EM164 light and heavy chains and of the human-adapted variants are shown here. However, the scope of the present invention is not limited to antibodies and fragments comprising such sequences. On the other hand, all antibodies and fragments that specifically bind to insulin-like growth factor-I receptors and antagonize the biological activity of the receptor but have little agonist activity are within the scope of the present invention. Thus, antibodies and fragments may differ from the antibody EM164 or human-derived derivatives in the amino acid sequences of their backbone, CDR, light and heavy chains, which is also within the scope of the present invention.

CDRs of antibody EM164 have been confirmed by modeling and molecular structures have been expected. Again, while CDRs are important for epitope recognition, they are not essential to the antibodies and fragments of the invention. Thus, for example, antibodies and fragments having improved properties produced by the affinity maturation of the antibodies of the invention are provided.

Antibody mimetics, as well as various antibodies and antibody fragments, can be made by insertion within a variety of constant region sequences flanked by mutations, deletions and / or particular sets of CDRs. Thus, for example, different classifications of Ab are possible with a given set of CDRs by substitution of other heavy chains, while, for example, IgG1-4, IgM, IgA1-2, IgD, IgE antibody types and isotypes Can be produced. Similarly, artificial antibodies within the scope of the present invention may be produced by embedding a given set of CDRs entirely within the synthetic construct. The term "variable" is used in the present invention to describe a portion of the various regions that differ in the sequence between antibodies, and is used in the antibody specificity and association with each antigen. In general, however, diversity is not evenly distributed across the various regions of the antibody. In both the light and heavy chain variable regions, they are typically concentrated in three parts called complementarity determining regions (CDRs) or hypervariable regions. The better conserved parts of the variable region are called FRs. The variable regions of the light and heavy chains each comprise four FR regions, mainly employing a beta-sheet configuration, linked by three CDRs, forming loop connections, and in some cases forming part of the beta-sheet structure. . In each chain, the CDRs, closely adjacent by the FR region, are bound together with CDRs from the other chain and contribute to the formation of antigens by binding the sides of the antibody (EAKabatet al. Sequences of Proteins of Immunological Interest, fifth). edition, 1991, NIH). The constant region is not directly involved in binding the antibody to the antigen, but shows various effector functions such as the involvement of the antibody in antibody dependent cytotoxicity.

Antibodies adapted to the human body, or antibodies adapted without rejection by other mammals, can be produced using various techniques such as resurfacing and CDR tissue transplantation. In resurfacing techniques, molecular modeling, statistical analysis, and mutationsis combined to adapt to the non-CDR surface of the variable region to resemble the surface of a known antibody of the target group. Resurfacing strategies and methods of antibodies and methods for reducing immunodeficiency of antibodies in other groups are described in US Pat. No. 5,639,641, which is incorporated herein by reference. In CDR tissue transplantation techniques, rodent light and heavy chain CDRs are fully transplanted into human structural sequences.

The invention also encompasses the equivalent functions of the antibodies described in the specification. Equivalent function has binding properties compared to the binding properties of antibodies, and includes, for example, chimeric single chain antibodies and fragments thereof adapted to the human body. Methods of producing such equivalent functions are described in PCT application WO 93/21319, European Patent Application 239,400; PCT Application WO 89/09622; European Patent Application 338,745; And European Patent Application 332,424, each of which is incorporated by reference.

Functional equivalence includes polypeptides having amino acid sequences that are substantially identical to the amino acid sequences of the variable or hypervariable regions of the antibodies of the invention. The term "substantially the same" as applied to amino acid sequences is described in Pearson and Lipman, Proc. Natl. Acad. Sci. As determined by the FASTA search method according to USA 85, 2444-2448 (1988), it is defined as having at least about 90%, more preferably at least 95% sequence homology with another amino acid sequence.

Chimeric antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions, and variable regions derived substantially or exclusively from sequences of variable regions from mammals other than humans. Antibody forms adapted to the human body are made, for example, by replacing the complementarity determining regions of rodent antibodies with human structural regions (W092 / 22653). Antibodies adapted to the human body preferably comprise constant and variable regions other than complementarity determining regions (CDRs) derived substantially or exclusively from corresponding human antibody regions and CDRs derived substantially or exclusively from mammals other than humans. Have,

Functional equivalents include single-chain antibody fragments, also known as single-chain antibodies (scFvs). Such fragments include at least one fragment of a variable heavy chain amino acid sequence (VH) antibody bound to at least one fragment of a variable light chain sequence (VL) antibody with or without one or more interconnected links. These links are short, flexible peptides chosen to ensure that proper three-dimensional folding of the (VH) and (VL) regions occurs once linked to ensure that the single-chain antibody fragment retains the target molecule binding properties of all the antibodies to which it is derived. Can be. In general, the carboxyl ends of the (VH) and (VL) sequences are covalently bound to the amino acid ends of the complementary (VH) and (VL) sequences by such peptide linkers. Single-chain antibody fragments can be generated by molecular cloning, antibody phage display libraries or similar techniques. Such proteins can be produced in prokaryotic and eukaryotic cells, including bacteria.

Single-chain antibody fragments comprise an amino acid sequence having at least one of the variable or complementary determining regions (CDRs) of all antibodies described herein, but some or all of the constant regions of these antibodies are deleted. These constant regions are not essential for antigen binding but constitute a major part of the structure of all antibodies. Single-chain antibody fragments therefore must overcome some problems in using antibodies that include some or all of the constant regions. For example, single-chain antibody fragments have a propensity to be free from undesirable interactions or other undesirable biological activities between biological molecules and heavy chain constant regions. In addition, since single-chain antibody fragments are significantly smaller than all antibodies, they have higher capillary permeability compared to all antibodies, allowing the single-chain antibody fragments to bind more effectively to target antigen-binding aspects and to be restricted locally. In addition, antibody fragments can be produced on a relatively large scale in prokaryotic cells and thus promote their production. In addition, single-chain antibody fragments are relatively small, causing less immune response at the receptor than all antibodies.

Moreover, functional equivalence includes fragments of antibodies that have the same or similar binding properties to all antibodies. Such fragments may comprise one or both of Fab fragments or F (ab ′) 2 fragments. Although fragments comprising fewer than three regions, such as 3, 4 or 5 CDRs, are also functional, preferably antibody fragments include all six complementarity determining regions of all antibodies. In addition, functional equivalence may bind or bind to any of the following immunoglobulin families: IgG, IgM, IgA, IgD, or IgE, and subclasses thereof.

Knowledge of amino acid and nucleic acid sequences for anti-IGF-I receptor antibody EM164 and its humanized variants, as described herein, binds to human IGF-I receptors and inhibits the cellular function of IGF-I receptors. Can be used to develop other antibodies. There are several studies investigating the effects of varying one or more amino acids at various positions in the antibody sequence, based on knowledge of the sequence of the first antibody, ie binding and expression steps (Yang, WP et al., 1995, J. Mol. Biol., 254, 392-403; Rader, C. et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915; Vaughan, TJ et al., 1998, Nature Biotechnology, 16, 535-539.

In these studies, modification of the first antibody occurs by altering the sequence of heavy and light chain genes in CDR1, CDR2, CDR3, or constituent regions. Methods used at this time are oligonucleotide-mediated site-directed mutations, cassette mutations, error-pron PCR, DNA shuffling, or mutant strains of E. coli. (Vaughan, TJ et al., 1998, Nature Biotechnology, 16, 535-539; Adey, NB et al., 1996, Chapter 16, pp. 277-291, in "Phage Display of Peptides and Proteins", Eds. Kay , BK et al., Academic Press). The method of changing the sequence of these first antibodies results in increasing the affinity of the second antibody (Gram, H. et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 3576-3580; Boder, ET et al., 2000, Proc. Natl. Acad. Sci. USA, 97, 10701-10705; Davies, J. and Riechmann, L., 1996, Immunotechnolgy, 2, 169-179; Thompson, J. et. al., 1996, J. Mol. Biol., 256, 77-88; Short, MK et al., 2002, J. Biol. Chem., 277, 16365-16370; Furukawa, K. et al., 2001, J. Biol. Chem., 276, 27622-27628).

By a similarly managed strategy of changing one or more amino acid residues of an antibody, the antibody sequences described herein can be used to develop anti-IGF-I receptor antibodies with improved function.

Conjugations according to the invention include antibodies, fragments, and their analogs linked to the cytotoxic agents described herein. The cytotoxic factors are maytansinoids, taxanes and analogs of CC-1065. Conjugation can be prepared by in vivo methods. To link the cytotoxic factor to the antibody, a linking group is used. Suitable link groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, Peptidase labile groups, and esterase labile groups. For example, conjugation can be constructed using disulfide variable regions or by forming thioether linkages between antibodies and cytotoxic factors.

Maytansinoids and their analogs are one of the cytotoxic factors. Suitable maytansinoid examples include maytansinol and its analogs. Suitable maytansinoids are U.S. Patent number 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; And 5,846,545.

Taxanes are also cytotoxic factors. Suitable taxanes for use in the present invention are U.S. Patent Nos. 6,372,738 and 6,340,701.

CC-1065 and their analogs are also preferred as the cytotoxic drugs used in the present invention. CC-1065 and their analogues are described in U.S. Pat. Patent number 6,372,738; 6,340,701; 5,846,545 and 5,585,499.

An attractive candidate for such cytotoxic conjugate preparation is CC-1065, which is a potent anti-tumor antibiotic isolated from Streptomyces zelensis bacterial culture. CC-165 is in vivo than commonly used anticancer drugs such as doxorubicin, methotrexate and vincristine (BK Bhuyan et al., Cancer Res., 42, 3532-3537 (1982)). At about 1000 times more powerful.

Methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil Cytotoxic agents, such as, and calicheamicin, are also suitable for the conjugation preparations of the invention, and the drug molecule may also be linked to the antibody molecule via an intermediate carrier molecule such as serum albumin.

For application in diagnosis, the antibody according to the invention is typically labeled with a detectable moiety. The detectable portion may be any that can generate a detection signal directly or indirectly. For example, the detectable moiety is a radioisotope such as 3H, 14C, 32P, 35S, or 131I; Chemiluminescent compounds, such as fluorescent or fluorescein isothiocyanate, rhodamine, or luciferin; Or enzymes such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Hunter, et al., Nature 144: 945 (1962); David, et al., Biochemistry 13: 1014 (1974); Pain, et al., J. Immunol. Meth. 40: 219 (1981); Nygren, J. Histochem. And Cytochem. Several methods of conjugating an antibody to a detectable moiety known in the art, including those described by 30: 407 (1982), can be used.

Antibodies according to the present invention are characterized by competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147). -158 (CRC Press, Inc., 1987)) can be used.

The antibodies according to the invention are also useful for in vivo imaging, in which the antibody labeled with a detectable moiety such as a radio-opaque agent or radioisotope is administered to the subject and preferably It is administered intravascularly and the presence and location of labeled antibodies in the host are analyzed. This imaging technique is useful for staging and treatment of malignancies. Antibodies can be labeled with any part that can be detected in the host by nuclear magnetic resonance, radiation medicine, or other detection means known in the art.

Antibodies according to the invention are also useful as affinity purification agents. In this process, the antibody is immobilized with a suitable support such as Sephadex resin or filter paper using methods well known in the art.

The antibodies according to the invention are also useful as reagents in biological studies based on the inhibition of the function of the IGF-I receptor in cells.

For application in therapy, the antibody or conjugate according to the invention is administered to the subject in the form of a pharmacologically acceptable pill. They are intramuscular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical by pill or by prolonged infusion over time. administered intravenously by topical or inhalation route. Antibodies can also be administered by intratumoral, peritumoral, intralesional, or perilesional routes to obtain systemic as well as local therapeutic effects. Suitable pharmaceutically acceptable carriers, diluents, and additives are well known and can be determined by one skilled in the art. Suitable carriers, diluents and / or additives include (1) Dulbecco's phosphate buffered saline, approximately pH 7.4, containing about 1 mg / ml in 25 mg / ml of human serum albumin, (2) 0.9% salt (0.9% w / v NaCl), and (3) 5% (w / v) dextrose. The method according to the invention can be carried out in vitro, in vivo or ex vivo.

In another method of treatment, the antibody, antibody fragment or conjugation according to the invention is administered with one or more additional therapeutic agents. Suitable therapeutic agents include, but are not limited to, cytotoxic factors or cytostatic factors. Taxol is a preferred therapeutic and cytotoxic factor.

Cancer therapeutic agents are drugs that limit or kill the growth of cancer cells while causing minimal damage to the host. As such, these drugs take advantage of differences in cancer cell characteristics (eg, metabolism, vascularization, or cell-surface antigen presentation) that are different from healthy strain cells. Differences in tumor morphology are an important part of the intervention: for example, the second treatment can be an antibody, such as an anti-VEGF antibody, useful for the neovascularization of the interior of a single slow-growing tumor. Other treatments include adjuncts such as granisetron HCL, androgen inhibitors such as leuprolide acetate, antibiotics such as doxorubicin, antiestrogens such as tamoxifen, and interferon Antimetabolites such as alpha-2a, cytotoxic agents such as taxol, enzyme inhibitors such as ras farnesyl-transferase inhibitor, aldesleukin Immunomodulators such as, and nitrogen mustard derivatives such as melphalan HCl and the like, and the like, but are not limited thereto.

In the case of aqueous dosage forms, rather than lyophilized, antibodies will typically be formulated at concentrations of about 0.1 mg / ml to 100 mg / ml, although a wide range of alterations are possible. . Dosages suitable for antibody or conjugation to treat a disease, regardless of whether the antibody has been administered for prophylactic or therapeutic purposes, include the type of disease to be treated, the severity and course of the disease, the course of previous treatment, the history of the patient, Respond to antibodies, and the discretion of the attending physician. The antibody is appropriately administered to the patient at one time or through a series of treatments.

Depending on the type and extent of the disease, for example, between about 0.015 and 15 mg of antibody per kilogram of patient's body weight is the initial dose to be administered to the patient, regardless of whether one or more separate doses or sustained doses are administered. For repeated administrations over several days or long periods of time, treatment is repeated as appropriate until the condition is suppressed to the desired extent. However, other drug regimens may be useful and are not excluded.

The present invention will now be described in detail with reference to the following examples. This is merely an example and is not intended to limit the invention to this.

Example 1 Murine EM164 Antibodies

In a first embodiment, the complete first amino acid structure and cDNA sequence of a rodent antibody in the present invention is published along with binding properties and means for its recombination expression. Accordingly, a complete description of the antibodies of the present invention and their preparations is provided, and those antibodies can be prepared without undue experimentation by one of ordinary skill in the art of immunology.

A. Generation of anti-IGF-I receptor monoclonal antibody hybridomas

Cell lines expressing the human IGF-I receptor with the Y1251F mutation were used for immunization expressing the top number of the IGF-I receptor (˜107 per cell). Y1251F-mutation in the cytoplasmic region of the IGF-I receptor resulted in the loss of transformation and anti-apoptotic signals, but affected IGF-I association and mitogenic signals stimulated by IGF-I (O'Connor, R. et al., 1997, Mol. Cell. Biol., 17, 427-435; Miura, M. et al., 1995, J. Biol. Chem., 270, 22639-22644 ). Since the antibody in this example is bound to the extracellular region of the IGF-I receptor, the mutation did not significantly affect antibody production, which was the same for both the Y1251F mutation and the wild type receptor.

Cell lines expressing the human IGF-I receptor with the Y1251F mutation were transfected with the puromycin-resistance gene and the Y1251F-mutant human IGF-I receptor gene to 3T3- of IGF-I-receptor-deficient rodents. Were generated from similar cells and selected by FACS sorting for expression of the upper IGF-I receptor using puromycin (2.5 microgram / mL) (Miura, M. et al., 1995, J. Biol. Chem., 270, 22639-22644). Cell lines with higher levels of expression of the IGF-I receptor are further selected using high concentrations of puromycin, such as 25 microgram / mL, which is toxic to most cells. Survival populations were singled out and those showing higher level expression of the IGF-I receptor were selected.

Six-month-old CAF1 / J cancer rodents were immunized intraperitoneally using Y1251F-mutant-human-IGF-I-receptor-overexpressing cells (5 × 10 5 cells, suspended in 0.2 mL PBS) on the first day of the experiment. Animals were raised with 0.2 mL cell suspension as follows: 2 days, 1 × 10 6 cells; 5 days, 2 × 10 6 cells; 7, 9, 12, and 23 days, 1 × 10 7 cells. On day 26, rodents died and spleen was removed.

The spleen was crushed between two frozen glass slides to obtain a single cell suspension. This single cell suspension was washed using serum-free RPMI medium containing penicillin and streptomycin (SFM). Spleen cell pellets were resuspended for 10 minutes in a frozen state with 10 mL of 0.83% (w / v) ammonium chloride solution to lyse red blood cells, and then serum-free medium (SFM). Washed using. Spleen cells (1.2 × 108) are congested from non-secreting rodent myeloma cell line P3X63Ag8.653 (ATCC, Rockville, Md .; Cat. # CRL1580) into myeloma cells (4 × 107) in the coronary organs and serum free. Washed using RPMI-1640 (SFM) medium. The supernatant was removed and the cell pallet was resuspended in the remaining medium. Coronal organs were placed in a beaker of water at 37 ° C and 1.5 mL of polyethylene glycol solution (50% PEG (w / v), average molecular weight 1500 at 75 mM HEPES, pH 8) was 0.5 mL while the organ vibrated gently. Slowly added at a drop rate of / min. After 1 minute, 10 mL of SFM was added as follows: 1 mL for the first 1 minute, 2 mL for the next 1 minute, and 7 mL at 3 minutes. Another 10 mL was added slowly over the next 10 minutes. Cells were pelleted by centrifuge, washed in SFM, 5% Petal Bobbin Serum (FBS), Hypoxanthin / Aminopterin / Thymidine (HAT), Penicillin, Streptomycin, and 10% Hybridoma Cloning supplement (HCS) was resuspended in RPMI-1640 growth medium added. Cells were planted into 96-well flat-bottom tissue culture plates in 2 × 105 spleen cells at 200 μL per well. After 5-7 days, 100 μL per well was removed and replaced with growth medium added hypoxanthine / thymidine (HT) and 5% FBS. Common conditions used for immunization and hybridoma production are J. Langone and H. Vunakis (Eds., Methods in Enzymology, Vol. 121, "Immunochemical Techniques, Part I"; 1986; Academic Press, Florida) and E. Harlow and D. Lane ("Antibodies: A Laboratory Manual"; 1988; Cold Spring Harbor Laboratory Press, New York). Other techniques for immunity and hybridoma production may also be used, as is well known to those skilled in the art.

Culture supernatants from hybridoma clones were used to bind to human IGF-I receptors purified by ELISA, to specifically bind to cells overexpressing human IGF-I receptors, and as described below. Screening for lack of binding to cells overexpressing human insulin receptor by FACS screening. Clones that showed higher binding affinity for cells that overexpress human IGF-I receptors than cells overexpressing human insulin receptors were augmented and subcloned. Culture supernatants of subclones were further screened by the above binding assay. By this procedure, subclones 3F1-C8-D7 (EM164) were selected and the heavy and light chain genes were cloned and sequenced as follows.

Human IGF-I receptors were isolated by the following method for use in screening supernatants from hybridoma clones and binding to IGF-I receptors. IGF-I was prepared by modifying recombination IGF-I using a biotinylation reagent such as sulfo-NHS-LC-biotin, sulfo-NHS-SS-biotin, or NHS-PEO4-biotin. Biotin-labeled IGF-I was incubated with lysates from human wild type or Y1251F mutant IGFR cells absorbed and overexpressed in streptavidin-agarose beads. Beads were washed and eluted using buffers containing 2-4 M urea and surfactants such as Triton X-100 or oroctyl-β-glucoside . The eluted IGF-I receptor was dialyzed with PBS and purified by SDS-PAGE under reduced conditions. The alpha and beta chain rings of the molecule's IGF-I receptor weighed about 135 kDa and 95 kDa, respectively.

To check for binding of the hybridoma supernatant to purified IGF-I receptor, Imulon-4HB ELISA plate (Dynatech) was prepared in 50 mM CHES buffer (100 μL; 4 ° C., 1 day) at pH 9.5. It was coated with a diluted, purified human IGF-I receptor sample (prepared by dialysis from urea / octyl-β-glucoside elution of an affinity purified sample). Wells were closed with 200 μL of blocking buffer (10 mg / mL BSA in TBS-T buffer containing 50 mM Tris, 150 mM NaCl, pH 7.5, and 0.1% tween-20) and hybridoma clones (100 μL) (Diluted in blocking buffer) from the supernatant for 1 to 12 hours, washed with TBS-T buffer, and goat-anti-mouse-IgG-Fc-antibody-horseradish peroxidase (HRP) conjugation (100 μL; 0.8 μg / mL in blocking buffer; Jackson ImmunoResearch Laboratories) and then ABTS / H ₂ O ₂ substrate at 405 nm (0.5 mg / mL ABTS, 0.03% H ₂ O ₂ in 0.1 M citrate buffer, pH 4.2) It was washed and detected using. Typically, the supernatant from the 3F1 hybridoma subclone signaled about 1.2 absorption units within 3 minutes of production, unlike some other hybridoma clones that obtained 0.0 value in the supernatant. The general state of this ELISA is similar to the standard ELISA conditions for antibody binding and detection described by E. Harlow and D. Lane ("Using Antibodies: A Laboratory Manual"; 1999, Cold Spring Harbor Laboratory Press, New York). However, the above conditions can also be used.

Screening of hybridoma supernatants to specifically bind to human IGF-I receptors rather than human insulin receptors uses ELISAs in the overexpressed human Y1251F-IGF-I receptor cell line and in the overexpressed human insulin receptor cell line. Was performed. These bilateral cell lines were produced from rodent 3T3-like cells lacking the IGF-I receptor. IGF-I receptors overexpressing cells and insulin receptors overexpressing cells were harvested from tissue culture flasks by rapid trypsin / EDTA treatment, suspended in growth media containing 10% FBS, and pelletized by centrifugation. And washed with PBS. Coating washed cells (100 .mu.L of about 1-3.times.10.sup.6 cells / mL) with phytohemagglutinin (100 .mu.L of 20 .mu.g / mL PHA) Wells of prepared Imulon-2HB plates were added, centrifuged and attached to PHA-coated wells for 10 minutes. Plates containing cells were patted to remove PBS and then dried at 37 ° C. overnight. The wells were blocked for 1 hour at 37 ° C. with 5 mg / mL BSA solution in PBS and then gently washed using PBS. Then, aliquots of the supernatant from the hybridomas (100 μL; diluted in blocking buffer) contain wells containing IGF-I-receptor-overexpressing cells and wells containing insulin receptor-overexpressing cells. And incubated at room temperature for 1 hour. Wells were washed with PBS, incubated with goat-anti-mouse-IgG-Fc-antibody-horseradish peroxidase conjugate (100 μL; 0.8 μg / mL in blocking buffer) for 1 hour, then washed and Binding was then detected using an ABTS / H ₂ O ₂ substrate. The formal supernatants obtained from 3F1 hybridoma subclone cultured with overexpressing IGF-I receptor cells signaled 0.88 uptake units within 12 minutes of production, as opposed to obtaining 0.22 uptake unit values from overexpressing human insulin receptors.

Hybridomas were grown in Integra CL 350 flasks (Integra Biosciences, Maryland) according to manufacturer's specifications to provide purified EM164 antibodies. An antibody of about 0.5-1 mg / mL was obtained from the Integra flask in the harvested supernatant using antibody criteria based on quantification by ELISA and SDS-PAGE / Coomassie blue staining. The antibody was purified by affinity chromatography on a protein agarose bead column under standard purification conditions of loading and washing in pH 8.9, 100 mM Tribuffer containing 3M NaCl, followed by 150 mM NaCl. Eluted in 100 mM acetic acid solution. Eluted fragments containing the antibody were neutralized using cold 2M K2HPO4 solution and dialyzed in PBS at 4 ° C. Antibody concentrations were determined by measuring absorbance at 280 nm (death rate = 1.4 mg-1 mL cm-1). Purified antibody samples were analyzed by SDS-PAGE under reducing conditions and Coomassie blue staining, showing only the heavy and light chains of the antibody at about 55 kDa and 25 kDa, respectively. The reference sample of the purified antibody was IgGI with Kappa light chain.

B. Binding Specificity of EM164 Antibodies

Specific binding of purified EM164 antibodies was performed by fluorescence excitation cell sorting (FACS) using cells that overexpress human IGF-I receptors and cells that overexpress human insulin receptor cells (FIG. 1). Culture of EM 164 antibody (50-100 nM) in 100 μL cold FACS buffer (1 mg / mL BSA in Dulbecco's MEM medium) was performed using cells overexpressing IGF-I receptor and insulin receptors (2 × 105 cells / mL). Cells were overexpressed) for 1 hour on a round-bottom 96-well plate. Cells were pelleted by centrifuge and washed with cold FACS buffer by gentle vibration. The goat-anti-mouse-IgG-antibody-FITC conjugate (100 .mu.L; 10 .mu.g / mL in FACS buffer) was then incubated for 1 hour in the frozen state. Cells were pelleted, washed and resuspended in 120 μL of 1% formaldehyde solution in PBS. Plates were analyzed using a FACS Calibur reader (BD Biosciences).

In contrast to insignificant metastasis in culturing insulin receptors overexpressing cells with EM164 antibody (Fig. 1), strong fluorescence shift in culture of IGF-I receptors overexpressing cells with EM164 antibody. ), Which demonstrated that the EM 164 antibody selectively binds to the IGF-I receptor and does not bind to the insulin receptor. Control antibodies, anti-IGF-I receptor antibody 1H7 (Santa Cruz Biotechnology) and anti-insulin receptor alpha antibody (BD Pharmingen Laboratories), respectively, have fluorescent transitions in culture of cells overexpressing IGF-I and insulin receptors. (FIG. 1). Strong fluorescence transfer was also observed by AFACS analysis using EM 164 antibody and human breast cancer MCF-7 cells, which showed IGF-I receptors (Dufourny, B. et al., 1997, J. Biol. Chem., 272, 31163-31171) and show that the EM164 antibody binds to human IGF-I receptor on the surface of human tumor cells.

The separation constant (Kd) for binding the EM164 antibody to the human IGF-I receptor is either directly coated IGF-I receptor (affinity purified using biotin-labeled IGF-I as above) or indirectly captured. Biotin-labeled IGF-I receptors were used to measure ELISA titration of antibody binding at several concentrations. Biotin-labeled IGF-I receptors are characterized by the use of PEO-maleimide-biotin reagents (Pierce, Molecular Biosciences) to convert bioactive soluble lysates into biotin from IGF-I receptors that overexpress cells. Prepared by being labeled, which was purified affinity using an anti-IGF-I receptor beta chain antibody immobilized on NHS-agarose beads, eluted with 2-4 M urea in a buffer containing NP-40 surfactant. Dialysis in PBS.

Kd measurement for binding of EM164 antibody with biotin-labeled IGF-I receptor was performed overnight at 1 ° C. in 1 g of Imulon-2HB plate in carbonate buffer (150 mM sodium carbonate, 350 mM sodium bicarbonate). / mL streptavidin was performed by coating with 100L. Wells coated with streptavidin were blocked with 200 L of blocking buffer (10 mg / mL BSA in TBS-T buffer), washed with TBS-T buffer and with biotin-labeled IGF-I receptor (10 to 100 ng) Incubated at room temperature for 4 hours. The wells containing the indirectly captured biotin-labeled IGF-I receptor are then washed and incubated with EM164 antibody in blocking buffer at various concentrations (5.110-13 M to 200 nM) for 2 hours at room temperature followed by 4 ° C. Incubated overnight. Wells were then washed with TBS-T buffer and incubated with goat-anti-mouse-IgGH + L-antibody-horseradish peroxidase conjugate (100L; 0.5 g / mL in blocking buffer), followed by 405 nm. Washed and detected using ABTS / H ₂ O ₂ substrate. Kd values were measured by non-linear regression for one-site binding.

Similar binding titrations were performed using Fab fragments of EM164 antibodies and described by E. Harlow and D. Lane ("Using Antibodies: A Laboratory Manual"; 1999, Cold Spring Harbor Laboratory Press, New York). Prepared by papain digestion of antibody as described.

Binding titration curves for the binding of EM164 antibody to the human IGF-I receptor labeled with biotin yielded a Kd value of 0.1 nM (FIG. 2). Fab fragments of the EM164 antibody also bound very tightly to the human IGF-I receptor with a 0.3 nM Kd value, indicating that the single binding of the EM164 antibody with the IGF-I receptor was also very strong.

The reason why the separation constant for binding of the IGF-I receptor is extremely low due to the EM164 antibody is evidenced by the strong binding signal observed after a 1-2-day extension of the antibody bound to the immobilized IGF-I receptor. As it is, partly due to the very slow koff ratio.

EM164 antibodies are demonstrated by culturing surfactant soluble lysates of human breast cancer MCF-7 cells with EM164 antibodies immobilized on protein G-agarose beads (Pierce Chemical Company). Can be used for immunoprecipitation. Western blots of EM164 antibody immunoprecipitations consisted of rabbit polyclonal anti-IGF-I receptor beta chain (C-terminus) antibodies (Santa Cruz Biotechnology) and goat-anti-rabbit-IgG-antibody-horseradish furs. It was detected using an oxidase conjugate, then washed and detected with ECL (chemiluminescence). Western blot of EM164 immunoprecipitation from MCF-7 cells showed a ring corresponding to the beta chain of the IGF-I receptor at about 95 kDa and pro-IGF-I receptor at about 220 kDa. Similar immunoprecipitation against other cell types was performed to check for species specificity in the binding of EM164 antibodies, which also bound to IGF-I receptors from cos-7 cells (African green monkeys), but with 3T3 cells ( Mice), CHO cells (chinese hamster) or goat fibroblasts did not bind to the IGF-I receptor. The EM164 antibody did not detect SDS-denatured human IGF-I receptors in western blots of lysate from MCF-7 cells, which bound to the conformaional epitopes of the original undenatured human IGF-I receptors. It is shown.

The binding region of the EM164 antibody was further characterized using a truncated alpha chain construct, cysteine flanked by the fused L1 and L2 regions (residues 1-468) along with the 16-mer-C-terminus (residues 704-719). The rich region was terminated by a C-terminal epitope tag. This smaller IGF-I receptor, lacking residues 469-703, has been known to bind to IGF-I, although looser than the original full length IGF-I receptor (Molina, L. et al., 2000, FEBS Letters, 467, 226-230; Kristensen, C. et al., 1999, J. Biol. Chem., 274, 37251-37356). Thus, a truncated IGF-I receptor alpha chain construct was prepared comprising residues 1-468 flanking the C-terminal with the C-terminal myc epitope tag flanking and residues 704-719. A stable cell line was constructed that expresses this construct and also transiently expresses the structure in kidney 293T cells of human embryos. Strong binding of this cleaved IGF-I receptor alpha chain construct with EM164 antibody was observed. Of the two antibodies tested, IR3 (Calbiochem) was also bound to this truncated alpha chain, but the 1H7 antibody (Santa Cruz Biotechnology) was not bound, indicating that the epitope of the EM164 antibody was clearly distinguished from the epitope of the 1H7 antibody. .

C. Inhibition of IGF-I Binding to MCF-7 Cells by EM164 Antibody

Binding of IGF-I cells to human breast cancer MCF-7 cells was inhibited by EM164 antibody (FIG. 3). MCF-7 cells were incubated with 5 μg / mL EM164 antibody for 2 hours or without EM164 antibody in serum-free medium and then incubated at 37 ° C. for 20 minutes with 50 ng / mL of IGF-I labeled with biotin. It became. Cells were then washed twice in serum-free medium to remove biotin-unbound -IGF-I and then lysed in 50 mM HEPES containing pH 7.4, 1% NP-40 and protein inhibitors. Imulon-2HB ELISA plates were coated with rodent monoclonal anti-IGF-I receptor beta chain antibodies and used to capture IGF-I receptor and biotin-bound -IGF-I from lysate. . Binding of the coated antibody to the cytoplasmic C-terminal region of the beta chain of the IGF-I receptor did not interfere with binding of the biotin-IGF-I to the extracellular region of the IGF-I receptor. Wells were washed, incubated with streptavidin-horseradish peroxidase conjugate, washed again and then detected using an ABTS / H ₂ O ₂ substrate. It is essentially quantitative that the 5 μg / mL EM164 antibody inhibits binding of IGF-I and MCF-7 cells and is equivalent to the inhibition of the ELISA background obtained using biotin-IGF-I deficiency control.

In addition to the assay described above for the EM164 antibody inhibiting binding of IGF-I to MCF-7 cells, endogenous physiological ligands (such as IGF-I or IGF-II) As required under physiological conditions for conflicting anti-IGF-I receptor antibodies for substitution, the following analysis shows that EM164 antibodies are quite effective against substituted region IGF-I from MCF-7 cells. In this IGF-I substitution assay, MCF-7 cells cultured in 12-well plates were biotin-labeled IGF-I in serum deficient and serum depleted at 37 ° C. (or 4 ° C.) for 1-2 hours. Incubate with (20-50 ng / ml). Biotin-labeled IGF-I binding cells are treated with EM164 antibody or regulatory antibody (10-100 μg / ml) at 37 ° C. (or 4 ° C.) for 30 minutes to 4 hours. Cells are then washed with PBS and lysed at 4 ° C. in lysis buffer containing 1% NP-40. An ELISA is performed to capture the IGF-I receptor from lysate as described above, followed by detecting the biotin-labeled IGF-I binding to the receptor using a streptavidin-horseradish peroxidase conjugate. This ELISA is almost complete (90% in 30 minutes and ~ 100% for 4 hours) at 37 ° C and about 50% for 2 hours at 4 ° C. EM164 is capable of pre-binding biotin-labeled IGF-I from cells. Prove that you can eliminate. In another experiment, NCl-H838 lung cancer cells were incubated with biotin-IGF-I, then washed and incubated with EM164 antibody for 2 hours at 4 ° C., resulting in an 80% reduction in bound biotin-IGF-I. . Therefore, EM164 antibody is quite effective in removing pre-binding-IGF-I from cancer cells. This will be an important treatment for the antagonism of IGF-I receptors by substitution of endogenous physiological ligand binding.

Longer incubation with EM164 antibody for 2 hours at 37 ° C downregulated IGF-I receptor 25%, but based on Western blot analysis using anti-IGF-I receptor beta chain antibody (Santa Cruze Biotechnology; sc713) Incubating MCF-7 cells with EM164 antibody for 2 hours at 4 ° C. (or 30 minutes at 37 ° C.) resulted in no significant downregulation of the IGF-I receptor. Therefore, in these short duration experiments, inhibition of IGF-I binding by EM164 antibody and removal of bound IGF-I at both 4 ° C. and 37 ° C. is not explained by downregulation of receptors due to binding of EM164 antibody. The strong inhibition of the binding of IGF-I receptors to IGF-I and the removal of pre-bound IGF-I by the EM164 antibody may be due to covalent or steric closure of the binding moiety or to allosteric effects. Either way, there is a possibility of a competitive competition.

D. Inhibition of IGF-I Receptor Mediated Cell Signaling by EM164 Antibody

Treatment of breast cancer MCF-7 cells and osteosarcoma SaOS-2 cells with EM164 antibody was performed by autophosphorylation of the IGF-I receptor and by insulin receptor substrate-1 (IRS-1), Akt and Erk1 / 2 (FIGS. 4-6). As shown by the inhibition of photophosphorylation of downstream effectors such as), it almost completely inhibited intracellular IGF-I receptor signaling.

In FIG. 4, MCF-7 cells were grown in 12-well plates in normal medium for 3 days and then treated with 20 μg / mL EM164 antibody (or anti-B4 regulatory antibody) in medium without serum for 3 hours, and then Stimulated with 50 ng / mL IGF-I for 20 minutes at 37 ° C. The cells were then protein and phosphorylation inhibitor (50 mM HEPES buffer, pH 7.4, 1% NP-40, 1 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate). (sodium pyrophosphate), 2.5 mM EDTA, 10 .mu.M leupeptin, 5 .mu.M pepstatin, 1 mM PMSF, 5 mM benzamidine, and 5.mu.g lysis in ice-cold lysis buffer containing / mL aprotinin). ELISA plates were precoated using anti-IGF-I receptor beta chain C-terminal monoclonal antibody TC123 and incubated with lysis samples for 5 hours at room temperature to capture IGF-I receptor. The wells containing the captured IGF-I receptor were then washed and incubated with biotin-labeled anti-phosphotyrosine antibody (PY20; 0.25 .mu.g / mL; BD Transduction Laboratories) for 30 minutes, followed by washing Incubated with streptavidin-horseradish peroxidase conjugate (0.8.mu.g / mL) for 30 minutes. Wells were washed and detected using an ABTS / H ₂ O ₂ substrate. The use of regulatory anti-B4 antibodies shows no inhibition of IGF-I stimulating autophosphorylation of IGF-I receptors. In contrast, complete inhibition of autophosphorylation stimulated by IGFI of the IGF-I receptor was obtained upon treatment with EM164 antibody (FIG. 4).

To demonstrate inhibition of phosphorylation of insulin receptor substrate-1 (IRS-1), an ELISA using immobilized anti-IRS-1 antibody to capture IRS-1 from lysate was used followed by phosphorylated-IRS-1 The associated p85 subunit of phosphatidylinositol-3-canase (PI-3-kinase) to be bound to was determined (Jackson, JG et al., 1998, J. Biol. Chem., 273, 9994-10003). .

In FIG. 5, MCF-7 cells were treated with 5 pg / mL antibody (EM164 or IR3) for 2 hours in serum-free medium and then stimulated with 50 μg / mL IGF-I at 37 ° C. for 10 minutes. Anti-IRS-1 antibody (rabbit polyclonal; Upstate Biotechnology) was indirectly captured by incubation with a goat-anti-rabbit-IgG antibody coated on an ELISA plate and then incubated overnight at 4 ° C. to obtain IRS from cell lysate samples. It was used to capture -1. Wells were then incubated with mouse monoclonal anti-p85-PI-3-kinase antibody (Upstate Biotechnology) for 4 hours and then treated with goat-anti-mouse-IgG-antibody-HRP conjugate for 30 minutes. Wells were then washed and detected using ABTS / H ₂ O ₂ substrate (FIG. 5). As shown in FIG. 5, EM164 antibody is more effective than IR3 antibody in inhibiting IRS-1 photophosphorylation stimulated by IGF-I, while EM164 antibody is incubated with cells in the absence of IGF-I. No negative effects were seen on monophosphorylation.

The activity of other downstream agonists such as Akt and Erk1 / 2 can also be assessed by rabbit polyclonal anti-phospho-Ser.sup. 473 Akt polyclonal and anti-phospho-ERK1 / 2 antibodies; Cell Signaling As shown using Western blot of the technology, SaOS-2 cells (FIG. 6) and MCF-7 cells were inhibited in a dose-dependent method by EM164 antibody. Pan-ERK antibodies demonstrated the same protein loading in all passages (FIG. 6). Treatment of SaOS-2 cells with EM164 antibody did not inhibit EGF-stimulated phosphorylation of Erk1 / 2, which showed inhibition specificity of the IGF-I receptor signaling pathway by EM164 antibody.

E. Inhibition of IGF-I-, IGF-II- and Serum-stimulated Growth and Survival of Human Tumor Cells by EM164 Antibody

Several human tumor cell lines have been tested for their growth response to IGF-I under serum removal conditions. These cell lines were treated with EM164 antibody in the presence of IGF-I, IGF-II, or serum and their growth responses were measured using MTT assay after 2-4 days. Approximately 1500 cells were plated in 96-well plates in regular medium with serum and replaced with serum-free medium the next day (either serum-free RPMI medium supplemented with transferrin and BSA, or phenol-red free medium as specified by Dufourny , B. et al., 1997, J. Biol. Chem., 272, 31163-31171). After growing one day in serum removal medium, cells were incubated with about 75 μL of 10 μg / mL antibody for 30 minutes to 3 hours followed by the addition of 25 μL of IGF-I (or IGF-II or serum) solution to 10 ng / mL IGF- A serum final concentration of I, or 20 ng / mL IGF-II, or 0.04-10% was obtained. In some experiments, cells were first stimulated with IGF-I for 15 minutes before adding EM164 antibody or both IGF-I and EM164 antibodies were added. The cells then grew for 2-3 days. Then MTT solution (3- (4,5) -dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; 25 .mu.L of a 5 mg / mL solution in PBS) was added and the cells were reincubated for 2-3 hours. The medium was then removed and replaced with 100 μL DMSO, mixed and the absorption of the plate was measured at 545 nm. Many human tumor cell lines are markedly inhibited by the EM164 antibody, whether the antibody was added prior to IGF-I or IGF-I prior to the antibody, or both IGF-I and the antibody were added. Growth and survival responses are shown with addition of -I or IGF-II or serum (Table 1).

Table 1 Inhibition of EM164 Antibodies on Growth and Survival of Tumor Cells Stimulated by IGF-I

^a MTT assay for daily growth / cell viability up to 3-4 days in response to 10 ng / mLIGF-I in serum-free medium containing 5-10ug / mL EM164 antibody

^b Colonization or cell growth inhibition by EM164 antibody in 1.25-10% serum in the presence of 5-10 μg / mL EM164 antibody based on comparison with control (antibodies without serum, and serum without antibodies);

MCF-7, NCI-H838, and SK-N- based on comparison with control (antibodies without serum, and serum without antibodies) to explain autocrine / paracrine IGF-stimulation by cells The degree of inhibition on SH cells was measured quantitatively.

EM164 antibody strongly inhibited the growth of breast cancer MCF-7 cells promoted by IGF-I or serum (FIG. 7,8). In a separate experiment, the EM164 antibody strongly inhibited the growth of IGF-II- promoted MCF-7 cells. Previous reports using commercially available antibodies, such as IR3 antibodies, showed only mild inhibition on the growth and survival of serum-promoted MCF-7 cells, as confirmed for IR3 and 1H7 in FIG. Cullen, KJ et al., 1990, Cancer Res., 50,48-53). In contrast, the EM164 antibody is a potent inhibitor of growth of serum or IGF-promoted MCF-7 cells. As shown in Figure 8, EM164 antibody equally effectively prevented the growth of MCF-7 cells at a wide range of serum concentrations (0.04-10% serum).

Growth inhibition of MCF-7 cells by EM164 antibody was measured by counting cell numbers. Thus, in a 12-well plate, approximately 7,500 cells were attached to the surface in an RPMI environment with 10% FBS, with or without 10 pg / mL of the EM164 antibody. After 5 days of growth, the cell count in the EM164 treated sample was only 1.7 × 10 ⁴ , whereas the cell count in the untreated control sample was 20.5 × 10 ⁴ . Treatment with EM164 antibody inhibited the growth of MCF-7 cells by about 12-fold for 5 days. This inhibition by the EM164 antibody is significantly greater than the reports of 2.5-fold inhibition using IR3 antibody in 6-day experiments on MCF-7 cells (Rohlik, QT et al., 1987, Biochem. Biophys. Res. Commun., 149,276-281).

Growth and survival of the small lung cancer cell line NCI-H838 stimulated by IGF-I- and serum was also strongly inhibited by the EM164 antibody compared to anti-B4 antibody control (FIG. 9). Treatment with EM164 antibodies in a serum-free environment results in a smaller signal than in both NCI-H838 and MCF-7 cell samples that were not treated with EM164 antibodies, possibly EM 164 antibodies also autocrine and release of these cells. This is because it inhibits the secretion (paracrine) IGF-I and IGF-1I promotion (Fig. 7,9). The colony size of HT29 colon cancer cells was also significantly reduced by EM164 antibody treatment.

Therefore, EM164 antibody is more than 80% superior to all known anti-IGF-I receptor antibodies in the efficacy of suppressing the growth of tumor cells such as MCF-7 cells and NCI-H838 cells by serum promotion.

The EM164 antibody triggered a slowing of cell growth during the G0 / G1 phase of the cell cycle and abolished the cleavage-inducing effect of IGF-I. For cell cycle analysis, MCF-7 cells were tested with IGF-I (20 ng / mL) in the presence or absence of EM164 (20 ug / mL) for 1 day, followed by propidium iodine staining and flow cytometry (flow cytometry). As shown in FIG. 25, one cycle of cells responding to IGF-I promotion in the absence of EM164 (with 41% S phase cells and 50% G0 / G1 phase cells) was performed on EM164-treated cells (S phase cells 9 % And 77% of G0 / G1 phase cells).

In addition to disrupting cell proliferation, EM164 antibody treatment results in cell death. For the measurement of apoptosis, cleavage of cytokeratin CK18 protein by caspase in NCI-H838 lung cancer cells incubated with IGF-I or serum in the presence or absence of EM164 for 1 day (FIG. 26). The addition of IGF-I or serum in the absence of EM164 weakened the cleavage of CK18 protein by caspase as compared to the absence of control for IGF-I, which suggests that IGF-I and serum may act as caspase. Inhibit. As indicated by the more cleavage of CK18 protein in the presence of EM164 than in the absence of EM164, EM164 treatment inhibited the anti-apoptotic effects of IGF-I and serum.

F. Synergistic Inhibition by EM164 Antibodies on Growth and Survival of Human Tumor Cells in Binding to Other Cytotoxic and Cytotoxic Inhibitors

Combined administration of EM164 antibody and Taxol inhibited the growth and survival of non-small cell lung cancer Calu6 cells more significantly than administration of Taxol alone. Similarly, combined administration of the EM164 antibody and camptothecin inhibited the growth and survival of colon cancer HT29 cells more than camptothecin alone. Since only the EM164 antibody could not be expected to be as toxic to cells as an organic toxic drug, the synergistic effect between the significant cell proliferation inhibitory effect of the EM164 antibody and the cytotoxic effect of the chemotoxic drug was a complex cancer in clinical prescription. There may be a high effect in therapy.

Combined administration of anti-EGF receptor antibody (KS77) and EM164 antibody is more like HT-3 cells, RD cells, MCF-7 cells, and A431 cells than administration of anti-EGF receptor antibody (KS77) or EM164 antibody alone. The growth and survival of some tumor cell lines was suppressed with more pronounced effects. Thus, synergistic effects of binding of neutralizing antibodies to two growth factor receptors, such as the IGF-I receptor and the EGF receptor, may be useful in clinical cancer experiments.

Combinations of EM164 antibodies and cytotoxic drugs are also valuable in delivering cytotoxic drugs by targeting tumors that overexpress IGF-I receptors. Binding of radioisotopes or other markers with EM164 antibodies can be used for the experimentation and imaging of tumors that overexpress IGF-I receptors.

G. Effect of EM164 treatment on human cancer xenograft in immunodeficient mice as mono- or in combination with anti-cancer drugs

Human non-small cell lung cancer Calu-6 xenografts are established by subcutaneous injection of 1 × 10 7 Calu-6 cells in immunodeficient mice. As shown in FIG. 10, five mice per treatment group were administered with EM164 antibody alone (6 0.8 mg injections per mouse, twice a week iv) to these mice containing established 100 mm ³ Calu-6 xenografts. ) Or only Taxol (5 injections of Taxol, ip every 2 days; 15 mg / Kg), or a combination of Taxol and EM164 antibody treatment, or PBS only (200 uL per mouse, 6 injections, 2 per week) Times, iv).

Tumor growth was significantly slowed down by EM164 antibody treatment compared to PBS control. Non-toxicity of EM164 antibody was observed based on the measurement of mouse weight. Treatment with Taxol alone was effective until day 14, but then tumors began to grow again. However, tumor growth was significantly retarded in the combined treatment group of Taxol and EM164 antibodies compared to the group treated with Taxol alone.

Human pancreatic cancer xenografts were established in female SCID / ICR mice (Taconic) 5 weeks old by subcutaneous injection of 107 BxPC-3 cells under PBS on the first day.

Mice suffering from established 80 mm ³ tumors were then tested with EM164 alone using five treatment groups, five per group (13 injections of 0.8 mg per iv, iv lateral tail vein, 12, 16, 19, 23, 26, 29, 36, 43, 50, 54, 58, 61 and 64 days) and gemcitabine alone (150 mg twice a kilogram per kg per shot, ip 12 and day 19) According to the above schedule, the test was conducted with gemcitabine and EM164 complex, and only with PBS and with control antibody (same as EM164 schedule). As shown in FIG. 27, the combined treatment with EM164 alone or with gemcitabine initially resulted in total degeneration of xenograft tumors in four out of five in the EM164 treatment group and in all five in the combined treatment group. Measurable tumor regrowth was observed in at least one animal at day 43 in the EM164 treatment group and at day 68 in the combination treatment group, and in control experiments (P = 0.029 and 0.002, respectively; two-tailed T-test; The mean tumor volume was significantly smaller at day 74 compared to FIG. 27).

In another study, EM164 antibody treatment (binding with a single or anti-EGF receptor antibody; intraperitoneal injection) inhibited the growth of established BxPC-3 xenografts in mice.

H. Cloning and Sequencing Light and Heavy Chains of EM164 Antibodies

Total RNA was purified from EM164 hybridoma cells. Reverse transcriptase reactions were performed using either 4-5 ug of total RNA and oligo dT or any of the hexamer primers.

PCR reactions were performed by Co et al. Using the RACE method described in (J. Immunol., 148, 1149-1154 (1992)) and the degenerated primers described in Wang et al., (J. Immunol. Methods, 233,167-177 (2000)). It became.

The RACE PCR method requires an intermediate step to add a poly G tail to the third end of the first chain of cDNA. RT reaction was purified with Qiagen column and eluted in 50 ul 1 × NEB buffer 4.

The dG tailing reaction is performed by lysis with 0.25 mM CoCl ₂ , 1 mM dGTP, and 5 unit end transferase (NEB) in 1 × NEB buffer 4.

The mixture was incubated at 37 ° C. for 30 minutes and then one fifth (10ul) of the reaction was added directly to the PCR reaction to serve as template DNA.

RACE and degraded PCR reactions are identical except for differences in primers and templates. Terminal transferase reactions were used directly for the RACE PCR template, while RT mixing was used directly for the degraded PCR reaction.

In both RACE and degraded PCR reactions, the same 3 'light chain primer HindKL-tatagagctcaagcttggatggtgggaagatggatacagttggtgc (SEQ ID NO: 14) and 3' light chain primer Bgl2IgGl-ggaagatctatagacagatgggggtgtcgttttggc (SEQ ID NO: 15) were used.

In RACE PCR, one poly C 5 'primer was used in both heavy and light chains:

EcoPolyC-TATATCTAGAATTCCCCCCCCCCCCCCCCC (SEQ ID NO: 16),

On the other hand, the degraded 5 'terminal PCR primers were SaclMK-GGGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO: 17) for the light chain, and EcoRlMHI-CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQID NO: 18) and EcoRlMHCTCS: NOGCGGG of the heavy chain. It was a mix of proportions.

In the above primer sequences, the mixed bases are defined as follows.

H = A + T + C, S = g + C, Y = C + T, K = G + T, M = A + C, R = A + g, W = A + T, V = A + C + G.

PCR reactions are performed according to the following program.

1) 94 ° C 3 minutes, 2) 94 ° C 15 seconds, 3) 45 ° C 1 minute, 4) 72 ° C 2 minutes, 5) 29 cycles in step # 2, 6) 10 ° C at 72 ° C for the final expansion step End.

PCR products are cloned into pBluescript II SK + (Stratagene) using restriction enzymes generated by PCR primers.

Several individual light and heavy chain clones were sequenced by conventional means to identify and prevent polymerase from generating sequence errors (FIGS. 12 and 13).

 Three light and heavy chain CDRs are defined using Chothia baseline definition (FIGS. 12-14).

Searching the NCBI IgBlast database showed that the light chain variable region of the anti-IGF-I receptor antibody was probably derived from the mouse IgVk Crl germination system gene, while the heavy chain variable region was probably derived from the mouse IgVhJ558.c germination system gene. (Figure 15).

Protein sequencing of the EM164 antibody in rodents was performed to confirm the sequences shown in FIGS. 12 and 13. Heavy and light chain protein bands of purified EM164 antibody were transferred from the gel (SDS_PAGE, reducing conditions) to the PVDF membrane, removed from the PVDF membrane, and analyzed by protein sequencing. The N-terminal sequence of the light chain was determined to be DVLMTQTPLS (SEQ ID NO: 20) by Edman sequencing, which was consistent with the N-terminal sequence of the cloned light chain obtained from EM164 hybridomas.

It has been found that the N-terminus of the heavy chain must be blocked for the Edman protein sequence.

The peptide fragment digested by trypsin of the heavy chain of mass1129.5 (M + H +, single isotope) is degraded via post-source decay (PSD) and its sequence is GRPDYYGSSK (SEQ ID NO: 21 Was decided. Peptide fragments digested by trypsin of the heavy chain of another mass2664.2 (M + H +, single isotope) were also degraded via post-source decay and its sequence was characterized by SSSTAYMQLSSLTSEDSAVYYFAR (SEQ ID NO: 22). These two sequences are completely identical to the sequences of CDR3 and construct 3 (FR3) of the cloned heavy chain gene obtained from EM164 hybridomas.

I. Recombinant Expression of EM164 Antibody

The binding sequence of the light and heavy chains was cloned into a single mammalian expression vector (FIG. 16). PCR primers for human variable sequences resulted in restriction regions allowing human signal base sequences to be attached to the pBluescriptII cloning vector, and the variable sequences were expressed in mammalian expression plasmids using EcoRI and BsiWI or HindIII and ApaⅠ for light or heavy chains, respectively. It was cloned (Figure 16). The light chain variable region was cloned into the human IgK constant region in the structure and the heavy chain variable region was cloned into the human Ig gamma constant region sequence. The human CMV promoter in the final expression plasmid resulted in the expression of both the light and heavy chain cDNA sequences. Expression and purification of recombinant mouse EM164 antibody is performed according to methods known in the art.

Example 2 Humanized Versions of EM164 Antibody

Resurfacing of EM164 antibodies to provide humanized variants suitable as therapeutic or diagnostic agents generally proceeds according to the principles and methods disclosed in US Pat. No. 5,639,641, as follows.

A. Surface Prediction

Solute accessibility of variable region residues of a series of antibodies with lysed structures is used to predict surface residues for the variable regions of rodent anti-IGF-I receptor antibodies (EM164).

Amino acid lysis accessibility for 127 unique antibody structure files (Table 2) is calculated using the MC software package (Pedersen et al., 1994, J. Mol. Biol., 235,959-973). The sequence alignment from the above 127 structure sets determines the ten most similar light and heavy chain amino acid sequences. The average solubility access for each variable region residue is calculated, and positions larger than the average accessibility of 30% are taken into account, resulting in surface residues. Positions with average accessibility between 25% and 35% are further checked by calculating individual residue accessibility only for structures with two identical side residues.

TABLE 2 127 Antibody Structure Referencing Brookhaven Database Used to Predict Surface of Anti-IGF-I-Receptor Antibody (EM164)

B. Molecular Modeling

Molecular models of rodent EM164 were made using the Oxford molecular software package AbM. The antibody structure was made from the structural file for the antibody with the most similar amino acid sequence, 2jel for the light chain and 1nqb for the heavy chain. Atypical CDRs are made by searching a C-α structure database that contains dissolved structures. Residues located within 5 of one CDR were determined.

C. Human Ab Selection

The surface position of rodent EM164 is compared with the corresponding position in the human antibody sequence in the Kabat database (Johnson, G. and Wu, T. T. (2001) Nucleic Acids Research, 29: 205-206).

Antibody database operating software SR (Searle 1998) was used to naturally align and derive antibody surface residues from heavy and light chain human antibody pairs.

With particular consideration given to positions within 5 of the CDRs, the human antibody surface with the most consistent surface residues was chosen to replace rodent anti-IGF-I receptor antibody surface residues.

D. PCR Mutations

PCR mutations were performed on these rodent EM164 cDNA clones to form resurfaced human EM164 (hereinafter huEM164).

Primer sets were designed to make the 8 amino acid mutations required for all experimental versions of huEM164, and additional primers were designed to alternately replace two 5 residues (Table 3).

PCR reactions were performed according to the following scheme. 1) 94 ° C 1 minute, 2) 94 ° C 15 seconds, 3) 55 ° C 1 minute, 4) 72 ° C 1 minute, 5) 29 cycles with # 2, 6) final expansion at 72 ° C for 4 minutes . PCR products were digested with the corresponding restriction enzymes and cloned into pBluescript replication vectors as mentioned above. Replication takes place sequentially to demonstrate the required amino acid replacement.

Table 3 PCR-primers used to generate four humanized EM164 antibodies

E. Variable Region Surface Residues

Pedersen et al. The antibody resurfacing techniques described in (J. Mol. Biol., 235, 959-973, 1994) and Roguska et al. (Protein Eng., 9,895-904, 1996) begin by predicting the surface residues of rodent antibody variable regions. do.

Surface residues are defined as amino acids whose water molecules have access to at least 30% of the total surface area of the amino acids.

Of the 127 antibody structure files, the ten most similar antibodies were identified (Figures 17 and 18). The lysis accessibility for each Kabat position was averaged for these aligned sequences and the distribution of relative accessibility for each residue is shown in FIG. Both light and heavy chains have 26 residues with at least 30% relative average accessibility (FIG. 19); Thus these residues are the surface residues predicted for EM164. Some residues have average accessibility between 25% and 35%, which are further checked by averaging only antibodies with two matching residues located on either side of the residue (Tables 4, 5). After this additional analysis, the original surface residues identified above remain unchanged.

TABLE 4 Surface residues and average accessibility for light and heavy chain variable sequences of EM164 antibody (ave.acc.)

TABLE 5

Residues having an average access between 25% and 35% are further analyzed by averaging a subset of antibodies with two identical residues located on either side of the residue in question. These landmark locations and their new average accessibility are given. NAs refer to residues that do not have the same side residues in the ten most similar antibodies.

F. Molecular modeling to determine which residues fall within 5 of the CDR

The molecular model generated by the AbM software package is analyzed to determine which EM164 surface residues are within 5 of the CDRs. In order to resurface the rodent EM164 antibody, all surface residues outside the CDRs must be modified into human counterparts, but residues within 5 of the CDRs are also treated with special protection as they may contribute to antigen specificity. Therefore, these latter residues must be identified and carefully considered throughout the humanization process. The CDR definitions used for resurfacing combine the AbM definitions for the heavy chain CDR2 and the Kabat definitions for the remaining 5 CDRs (FIG. 14).

Table 6 shows residues present within 5 of any CDR residues in the light or heavy chain sequence of the EM164 model.

TABLE 6 EM164 antibody structure surface residues within 5 ms of one CDR

G. Identification of the most similar human surface

Candidate human antibody surfaces for EM164 resurfacing have been identified using SR software within the Kabat antibody sequence database, where the SR software provides a search for only specified residue positions against the antibody database. In order to maintain a natural pair, the surface residues of both the light and heavy chains are compared together.

The most corresponding human surface from the Kabat database was sorted by sequence homology rank. The top five surfaces are given in Table 7. These surfaces are compared for identification of which of them requires minimal modification within 5 of the CDR. The leukemia B-cell antibody, CLL 1.69, requires a minimum number of surface residue changes (10 total) and only two of these residues are within 5 of the CDRs.

Full length variable region sequences for EM164 were also aligned corresponding to the Kabat human antibody database and CLL 1.69 was identified as the most similar human variable region sequence. In addition, this sequence comparison revealed that the human leukemia B-cell antibody CLL 1.69 was selective, as much as the human surface for EM164.

Table 7 Top Human Sequences Extracted from the Kabat Database

Alignment is caused by (Pederson1993). EM164 surface residues from within 5 underlined in one CDR.

H. Construction of the Humanized EM164 Gene

Ten surface residue changes for EM164 (Table 7) are made using PCR mutation techniques as mentioned above. Since 8 of the surface residues for CLL 1.69 are not within 5 of the CDRs, these residues are mutated in all humanized variants of EM164 from rodents to humans (Tables 8 and 9). Two light chain surface residues present within 5 of the CDRs (Kab at positions 3 and 45) are either mutated to human or maintained as rodents. Together these selections result in four humanized variants of the EM164 formed (FIGS. 22 and 23).

Of the four humanized variants, variant 1.0 has all ten human surface residues. The most conservative variant taking into account changes near the CDRs is variant 1.1, which preserves both rodent surface residues present within 5 of the CDRs. All four humanized EM164 antibody genes were cloned into antibody expression plasmids used for immediate and stable transformation (FIG. 16).

TABLE 8 Residue changes for variants 1.0 to 1.3 of humanized EM164 antibody

I. Comparison of affinity between humanized EM164 antibody variants and rodent EM164 antibodies for binding to full-length IGF-I receptors and truncated IGF-I receptor alpha chains

As mentioned above, the affinity of antibody variants 1.0-1.3 of humanized EM164 is competing for binding using the full-length human IGF-I receptor labeled with biotin or the truncated IGF-I receptor alpha chain labeled with myc-epitope. The analysis was compared to the affinity of rodent EM164 antibodies. Humanized EM164 antibody samples were obtained by transient transformation of appropriate expression vectors in kidney 293T cells of human embryos, and antibody concentrations were determined by ELISA using purified humanized antibody standards. For ELISA binding competition measurements, mixtures of humanized antibody samples with various concentrations of rodent Em164 antibodies were indirectly taken biotin-labeled full-length IGF-I receptors or truncated IGF-I receptors labeled with myc-epitope. Incubated with alpha chains. After equilibration, the bound humanized antibody was detected using a goat-anti-human-Fab'2-antibody-horseradish peroxidase conjugate. ((Rodent Ab] / [Coupled Humanized Ab]) vs ([Rodent Ab] / [Humanized Ab]) is theoretically based on a straight line with slope = (Kd humanized Ab / Kd rodent Ab) It was generated and used to determine the relative affinity of humanized and rodent antibodies.

A typical competitive analysis is shown in FIG. Imulon-2HB ELISA plates were coated for 7 hours at ambient temperature in carbonate buffer at 100 uL of 5 ug / mL streptavidin per well. Streptavidin-coated wells were blocked with 200 uL of blocking buffer (10 mg / mL BSA in TBS-T buffer) for 1 hour, washed with TBS-T buffer, and biotin-labeled IGF-I overnight at 4 ° C. Incubated with receptors (5 ng per well). The wells containing the indirectly harvested biotin-labeled IGF-I receptors were then washed and humanized EM164 antibody (15.5ng) and rodent antibodies (0ng, 16.35ng, 32.7ng) in 100uL blocking buffer for 2 hours at ambient temperature. , 65.4 ng, or 163.5 ng), followed by incubation at 4 ° C. overnight. Wells were then washed with TBS-T buffer and incubated with goat-anti-human-Fab'2-antibody-horseradish peroxidase conjugate for 1 hour (100 uL in blocking buffer; 1 ug / mL), Washed and detected using ABTS / H202 substrate at 405 nm.

([Rodent Ab] / [Bound Humanized Ab]) vs ([Rodent Ab] / [Humanized Ab]) shows a slope of 0.52 (= K _{d humanized Ab} / K _{d rodent Ab} ) The straight line (r ² = 0.996) was calculated. Therefore, humanized antibody variant 1.0 binds to the IGF-I receptor more tightly than rodent EM164 antibodies. Similar slopes ranging from approximately 0.5 to 0.8 compete for humanized EM164 antibody variants 1.0, 1.1, 1.2, and 1.3 with rodent EM164 to bind to full-length IGF-I receptors or to truncated IGF-I receptor alpha chains. The slope of the EM164 antibody of all humanized variants showed similar affinity, all better than that of the parental rodent EM164 antibody. The chimeric variant of EM164 with the 92F → C mutation in the heavy chain showed that in a similar binding competition with the rodent EM164 antibody, the 92F → C mutation of EM164 had three times lower affinity than the rodent EM164 antibody for binding to the IGF-I receptor. A tilt of about 3 was shown. Humanized EM164 vl.0 antibody showed similar inhibition on the growth and survival of MCF-7 cells by IGF-I-stimulation as did rodent EM164 antibody. Inhibition of serum-stimulated growth and survival of MCF-7 cells by humanized EM164 vl.0 antibody was similar to that by rodent EM164 antibody.

TABLE 9

The Kabat numbering structure is used for the light and heavy chain variable region polypeptides of the different variants of EM164Ab. Amino acid residues are grouped into structures (FR) and complementarity determining regions (CDRs), depending on their position in the polypeptide chain.

Kabat et al. Sequences of Proteins of Irnnmunological Interest, Fifth Edition, 1991, NIH Publication No. Reference is made from 91-3242.

J. Process for preparing enhanced anti-IGF-I-receptor antibodies starting from rodent and humanized antibody sequences

The amino acid and nucleic acid sequences of the anti-IGF-I receptor antibody EM164 and its humanized variants are within the scope of the present invention and used to develop other antibodies that improve properties. Such improved properties include improved affinity for IFG-I receptors. Numerous studies have investigated the effects of introducing various phase changes of one or more amino acids in the arrangement of antibodies based on primary antibody sequence knowledge and characteristics such as binding and concentration of expression (Yang, WP et al., 1995, J. Mol. Biol., 254,392-403; Rader, C. et al., 1998, Proc. Natl. Acad. Sci. USA, 95,8910-8915; Vaughan, TJ et al., 1998, Nature Biotechnology, 16,535 -539).

In this study, variants of various primary antibodies were identified using CDR1, CDR2, CDR3 using methods such as oligonucleotide-mediated site-directed mutations, cassette mutations, error-prone PCR, DNA shuffling or E. coli variant strains. Or by altering the sequence of heavy and light chain genes in the construct region (Vaughan, TJ et al., 1998, Nature Biotechnology, 16,535-539; Adey, NB et al., 1996, Chapter 16, pp. 277- 291, in "Phage Display of Peptides and Proteins", Eds. Kay, BK et al., Academic Press). These primary antibody sequence changes methods resulted in improved affinity of such secondary antibodies using standard screening techniques (Gram, H. et al., 1992, Proc. Natl. Acad. Sci. USA, 89,3576). -3580; Boder, ET et al., 2000, Proc. Natl. Acad. Sci. USA, 97,10701-10705; Davies, J. and Riechmann, L., 1996, Immunotechnolgy, 2,169-179; Thompson, J. et al., 1996, J. Mol. Biol., 256,77-88; Short, MK et al., 2002, J. Biol. Chem., 277, 16365-16370; Furukawa, K. et al., 2001 J. Biol. Chem., 276,27622-27628).

By a similar direct strategy of altering one or more amino acid residues of an antibody, the antibody sequences described herein are free amino groups or thiols, such as thiols, at convenient addition points for sharing, for example, for use in therapeutic addition. It can be used to develop anti-IGF-I receptor antibodies with enhanced functions such as antibodies with appropriate groups.

K. Alternative Expression Systems of Rodent, Chimeric, and Other Anti-IGF-I Receptor Antibodies

Rodent anti IGF-I receptor antibodies were also expressed in mammalian expression plasmids, similar to the method used to express humanized antibodies (above). Expression plasmids are known to have constant regions of rodents, including light chain kappa and heavy chain gamma-1 chains (McLean et al., 2000, Mol Immunol., 37,837-845). These plasmids were designed by simple restriction digestion and replication to accommodate the variable regions of any antibody, for example rodent anti-IGF-I receptor antibodies. Additional PCR of anti-IGF-I receptor antibodies is usually necessary to generate restrictions compatible with those in the expression plasmid.

An alternative approach to expressing fully rodent anti-IGF-I receptor antibodies has been to replace human constant regions in chimeric anti-IGF-I receptor antibody expression plasmids. Chimeric expression plasmids (FIG. 16) can be constructed using cassettes for both variable and light and heavy chain constant regions. Just as antibody variable sequences are replicated into expression plasmids by restriction digest, isolated restriction digests have been used to replicate any constant region sequences. For example, kappa light chain and gamma-1 heavy chain cDNAs were cloned from rodent hybridoma RNAs such as the RNAs described herein for replication of anti-IGF-1 antibody variable regions. Similarly, suitable primers were designed from sequences available in the Kabat database (Table 10). For example, RT-PCR was used to generate constant sites needed to replicate constant region sequences and to replicate these fragments into chimeric anti-IGF-I receptor antibody expression plasmids. This plasmid was then used to embody a complete rodent anti-IGF-I receptor antibody in a standard mammalian expression system such as the CHO cell line.

TABLE 10 Primers designed for replication of rodent gamma-1 constant region and rodent kappa constant region, respectively.

Primers were designed from sequences available in the Kabat database (Johnson, G and Wu, T. T. (2001) Nucleic Acids Research, 29: 205-206).

Hybridomas making rodent EM164 antibodies have been deposited with the Accession No. PTA-4457 at 2002, 6.14 to the American Type Culture Collection, PO Box 1549, Manassas, VA 20108 (ATCC) in accordance with the provisions of the Budapest Treaty.

Related patents and published publications are mentioned in this document, the contents of which are incorporated herein by reference in their entirety.

While the invention has been described in conjunction with the description and specific implementation thereof, it is apparent that various changes and modifications can be made by those skilled in the art without departing from the scope and spirit of the invention.

Antibodies that specifically bind or inhibit insulin-like growth factor-I receptors, human-adapted antibodies, resurfaced antibodies, antibody fragments, induced antibodies, and combinations thereof with cytotoxic factors may be associated with growth of tumor cells. It antagonizes the effects of IGF-I, IGF-II and serum in survival, with virtually no agonist activity. The antibodies and fragments thereof according to the present invention can be used for the treatment of tumors such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma, pancreatic cancer expressing elevated concentrations of IGF-I receptor, Derived antibodies can be used for the diagnosis and imaging of tumors expressing elevated concentrations of IGF-I receptors.

SEQUENCE LISTING <110> ImmunoGen, Inc. <120> ANTI-IGF-I RECEPTOR ANTIBODY <130> F170822 <140> PCT / US03 / 16211 <141> 2003-06-12 <150> 10 / 170,390 <151> 2002-06-14 <160> 96 <170> PatentIn version 3.3 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> antibody heavy chain complementarity determining region <400> 1 Ser Tyr Trp Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> antibody heavy chain complementarity determining region <400> 2 Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Arg <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> antibody heavy chain complementarity determining region <400> 3 Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp Val 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> antibody light chain complementarity determining region <400> 4 Arg Ser Ser Gln Ser Ile Val His Ser Asn Val Asn Thr Tyr Leu Glu 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> antibody light chain complementarity determining region <400> 5 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antibody light chain complementarity determining region <400> 6 Phe Gln Gly Ser His Val Pro Pro Thr 1 5 <210> 7 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> antibody heavy chain <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> antibody light chain <400> 8 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized light chain variable region <400> 9 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 10 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized light chain variable region <400> 10 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 11 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized light chain variable region <400> 11 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 12 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized light chain variable region <400> 12 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 13 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> humanized heavy chain variable region <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 14 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> degenerate 3 'light chain PCR primer-HindKL <400> 14 tatagagctc aagcttggat ggtgggaaga tggatacagt tggtgc 46 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> degenerate 3 'heavy chain PCR primer- Bgl2IgG1 <400> 15 ggaagatcta tagacagatg ggggtgtcgt tttggc 36 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> poly C 5 'PCR primer-EcoPolyC <400> 16 tatatctaga attccccccc cccccccccc 30 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> degenerate 5 'light chain PCR primer-Sac1MK <400> 17 gggagctcga yattgtgmts acmcarwctm ca 32 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> degenerate 5 'heavy chain PCR primer-EcoR1MH1 <220> <221> misc_feature (222) (18) .. (18) <223> "n" may be any nucleic acid <400> 18 cttccggaat tcsargtnma gctgsagsag tc 32 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> degenerate 5 'heavy chain PCR primer-EcoR1MH2 <220> <221> misc_feature (222) (18) .. (18) <223> "n" may be any nucleotide <400> 19 cttccggaat tcsargtnma gctgsagsag tcwgg 35 <210> 20 <211> 10 <212> PRT <213> Mus musculus <400> 20 Asp Val Leu Met Thr Gln Thr Pro Leu Ser 1 5 10 <210> 21 <211> 10 <212> PRT <213> Mus musculus <400> 21 Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys 1 5 10 <210> 22 <211> 24 <212> PRT <213> Mus musculus <400> 22 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 1 5 10 15 Ser Ala Val Tyr Tyr Phe Ala Arg 20 <210> 23 <211> 57 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 23 caggtgtaca ctcccaggtc caactggtgc agtctggggc tgaagtggtg aagcctg 57 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 24 caatcagaag ttccagggga aggccacac 29 <210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 25 ccttcccctg gaacttctga ttgtagttag tacg 34 <210> 26 <211> 37 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 26 caggtgtaca ctccgatgtt gtgatgaccc aaactcc 37 <210> 27 <211> 37 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 27 caggtgtaca ctccgatgtt ttgatgaccc aaactcc 37 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 28 gactagatct gcaagagatg gaggctggat ctccaagac 39 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 29 ttgcagatct agtcagagca tagtacatag taatg 35 <210> 30 <211> 48 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 30 gaatggtacc tgcagaaacc aggccagtct ccaaggctcc tgatctac 48 <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 31 gtggcagtgg agcagggaca gatttcac 28 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 32 gaaatctgtc cctgctccac tgccactg 28 <210> 33 <211> 19 <212> PRT <213> Homo sapiens <400> 33 Asp Leu Thr Leu Leu Gln Pro Gly Gln Lys Gly Asp Ser Arg Glu Lys 1 5 10 15 Lys Arg Ala <210> 34 <211> 18 <212> PRT <213> Homo sapiens <400> 34 Asp Val Thr Leu Leu Pro Pro Gly Gln Arg Gly Asp Ala Arg Glu Lys 1 5 10 15 Lys arg <210> 35 <211> 19 <212> PRT <213> Homo sapiens <400> 35 Asp Gln Ser Leu Ile Pro Pro Gly Gln Lys Gly Asp Ser Arg Asp Lys 1 5 10 15 Lys Arg Ala <210> 36 <211> 18 <212> PRT <213> Homo sapiens <400> 36 Asp Met Ser Ser Val Arg Pro Gly Gln Lys Gly Ser Ser Ser Asp Lys 1 5 10 15 Lys arg <210> 37 <211> 18 <212> PRT <213> Homo sapiens <400> 37 Glu Val Ser Gly Pro Arg Pro Gly Gln Arg Gly Asp Ser Arg Glu Lys 1 5 10 15 Lys arg <210> 38 <211> 18 <212> PRT <213> Homo sapiens <400> 38 Glu Val Ser Gly Pro Arg Pro Gly Gln Arg Gly Asp Ser Arg Glu Lys 1 5 10 15 Lys arg <210> 39 <211> 25 <212> PRT <213> Homo sapiens <400> 39 Gln Gln Gln Ala Leu Lys Pro Gly Lys Lys Thr Pro Gly Gln Glu Lys 1 5 10 15 Lys Arg Lys Ser Ser Ser Glu Ala Ser 20 25 <210> 40 <211> 25 <212> PRT <213> Homo sapiens <400> 40 Gln Gln Val Ala Val Lys Pro Gly Lys Lys Thr Pro Gly Gln Gln Lys 1 5 10 15 Gln Gly Lys Ser Ser Ser Glu Gln Ser 20 25 <210> 41 <211> 25 <212> PRT <213> Homo sapiens <400> 41 Gln Gln Gln Pro Leu Lys Pro Gly Lys Lys Thr Pro Gly Lys Asp Asp 1 5 10 15 Lys Gly Thr Ser Asn Asn Glu Gln Ser 20 25 <210> 42 <211> 25 <212> PRT <213> Homo sapiens <400> 42 Gln Gln Val Ala Val Lys Pro Gly Lys Lys Thr Pro Gly Gln Gln Lys 1 5 10 15 Lys Gly Lys Ser Ser Ser Glu Gln Ser 20 25 <210> 43 <211> 24 <212> PRT <213> Homo sapiens <400> 43 Gln Val Ala Val Lys Pro Gly Lys Lys Thr Pro Gly Gln Gln Lys Gln 1 5 10 15 Gly Lys Ser Ser Ser Glu Gln Ser 20 <210> 44 <211> 24 <212> PRT <213> Homo sapiens <400> 44 Gln Val Ala Val Lys Pro Gly Lys Lys Thr Pro Gly Gln Gln Lys Gln 1 5 10 15 Gly Glu Ser Ser Ser Glu Gln Ser 20 <210> 45 <211> 40 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 45 ttttgagctc ttatttacca ggagagtggg agaggctctt 40 <210> 46 <211> 37 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 46 ttttaagctt gccaaaacga cacccccatc tgtctat 37 <210> 47 <211> 32 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 47 ttttggatcc taacactcat tcctgttgaa gc 32 <210> 48 <211> 31 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 48 ttttgaattc gggctgatgc tgcaccaact g 31 <210> 49 <211> 396 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (396) <400> 49 atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct gct 48 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 tcc agt agt gat gtt ttg atg acc caa act cca ctc tcc ctg cct gtc 96 Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 agt ctt gga gat caa gcc tcc atc tct tgc aga tct agt cag agc att 144 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 35 40 45 gta cat agt aat gta aac acc tat tta gaa tgg tac ctg cag aaa cca 192 Val His Ser Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60 ggc cag tct cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct 240 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 ggg gtc cca gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca 288 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 ctc agg atc agc aga gtg gag gct gag gat ctg gga att tat tac tgc 336 Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys 100 105 110 ttt caa ggt tca cat gtt cct ccg acg ttc ggt gga ggc acc aag ctg 384 Phe Gln Gly Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 gaa atc aaa cgg 396 Glu Ile Lys Arg 130 <210> 50 <211> 132 <212> PRT <213> Mus musculus <400> 50 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 35 40 45 Val His Ser Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg 130 <210> 51 <211> 429 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (429) <400> 51 atg gga tgg agc tat atc atc ctc ttt ttg gta gca aca gct aca gaa 48 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Glu 1 5 10 15 gtc cac tcc cag gtc caa ctg cag cag tct ggg gct gaa ctg gtg aag 96 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys 20 25 30 cct ggg gct tca gtg aag ctg tcc tgt aag gct tct ggc tac acc ttc 144 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 acc agc tac tgg atg cac tgg gtg aag cag agg cct gga caa ggc ctt 192 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gag att aat cct agc aac ggt cgt act aac tac aat 240 Glu Trp Ile Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 gag aag ttc aag agg aag gcc aca ctg act gta gac aaa tcc tcc agc 288 Glu Lys Phe Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 aca gcc tac atg caa ctc agc agc ctg aca tct gag gac tct gcg gtc 336 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 tat tac ttt gca aga gga aga cca gat tac tac ggt agt agc aag tgg 384 Tyr Tyr Phe Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp 115 120 125 tac ttc gat gtc tgg ggc gca ggg acc acg gtc acc gtc tcc tca 429 Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 130 135 140 <210> 52 <211> 143 <212> PRT <213> Mus musculus <400> 52 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Glu 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Phe Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp 115 120 125 Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 130 135 140 <210> 53 <211> 10 <212> PRT <213> Mus musculus <400> 53 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> 54 <211> 10 <212> PRT <213> Mus musculus <400> 54 Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn 1 5 10 <210> 55 <211> 15 <212> PRT <213> Mus musculus <400> 55 Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp Val 1 5 10 15 <210> 56 <211> 100 <212> PRT <213> Mus musculus <400> 56 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro 100 <210> 57 <211> 98 <212> PRT <213> Mus musculus <400> 57 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala arg <210> 58 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 58 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 59 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 59 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Ser Ile Ser Ser Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Gln Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 60 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 60 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Thr His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 61 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 61 Asp Ile Glu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 62 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 62 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Phe Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Ser Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 63 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 63 Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser 20 25 30 Asn Gly Asp Thr Tyr Leu Asp Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 64 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 64 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Thr Ile Leu Leu Ser 20 25 30 Asp Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 65 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 65 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Ile Val His Ser 20 25 30 Ser Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Ile Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 66 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 66 Asp Val Leu Met Thr Gln Ile Pro Val Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 67 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 67 Asp Val Leu Met Thr Gln Thr Pro Val Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Ser His Ala Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 68 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 68 Asp Val Leu Met Thr Gln Ile Pro Val Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 69 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <220> <221> MISC_FEATURE (222) (28) .. (28) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (101) .. (101) <223> "X" may be any amino acid <400> 69 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Xaa Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Xaa Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 70 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 70 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 71 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 71 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Asp Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 72 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 72 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Asp Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 73 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 73 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Gly Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Pro Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Tyr Gly Thr Ser Tyr Gly Val Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 74 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 74 Gln Val Gln Leu Gln Gln Pro Gly Ser Val Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Trp Ile His Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile His Pro Asn Ser Gly Asn Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Val Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Tyr Gly Ser Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 75 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 75 Gln Val Gln Phe Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Leu Met His Trp Ile Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asn Val Val Thr Lys Phe Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Ala Tyr Cys Arg Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 76 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 76 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Lys Phe Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 77 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 77 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Phe 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Gly Thr His Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly His Ser Tyr Tyr Phe Tyr Asp Gly Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 78 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 78 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Met Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Lys Thr Thr Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ala 115 120 <210> 79 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 79 Gln Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Phe 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Gly Thr His Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly His Ser Tyr Tyr Phe Tyr Asp Gly Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 80 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 80 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr Trp 20 25 30 Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly 35 40 45 Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr His Glu Arg Phe Lys 50 55 60 Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Gly Val Tyr Tyr Cys Leu 85 90 95 His Gly Asn Tyr Asp Phe Asp Gly Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 81 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <220> <221> MISC_FEATURE (222) (20) .. (20) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (34) .. (34) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (43) .. (43) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (50) .. (50) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (52) .. (52) <223> "X" may be any amino acid <220> <221> MISC_FEATURE <222> (54) .. (54) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (57) .. (57) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (59) .. (59) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (99) .. (99) <223> "X" may be any amino acid <220> <221> MISC_FEATURE <222> (100) .. (100) <223> "X" may be any amino acid <220> <221> MISC_FEATURE <222> (103) .. (108) <223> "X" may be any amino acid <220> <221> MISC_FEATURE 116 (116) .. (116) <223> "X" may be any amino acid <400> 81 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Xaa His Trp Val Lys Gln Arg Pro Gly Xaa Gly Leu Glu Trp Ile 35 40 45 Gly Xaa Ile Xaa Pro Xaa Ser Gly Xaa Thr Xaa Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Val Tyr Cys 85 90 95 Ala Arg Xaa Xaa Tyr Tyr Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Xaa Val Thr Val Ser Ser 115 120 <210> 82 <211> 113 <212> PRT <213> Mus musculus <400> 82 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 83 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 83 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 84 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 84 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 85 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 85 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 86 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 86 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 87 <211> 123 <212> PRT <213> Mus musculus <400> 87 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser 115 120 <210> 88 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 88 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 <210> 89 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> variable region of humanized EM164 antibody-light chain <220> <221> CDS (222) (1) .. (339) <400> 89 gat gtt gtg atg acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat cca gcc tcc atc tct tgc aga tct agt cag agc ata gta cat agt 96 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 aat gta aac acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca agg ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga gca ggg aca gat ttc aca ctc agg atc 240 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga att tat tac tgc ttt caa ggt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 tca cat gtt cct ccg acg ttc ggt gga ggc acc aaa ctg gaa atc aaa 336 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 cgt 339 Arg <210> 90 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 90 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 91 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> variable region of humanized EM164 antibody-heavy chain <220> <221> CDS (222) (1) .. (369) <400> 91 cag gtc caa ctg gtg cag tct ggg gct gaa gtg gtg aag cct ggg gct 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 tca gtg aag ctg tcc tgt aag gct tct ggc tac acc ttc acc agc tac 96 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 tgg atg cac tgg gtg aag cag agg cct gga caa ggc ctt gag tgg att 144 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gag att aat cct agc aac ggt cgt act aac tac aat cag aag ttc 192 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60 cag ggg aag gcc aca ctg act gta gac aaa tcc tcc agc aca gcc tac 240 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 atg caa ctc agc agc ctg aca tct gag gac tct gcg gtc tat tac ttt 288 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe 85 90 95 gca aga gga aga cca gat tac tac ggt agt agc aag tgg tac ttc gat 336 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110 gtc tgg ggc caa ggg acc acg gtc acc gtc tcc 369 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 <210> 92 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 92 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 <210> 93 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> light chain variable region of humanized EM164 v1.1 antibody <220> <221> CDS (222) (1) .. (339) <400> 93 gat gtt ttg atg acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat cca gcc tcc atc tct tgc aga tct agt cag agc ata gta cat agt 96 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 aat gta aac acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga gca ggg aca gat ttc aca ctc agg atc 240 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga att tat tac tgc ttt caa ggt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 tca cat gtt cct ccg acg ttc ggt gga ggc acc aaa ctg gaa atc aaa 336 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 cgt 339 Arg <210> 94 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 94 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 95 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> light chain variable region of humanized EM164 v1.2 antibody <400> 95 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tccagcctcc 60 atctcttgca gatctagtca gagcatagta catagtaatg taaacaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaagg ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggagcaggga cagatttcac actcaggatc 240 agcagagtgg aggctgagga tctgggaatt tattactgct ttcaaggttc acatgttcct 300 ccgacgttcg gtggaggcac caaactggaa atcaaacgt 339 <210> 96 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> light chain variable region of humanized EM164 v1.3 antibody <400> 96 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tccagcctcc 60 atctcttgca gatctagtca gagcatagta catagtaatg taaacaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggagcaggga cagatttcac actcaggatc 240 agcagagtgg aggctgagga tctgggaatt tattactgct ttcaaggttc acatgttcct 300 ccgacgttcg gtggaggcac caaactggaa atcaaacgt 339

Claims (61)

An antibody or antibody fragment that specifically binds to an insulin-like growth factor-I receptor (IGF-IR) that is an antagonist of a receptor and is substantially free of agonist activity on the receptor. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is resurfaced. The antibody or antibody fragment according to claim 1, wherein the antibody or antibody fragment is humanized or humanized. Insulin-like growth factor-I receptor, which may inhibit the growth of tumor cells by at least 80% in the presence of a growth promoter selected from the group consisting of serum, insulin-like growth factor-I receptor and insulin-like growth factor-II receptor An antibody or antibody fragment that specifically binds (IGF-IR). The antibody of claim 4, wherein the tumor cells are human tumor cells. An antibody or antibody fragment comprising at least one complementarity determining region having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-6: SYWMH (SEQ ID NO: 1), EINPSNGRTNYNEKFKR (SEQ ID NO: 2), GRPDYYGSSKWYFDV (SEQ ID NO: 3), RSSQSIVHSNVNTYLE (SEQID NO: 4), KVSNRFS (SEQ ID NO: 5), FQGSHVPPT (SEQ ID NO: 6). The heavy chain region comprises three consecutive complementarity determining regions each having an amino acid sequence represented by SEQ ID NOS: 1-3, SYWMH (SEQ ID NO: 1), EINPSNGRTNYNEKFKR (SEQ ID NO: 2), GRPDYYGSSKWYFDV (SEQ ID NO: 3), The light chain region comprises three consecutive complementarity determining regions each having an amino acid sequence represented by SEQ ID NOS: 4-6, RSSQSIVHSNVNTYLE (SEQ ID NO: 4), KVSNRFS (SEQ ID NO: 5), FQGSHVPPT (SEQ ID NO: 6), An antibody or antibody fragment comprising at least one heavy chain region and at least one light chain region. 8. The antibody or fragment of claim 7, wherein the heavy chain region has at least 90% nucleotide homology with the amino acid sequence represented by SEQ ID NO: 7. QVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSS KWYFDVWGAGTTVTVSS (SEQ ID NO: 7). 9. The antibody or antibody fragment of claim 8, wherein the heavy chain region has at least 95% homology with the amino acid sequence represented by SEQ ID NO: 7. The antibody or antibody fragment according to claim 8, wherein the heavy chain region has an amino acid sequence represented by SEQ ID NO: 7. 8. The antibody or fragment of claim 7, wherein the light chain region has at least 90% nucleotide sequence homology with the amino acid sequence represented by SEQ ID NO: 8. DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 8). 12. The antibody or antibody fragment of claim 11, wherein the light chain region has at least 95% nucleotide sequence homology with the amino acid sequence represented by SEQ ID NO: 8. The antibody or antibody fragment of claim 11, wherein the light chain region has an amino acid sequence represented by SEQ ID NO: 8. 13. The antibody or antibody fragment according to claim 1, comprising at least one complementarity determining region having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-6. SYWMH (SEQ ID NO: 1), EINPSNGRTNYNEKFKR (SEQ ID NO: 2), GRPDYYGSSKWYFDV (SEQ ID NO: 3), RSSQSIVHSNVNTYLE (SEQ ID NO: 4), KVSNRFS (SEQ ID NO: 5), FQGSHVPPT (SEQ ID NO: 6). The method according to claim 1, wherein the heavy chain region comprises three consecutive complementarity determining regions each having an amino acid sequence represented by SEQ ID NOS: 1-3, SYWMH (SEQ ID NO: 1), EINPSNGRTNYNEKFKR (SEQ ID NO: 2), GRPDYYGSSKWYFDV (SEQ ID NO: 3), The light chain region comprises three consecutive complementarity determining regions each having an amino acid sequence represented by SEQ ID NOS: 4-6, RSSQSIVHSNVNTYLE (SEQ ID NO: 4), KVSNRFS (SEQ ID NO: 5), FQGSHVPPT (SEQ ID NO: 6), An antibody or antibody fragment comprising at least one heavy chain region and at least one light chain region. The antibody or antibody fragment according to claim 1, comprising a heavy chain region having at least 90% nucleotide sequence homology with the amino acid sequence represented by SEQ ID NO: 7. QVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEI NPSNGRTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSS KWYFDVWGAGTTVTVSS (SEQ ID NO: 7). 17. The antibody or fragment of claim 16, wherein the heavy chain region has at least 95% homology with the amino acid sequence represented by SEQ ID NO: 7. The antibody or antibody fragment according to claim 16, wherein the heavy chain region has an amino acid sequence represented by SEQ ID NO: 7. The antibody or antibody fragment according to claim 1, comprising a light chain region having a nucleotide sequence homology of at least 90% to the amino acid sequence represented by SEQ ID NO: 8. DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 8). 20. The antibody or fragment of claim 19, wherein the light chain region has at least 95% nucleotide homology with the amino acid sequence represented by SEQ ID NO: 8. 20. The antibody or fragment of claim 19, wherein the light chain region has an amino acid sequence represented by SEQ ID NO: 8. The method of claim 1, DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIY KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 9); DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 10); DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIY KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 11); And DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 12) comprising a light chain variable region comprising a fragment or an antibody having a sequence selected from the group consisting of. The antibody or antibody fragment of claim 1 comprising a heavy chain variable region having a sequence expressed by SEQ ID NO: 13. QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEI NPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSS KWYFDVWGQGTTVTVSS (SEQ ID NO: 13). A pharmaceutical composition comprising the antibody or antibody fragment of claim 1 and a pharmaceutically acceptable carrier. A conjugate comprising the antibody or antibody fragment of claim 1 linked to a cytotoxic agent. 26. The group of claim 25, wherein the cytotoxic agent is a group consisting of maytansinoids, small drugs, prodrugs, taxoids, CC-1065 and CC-1065 analogs. Combination characterized in that the selected from. A pharmaceutical composition comprising the conjugate of claim 26 and a pharmaceutically acceptable carrier. A diagnostic reagent comprising the composition of claim 24 or 27 labeled with an antibody or antibody fragment. 29. The diagnostic reagents of claim 28, wherein the label is selected from the group consisting of radioisotope labels, fluorescent labels, luminescent labels, imaging media and metal ions. A method of inhibiting growth of tumor cells, comprising contacting tumor cells with the antibody or antibody fragment of claim 1. A method of treating cancer patients, comprising administering to a cancer patient an effective amount of the antibody or antibody fragment of claim 1. 32. The method of claim 31 further comprising administering a therapeutic agent to said patient. 33. The method of claim 32, wherein said therapeutic agent is a cytotoxic agent. A method of treating cancer patients comprising administering to a cancer patient an effective amount of the conjugate of claim 25. 32. The method of claim 31, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, ovarian cancer, malignant bone marrow tumor, cervical cancer, prostate cancer, lung cancer, synovial cancer, and pancreatic cancer. Administering the diagnostic reagent of claim 28 to a subject suspected of having cancer; And detecting a distribution of said reagent from said subject. The method of claim 36, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, ovarian cancer, malignant bone marrow tumor, cervical cancer, prostate cancer, lung cancer, synovial cancer, and pancreatic cancer. (a) providing a DNA encoding an antibody or antibody fragment comprising at least one sequence selected from the group consisting of SEQ ID NOS: 1-8; SYWMH (SEQ ID NO: 1), EINPSNGRTNYNEKFKR (SEQ ID NO: 2), GRPDYYGSSKWYFDV (SEQ ID NO: 3), RSSQSIVHSNVNTYLE (SEQ ID NO: 4), KVSNRFS (SEQ ID NO: 5), FQGSHVPPT (SEQ ID NO: 6), QVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLE WIGEINPSNGRTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVY YFARGRPDYYGSSKWYFDVWGAGTTVTVSS (SEQ ID NO: 7), DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNVNTYLEWYLQKPGQSP KLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSH VPPTFGGGTKLEIKR (SEQ ID NO: 8); (b) inducing at least one nucleotide variation, deletion or insertion into the DNA to change the amino acid sequence of the antibody or antibody fragment encoded by the DNA; (c) expressing the antibody or antibody fragment; (d) An improved antibody or antibody fragment that specifically binds to an insulin-like growth factor-I receptor prepared by screening the antibody or antibody fragment for producing an improved antibody or antibody fragment. The antibody or antibody fragment of claim 38, wherein said improvement is enhanced affinity for the IGF-I receptor. The method of claim 38, wherein the at least one nucleotide variant, deletion or insertion comprises oligonucleotide mediated site-directed variation, cassette variation, error-prone PCR, DNA shuffling ( and an antibody fragment, characterized in that formed by a method selected from the group consisting of shuffling) and the use of variant strains of E. coli . A polynucleotide encoding the antibody or antibody fragment of any one of claims 6, 7, 8 or 11. A polynucleotide encoding the light chain or heavy chain region of the antibody or fragment of any one of claims 6, 7, 8 or 11. A vector comprising the polynucleotide of claim 41. A vector comprising the polynucleotide of claim 42. The vector of claim 43, wherein the vector is an expression vector capable of expressing the antibody or antibody fragment. 45. The vector of claim 44, wherein the vector is an expression vector capable of expressing the antibody or antibody fragment. The hybridoma cell line deposited with ATCC Accession No. PTA-4457, or an antibody or antibody fragment, is an antagonist of insulin-like growth factor-I receptors and is substantially free of agonist activity on the receptor. Rodent antibody EM164 produced by an antibody fragment that specifically binds to an I receptor. The hybridoma cell line deposited with ATCC Accession No. PTA-4457, or an antibody or antibody fragment, is an antagonist of insulin-like growth factor-I receptors and is substantially free of agonist activity on the receptor. Humanized or resurfaced antibody derived from EM164 prepared by an antibody fragment that specifically binds to a receptor. The antibody of claim 1, wherein the IGF-IR is human IGF-IR. Hybridoma cell line EM164 deposited with ATCC accession number PTA-4457. A method for inducing apoptosis in a cell, comprising contacting the cell with the antibody or antibody fragment of claim 1. A method of inducing apoptosis in a cell comprising contacting said cell with the antibody or antibody fragment of claim 47. A method of inducing apoptosis in a cell, comprising contacting said cell with the antibody or antibody fragment of claim 48. 54. The method of any one of claims 51 to 53, wherein said cells are cancer cells. The method of claim 1, a) inhibiting the cellular function of IGF-IR without activating it; b) inhibiting tumor cell growth by at least 80% in the presence of serum; c) binds to IGF-IR with a K _D of less than or equal to 0.3 × 10 ⁻⁹ M; d) inhibits binding of IGF-I to IGF-IR; e) inhibits IGF-I mediated cells by signaling by IGF-IR; f) inhibits IGF-IR phosphorylation induced by IGF-I; g) inhibits IGF-I and IGF-II mediated cell growth and survival; h) African green monkeys bind to IGF-IR but not to mouse, Chinese hamster or goat IGF-IR; And i) inhibits the activity of IRS-1, Akt and Erk 1/2; An antibody or antibody fragment having at least one property selected from the group consisting of: 56. The antibody or antibody fragment according to claim 55, wherein said antibody or antibody fragment comprises all of the above properties. The method of claim 1, The antibody or antibody fragment a) immunoglobulin (Ig) G1-4, IgM, IgA1-2, IgD or IgE molecules, or derivatives thereof; b) an antibody or antibody fragment, characterized in that it is a single chain antibody, a single chain antibody fragment (scFvs), a Fab fragment, a F (ab ') ₂ fragment, or a fragment comprising at least three CDRs. A host cell comprising the vector of claim 43. A host cell comprising the vector of claim 44. Anti-IGF-IR antibody comprising culturing the host cell according to claim 58 under suitable conditions for expressing the anti-IGF-IR antibody or antibody fragment and obtaining the anti-IGF-IR antibody or antibody fragment. Or a method for producing an antibody fragment. Anti-IGF-IR antibody comprising culturing the host cell according to claim 59 under suitable conditions for expressing the anti-IGF-IR antibody or antibody fragment and obtaining the anti-IGF-IR antibody or antibody fragment. Or a method for producing an antibody fragment.