JP2008502589A - Anti-IGF-I receptor antibody - Google Patents

Abstract

  Specifically binds to and inhibits insulin-like growth factor I receptor, antagonizes the effects of IGF-I, IGF-II, and serum on tumor cell growth and survival, and provides substantial agonist activity Antibody, humanized antibody, resurfaced antibody, antibody fragment, derivatized antibody, and conjugate of the antibody with a cytotoxic agent. Antibodies and fragments thereof, optionally in combination with other therapeutic agents, to treat tumors with elevated IGF-I receptor expression levels, such as breast cancer, colon cancer, lung cancer, ovarian carcinoma, synovial sarcoma, prostate cancer and pancreatic cancer And derivatized antibodies can be used for diagnosis and imaging of tumors with elevated IGF-I receptor expression levels.

Description

  [01] This application is a continuation-in-part of the parent application 10 / 170,390, June 14, 2002, which is incorporated herein in its entirety.

FIELD OF THE INVENTION [02] The present invention relates to antibodies that bind to human insulin-like growth factor I receptor (IGF-I receptor). More particularly, the present invention relates to anti-IGF-I receptor antibodies that inhibit the cellular function of IGF-I receptors. Even more particularly, the present invention relates to anti-IGF-I receptor antibodies that antagonize the effects of IGF-I, IGF-II, and serum on tumor cell growth and survival and substantially lack agonist activity. About. The invention also relates to fragments of the antibody, humanized and resurfaced versions of the antibody, conjugates of the antibody, antibody derivatives, and their use in diagnostic, research and therapeutic applications. . The invention further relates to improved antibodies or fragments thereof made from the above-described antibodies and fragments thereof. In another aspect, the present invention relates to a polynucleotide encoding an antibody or fragment thereof and to a vector comprising the polynucleotide.

BACKGROUND OF THE INVENTION [03] Insulin-like growth factor I receptor (IGF-I receptor) is a transmembrane heterotetrameric protein having two extracellular alpha chains and two transmembrane beta chains, these The chains are disulfide linked to form a β-α-α-β configuration. When the ligands insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) bind to the extracellular domain of the IGF-I receptor, the intracellular tyrosine kinase domain is activated, Receptor autophosphorylation and substrate phosphorylation occur. The IGF-I receptor is homologous to the insulin receptor, has 84% high sequence similarity in the beta chain tyrosine kinase domain, and 48% lower sequence in the alpha chain extracellular cysteine-rich domain Similarities (Ulrich, A. et al., 1986, EMBO, 5, 2503-2512; Fujita-Yamaguchi, Y. et al., 1986, J. Biol. Chem., 261, 16727-16731; LeRoith, D. et al., 1995, Endocrine Reviews, 16, 143-163). IGF-I receptors and their ligands (IGF-I and IGF-II) play important roles in many physiological processes, including growth and development during embryogenesis, metabolism in adults, cell proliferation and cell differentiation (LeRoith, D., 2000, Endocrinology, 141, 1277-1288; LeRoith, D., 1997, New England J. Med., 336, 633-640).

  [04] IGF-I and IGF-II are present mainly in complex with IGF binding proteins in blood, both function as endocrine hormones, and locally produced paracrine and Functions as an autocrine growth factor (Humbel, RE, 1990, Eur. J. Biochem., 190, 445-462; Cohick, WS and Clemmons, DR, 1993, Annu. Rev. Physiol., 55, 131-153).

  [05] IGF-I receptor has been implicated in promoting tumor cell growth, transformation and survival (Baserga, R. et al., 1997, Biochem. Biophys. Acta, 1332, F105-F126; Blackesley, VA et al., 1997, Journal of Endocrinology, 152, 339-344; Kaleko, M., Rutter, WJ, and Miller, AD 1990, Mol Cell Biol., 10, 464-. 473). Thus, several types of tumors are known to express higher than normal levels of IGF-I receptors, including breast cancer, colon cancer, ovarian carcinoma, synovial sarcoma and pancreatic cancer (Khandwala, HM , Et al., 2000, Endocrine Reviews, 21, 215-244; Werner, H. and LeRoith, D., 1996, Adv. Cancer Res., 68, 183-223, Happerfield, LC, et al., 1997. Pathol., 183, 412-417; Frier, S. et al., 1999, Gut, 44, 704-708; van Dam, PA et al., 1994, J. Clin. Pathol., 47, 914-919; Y. et al., 1999, Cancer Res., 59, 3588-3591; Bergmann, U. et al., 1995, Cancer Res., 55, 2007-2011). IGF-I and IGF-II have been shown to be potent mitogens in several human tumor cell lines such as lung cancer, breast cancer, colon cancer, osteosarcoma and cervical cancer in vitro (Ankrappp , DP and 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, YS et al., 1995, J. Am. Coll. Surg., 181, 145-154; Kappel, CC et al., 1994, Cancer Res. , 54, 2 03-2807;. Steller, M.A. et al., 1996, Cancer Res, 56, 1761-1765). Some of these tumors and tumor cell lines also express high levels of IGF-I or IGF-II, which can stimulate proliferation in an autocrine or paracrine fashion (Quinn, K A. et al., 1996, J. Biol. Chem., 271, 11477-11484).

  [06] Epidemiological studies show that elevated IGF-I plasma levels (and low levels of IGF binding protein-3) correlate with increased risk of prostate, colon, lung and breast cancer (Chan, J.M. et al., 1998, Science, 279, 563-566; Wolk, 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 have been suggested to reduce IGF-I levels in plasma or inhibit IGF-I receptor function for cancer prevention (Wu, Y. et al., 2002, Cancer Res., 62, 1030-1035; Grimberg, A and Cohen P., 2000, J. Cell. Physiol., 183, 1-9).

  [07] IGF-I receptor protects tumor cells from apoptosis caused by growth factor depletion, anchorage independence, or cytotoxic drug treatment (Navarro, M. and Baserga, R., 2001). , Endocrinology, 142, 1073-1081; Baserga, R. et al., 1997, Biochem. Biophys. Acta, 1332, F105-F126). Domains critical for the mitogenic, transforming and anti-apoptotic activities of the IGF-I receptor have been identified by mutational analysis.

  [08] For example, the tyrosine 1251 residue of the IGF-I receptor has been identified as being critical for anti-apoptotic and transformation activity but not for 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). The ligand-activated intracellular signaling pathway of the IGF-I receptor involves phosphorylation of tyrosine residues of insulin receptor substrates (IRS-1 and IRS-2), thereby producing phosphatidylinositol-3-kinase. (PI-3-kinase) is recruited to the membrane. The membrane-bound phospholipid product of PI-3-kinase activates Akt, a serine / threonine kinase, and the Akt substrate includes pro-apoptotic protein BAD, which is phosphorylated Become inactive (Datta, SR, Brunet, A. and Greenberg, ME, 1999, Genes & Development, 13, 2905-2927; Kulik, G., Klippel, A. and Weber, M. J., 1997, Mol.Cell.Biol. 17, 1595-1606). Mitogenic signaling of the IGF-I receptor in MCF-7 human breast cancer cells requires PI-3-kinase and is independent of mitogen-activated protein kinase while differentiated rat brown Viability signaling in cytoma PC12 cells requires both the PI-3-kinase pathway and the mitogen-activated protein kinase pathway (Dufourny, B. et al., 1997, J. Biol. Chem., 272). , 31163-31171; Parrizas, M., Saltiel, AR and LeRoith, D., 1997, J. Biol. Chem., 272, 154-161).

  [09] Down-regulating IGF-I receptor levels through an antisense strategy results in several tumor cell lines such as melanoma, lung carcinoma, ovarian cancer, glioblastoma, neuroblastoma and rhabdomyosarcoma Tumorigenicity has been shown to decrease in vivo and in vitro (Resicoff, 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; Et al., 1998, Cancer Res., 58, 5432-. Shapiro, DN et al., 1994, J. Clin. Invest., 94, 1235-1242). Similarly, dominant negative mutants of the IGF-I receptor may reduce in vivo tumorigenicity and in vitro proliferation of transformed Rat-1 cells overexpressing the IGF-I receptor. (Prager, D. et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 2181-2185).

  [10] Large-scale apoptosis occurs when tumor cells expressing antisense to IGF-I receptor mRNA are injected into animals in a biodiffusion chamber. This observation makes the IGF-I receptor an attractive therapeutic target based on the hypothesis that tumor cells are more sensitive than normal cells to apoptosis due to inhibition of the IGF-I receptor. (Resnicoff, M. et al., 1995, Cancer Res., 55, 2463-2469; Baserga, R., 1995, Cancer Res., 55, 249-252).

  [11] Another strategy for inhibiting IGF-I receptor function in tumor cells is to bind anti-IGF-I receptor antibodies that bind to the extracellular domain of IGF-I receptor and inhibit its activation. I use it. Several attempts have been reported to generate murine monoclonal antibodies against the IGF-I receptor, of which two inhibitory antibodies, IR3 and 1H7 are available, and several IGF-I Their use has been reported in receptor studies.

  [12] IR3 antibodies have been generated using partially purified placental preparations of the insulin receptor to immunize mice, thereby producing antibodies IR1 selective for insulin receptor binding. And two antibodies, IR2 and IR3, which show preferential immunoprecipitation of the IGF-I receptor (somatomedin-C receptor) but also weak immunoprecipitation of the insulin receptor (Kull, F C. et al., 1983, J. Biol. Chem., 258, 6561-6666).

  [13] The 1H7 antibody has been generated by immunizing mice with a purified placental preparation of the IGF-I receptor, which yields three stimulatory antibodies in addition to the inhibitory antibody 1H7. (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).

  [14] In another report, immunization of mice with transfected 3T3 cells expressing high levels of IGF-I receptor yielded a population of mouse monoclonal antibodies specific for human IGF-I receptor. These antibodies have been classified into seven groups by binding competition studies and by inhibition or stimulation of IGF-I binding to transfected 3T3 cells (Soos, MA et al., 1992, J. Biol. Chem., 267, 12955-12963).

  [15] As mentioned above, the IR3 antibody is the most commonly used inhibitory antibody in in vitro IGF-I receptor studies, but transfected 3T3 cells expressing the human IGF-I receptor. And CHO cells have the disadvantage of exhibiting agonistic activity (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 antibody populations generated by Soos et al., The most inhibitory antibodies, 24-57 and 24-60, also showed agonistic activity in transfected 3T3 cells (Soos, M. et al. A. et al., 1992, J. Biol. Chem., 267, 12955-12963). IR3 antibodies have been reported to inhibit IGF-I binding to expressed receptors (but not IGF-II binding) in intact cells and after solubilization. However, in vitro cells have been shown to inhibit both the ability of IGF-I and IGF-II to stimulate DNA synthesis (Steel-Perkins, G. and Roth, RA, 1990). , Biochem. Biophys. Res. Commun., 171, 1244-1251). With the chimeric insulin-IGF-I receptor construct, it is speculated that the binding epitope of IR3 antibody is the region 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).

  [16] The MCF-7 human breast cancer cell line is typically used as a model cell line that exhibits proliferative responses of IGF-I and IGF-II in vitro (Dufourny, B. et al., 1997, J. MoI. Biol. Chem., 272, 31163-31171). In MCF-7 cells, the IR3 antibody incompletely blocks the stimulatory effects of exogenously added IGF-I and IGF-II in serum-free conditions by approximately 80%. In addition, IR3 antibody does not significantly inhibit the growth of MCF-7 cells in 10% serum (less than 25%) (Cullen, KJ et al., 1990, Cancer Res., 50, 48-53). The growth of in vitro serum-stimulated MCF-7 cells was only weakly inhibited by IR3 antibody in nude mice, even when treated with IR3 antibody, significantly increased MCF-7 xenograft growth. It may also be related to the results of in vivo studies that it is not inhibited (Arteaga, CL, et al., 1989, J. Clin. Invest., 84, 1418-1423).

  [17] IR3 and other reported antibodies have weak agonistic activity and serum stimulates (rather than stimulation with exogenously added IGF-I or IGF-II in serum-free conditions) In more physiological conditions, it is unable to significantly inhibit the growth of tumor cells such as MCF-7 cells, thus significantly inhibiting the growth of serum-stimulated tumor cells, but by itself a significant agonist There is a need for new anti-IGF-I receptor antibodies that do not exhibit sexual activity.

SUMMARY OF THE INVENTION [18] Accordingly, an object of the present invention is to inhibit the cellular activity of the receptor by specifically binding to and antagonizing the insulin-like growth factor I receptor, and also To provide antibodies, antibody fragments and antibody derivatives substantially lacking agonist activity for the receptor.

  [19] Therefore, in the first embodiment, the amino acid sequence of both the light and heavy chain variable regions, the cDNA sequence of the light and heavy chain variable region genes, identification of its CDR (complementarity determining region), The murine antibody EM164, which is fully characterized herein, is provided for the identification of surface amino acids, as well as their means of expression in recombinant form.

  [20] In a second embodiment, a resurfaced or humanized version of antibody EM164, such that the surface exposed residues of the antibody or fragments thereof more closely resemble a known human antibody surface. Said version is provided, which is substituted with both light and heavy chains. Such humanized antibodies may have increased utility as therapeutic or diagnostic agents compared to murine EM164. The humanized version of antibody EM164 also includes the respective amino acid sequences of both the light and heavy chain variable regions, the DNA sequence of the light and heavy chain variable regions, the identification of the CDRs, the identification of these surface amino acids, and The disclosure is fully characterized herein with respect to disclosure of its means of expression in recombinant form.

  [21] In a third embodiment, greater than about 80% can inhibit the growth of cancer cells in the presence of growth stimulants such as serum, insulin-like growth factor I and insulin-like growth factor II An antibody is provided.

  [22] In the fourth embodiment, SEQ ID NOs: 1 to 3, respectively:

A heavy chain comprising a CDR having the amino acid sequence shown in
And SEQ ID NOs: 4-6:

An antibody or antibody fragment having a light chain comprising a CDR having the amino acid sequence shown in
[23] In a fifth embodiment, SEQ ID NO: 7:

An antibody having a heavy chain having an amino acid sequence sharing at least 90% sequence identity with the amino acid sequence shown in FIG.
[24] Similarly, SEQ ID NO: 8:

An antibody having a light chain having an amino acid sequence sharing at least 90% sequence identity with the amino acid sequence shown in FIG.
[25] In a sixth embodiment, SEQ ID NOs: 9 to 12:

An antibody having a humanized or surface rearranged light chain variable region having an amino acid sequence corresponding to one of the above is provided.
[26] Similarly, SEQ ID NO: 13:

An antibody having a humanized or surface rearranged heavy chain variable region having an amino acid sequence corresponding to is provided.
[27] In a seventh aspect, there is provided an antibody or antibody fragment of the invention having improved properties. For example, affinity maturation of an antibody or fragment of the present invention prepares an antibody or antibody fragment with improved affinity for the IGF-I receptor.

  [28] The present invention further relates to a conjugate of the above antibody, wherein the cytotoxic agent is shared directly or via a cleavable linker or an uncleavable linker with the antibody or epitope-binding fragment of the antibody of the present invention. Provided is a conjugate of said conjugate. In a preferred embodiment, the cytotoxic agent is taxol, maytansinoid, CC-1065 or a CC-1065 analog.

  [29] The present invention further provides further labeled antibodies or fragments thereof for use in research or diagnostic applications. In a preferred embodiment, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent or a metal ion.

  [30] Also provided is a diagnostic method, wherein the labeled antibody or fragment is administered to a subject suspected of having cancer and the distribution of the label in the subject is measured or monitored .

  [31] In an eighth aspect, the present invention has cancer by administering the antibody, antibody fragment or antibody conjugate of the present invention alone or in combination with other cytotoxic agents or therapeutic agents. A method of treating a subject is provided. The cancer is determined, for example, from elevated breast cancer, colon cancer, ovarian carcinoma, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial carcinoma, pancreatic cancer, or IGF-I receptor levels. It can also be one or more of other cancers that may be.

  [32] In the ninth aspect, the present invention has cancer by administering the antibody, antibody fragment or antibody conjugate of the present invention alone or in combination with other cytotoxic agents or therapeutic agents. A method of treating a subject is provided. Particularly preferred cytotoxic and therapeutic agents include docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane, Goserelin, Oxaliplatin, Carboplatin, Cisplatin, Dexamethasone, Antide, Bevacizumab (Avastin), 5-Fluorouracil, Leucovorin, Levamisole, Irinotecan, Etoposide, Topotecan, Gemcitabine, Vinorelbine, Estramustrone, Mitroxantrone , Streptozocin, rituximab (rituxan), idarubicin, busulfan, chlorambucil Fludarabine, imatinib, cytarabine, ibritumomab (zevalin), tositumomab (bexal), interferon alfa-2b, melphalan, bortezomib (velcade), altretamine, asparaginase, gefitinib (Iressa), erlonitib (Tarceva antibody), E Cetuximab, Abx-EGF), and epothilone. More preferably, the therapeutic agent is a platinum agent (carboplatin, oxaliplatin, cisplatin, etc.), a taxane (paclitaxel, docetaxel, etc.), gemcitabine, or camptothecin.

  [33] Cancer is breast cancer, colon cancer, ovarian carcinoma, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial carcinoma, pancreatic cancer, melanoma, multiple myeloma, neuroblastoma, and striated muscle It can also be one or more of the tumors or other cancers from which it may be determined that elevated IGF-I receptor levels.

  [34] In a tenth aspect, the present invention provides a kit comprising one or more elements described herein, and instructions for using these elements. In a preferred embodiment, the kit of the present invention includes the antibody, antibody fragment or conjugate of the present invention, and a therapeutic agent. The instructions for this preferred embodiment include instructions for inhibiting cancer cell growth using the antibodies, antibody fragments or conjugates, and therapeutic agents of the invention, and / or the antibodies of the invention. , Antibody fragments or conjugates, and instructions for using the therapeutic agents to treat patients with cancer.

DETAILED DESCRIPTION OF THE INVENTION [62] The inventors of the present invention have discovered and improved a novel antibody that specifically binds to human insulin-like growth factor I receptor (IGF-IR) on the cell surface. The antibodies and fragments have the unique ability to inhibit the cellular function of the receptor without the ability to activate the receptor itself. Thus, antibodies previously known to specifically bind to and inhibit IGF-IR also activate the receptor, even in the absence of IGF-IR ligand. The antibodies or fragments of the present invention antagonize IGF-IR but substantially lack agonist activity. Furthermore, the antibodies and antibody fragments of the present invention inhibit the growth of human tumor cells, such as MCF-7 cells, in the presence of serum by more than 80%, which was previously known anti-IGF-IR. To a greater degree than the inhibition obtained with antibodies.

  [63] The present invention starts with a murine anti-IGF-IR antibody, EM164 herein, which comprises both light and heavy chain amino acid sequences, CDR identification, surface amino acid identification, and recombinant Is fully characterized with respect to its expression means.

  [64] FIG. 15 shows the germline sequence in parallel with the sequence of EM164. The comparison identifies possible somatic mutations in EM164, including one each in light chain CDR1 and heavy chain CDR2.

  [65] Disclosed herein are the primary amino acid and DNA sequences of antibody EM164 light and heavy chains and humanized versions. However, the scope of the present invention is not limited to antibodies and fragments comprising these sequences. Instead, all antibodies and fragments that specifically bind to the insulin-like growth factor I receptor and antagonize the biological activity of the receptor, but substantially lack agonist activity, are disclosed herein. Belongs to the range. Thus, antibodies and antibody fragments may differ from antibody EM164 or humanized derivatives in backbone amino acid sequence, CDRs, light and heavy chains and still belong within the scope of the invention.

  [66] Modeling has identified the CDRs of antibody EM164 and predicted its molecular structure. Again, CDRs are important for epitope recognition but are not essential for the antibodies and fragments of the invention. Thus, antibodies and fragments with improved properties produced, for example, by affinity maturation of the antibodies of the invention are provided.

  [67] A variety of antibodies and antibody fragments, and antibody mimetics can be readily produced by mutations, deletions and / or insertions within the variable and constant region sequences adjacent to a particular set of CDRs. Thus, for example, for a given set of CDRs, it is also possible to create different classes of Abs by substituting with different heavy chains, and thus for example IgG1-4, IgM, IgA1-2, IgD, IgE antibodies It is also possible to produce types and isotypes. Similarly, it is possible to produce artificial antibodies within the scope of the present invention by embedding a predetermined set of CDRs within a fully synthesized framework. The term “variable” is used herein to refer to a particular portion of a variable domain that differs in sequence between antibodies and is used in the binding and specificity of each particular antibody to an antigen. However, variability is usually not evenly distributed throughout the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more conserved part of the variable domain is called the framework (FR). The heavy and light chain variable domains each contain four framework regions and employ a beta sheet configuration primarily linked to three CDRs, which link the beta sheet structure, and In some cases, it forms a loop that forms part of the beta sheet structure. The CDRs of each chain are held together in close proximity by the FR region and together with the CDRs from the other chain contribute to the formation of the antigen binding site of the antibody (EA Kabat et al. Sequences of Proteins of Immunological Interest, 5th edition, 1991, NIH). Constant domains are not directly involved in antibody binding to antigen, but exhibit a variety of effector functions, such as antibody involvement in antibody-dependent cytotoxicity.

  [68] It is also possible to produce humanized antibodies, or antibodies adapted for non-rejection by other mammals, using several techniques such as surface reconstruction and CDR grafting. In surface reconstruction techniques, molecular modeling, statistical analysis, and mutagenesis are combined to modulate the variable region non-CDR surface to resemble the surface of a known antibody in the target host. Strategies and methods for reconstituting antibody surfaces, as well as other methods for reducing antibody immunogenicity in different hosts, are disclosed in US Pat. No. 5,639,641, incorporated herein in its entirety. . In CDR grafting techniques, murine heavy and light chain CDRs are grafted into framework sequences that are fully human.

  [69] The present invention also includes functional equivalents of the antibodies described herein. Functional equivalents have binding properties comparable to those of antibodies and include, for example, chimeric, humanized and single chain antibodies, and fragments thereof. Methods for producing such functional equivalents 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 EP 332,424. Each of which is incorporated herein by reference in its entirety.

  [70] Functional equivalents include polypeptides having substantially the same amino acid sequence as the variable or hypervariable region of the antibodies of the invention. “Substantially the same”, when applied to amino acid sequences, is herein referred to as Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988), defined as a sequence having at least about 90%, and more preferably at least about 95% sequence identity with another amino acid sequence, as determined by the FASTA search method. Is done.

  [71] Chimeric antibodies are preferably obtained substantially or exclusively from constant regions obtained substantially or exclusively from human antibody constant regions, and from non-human mammalian variable region sequences. Has a variable region. Humanized forms of antibodies are made, for example, by substituting the complementarity-determining regions of mouse antibodies into the human framework domain, see, eg, PCT Publication No. WO 92/22653. Humanized antibodies are preferably substantially constant in constant regions and variable regions other than complementarity determining regions (CDRs) and substantially from non-human mammals derived substantially or exclusively from the corresponding human antibody regions. Or have CDRs derived exclusively.

[72] Functional equivalents also include single chain antibody fragments, also known as single chain antibodies (scFv). These fragments are tethered to at least one fragment of the antibody variable light chain sequence (V _L ) with or without one or more interconnecting linkers, the antibody variable heavy chain amino acid sequence (V _H ). Containing at least one fragment. Such linkers allow these domains to be maintained such that once the (V _L ) and (V _H ) domains are linked, the target molecule binding specificity of the entire antibody from which the single chain antibody fragment is derived is maintained. It is also possible to be a short flexible peptide selected to ensure proper three-dimensional folding. Generally, the carboxyl terminus of the (V _L) sequences, or (V _H) sequences, by such a peptide linker to the amino terminus of a complementary (V _L) sequence and (V _H) sequences, also be covalently linked Is possible. Single chain antibody fragments can also be generated by molecular cloning, antibody phage display libraries or similar techniques. It is also possible to produce these proteins in either eukaryotic cells or prokaryotic cells including bacteria.

  [73] Single-chain antibody fragments contain amino acid sequences having at least one of the variable regions or complementarity determining regions (CDRs) of the entire antibody described herein, but to some extent the constant domains of these antibodies or Lack everything. These constant domains are not required for antigen binding but constitute a major part of the total antibody structure. Single chain antibody fragments can thus overcome some of the deficiencies associated with the use of antibodies containing some or all of the constant domains. For example, single chain antibody fragments tend not to have undesirable interactions between biological molecules and heavy chain constant regions, or other undesirable biological activities. In addition, single chain antibody fragments are much smaller than whole antibodies and are therefore more capillary permeable than whole antibodies, allowing them to localize more efficiently and bind to target antigen binding sites. Become. Antibody fragments can also be produced on a relatively large scale in prokaryotic cells and are therefore easy to produce. Furthermore, because single chain antibody fragments are relatively small in size, they are less likely to elicit an immune response in the recipient than the whole antibody.

[74] Functional equivalents further include fragments of antibodies having binding characteristics that are the same as or comparable to those of whole antibodies. Such fragments can also contain one or both of Fab fragments or F (ab ′) ₂ fragments. Preferably, an antibody fragment contains all six complementarity determining regions of the whole antibody, but fragments containing fewer than all these regions, such as three, four or five CDRs, are also functional. . In addition, the functional equivalent can be a member of any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and subclasses, and functional equivalents. Can be combined with these classes and subclasses.

  [75] Using knowledge of the amino acid and nucleic acid sequences of the anti-IGF-I receptor antibody EM164 and humanized variants thereof described herein, it also binds to the human IGF-I receptor and accepts IGF-I receptor Other antibodies can be made that inhibit the body's cellular functions. Several studies have investigated the impact on properties such as binding and expression levels of introducing one or more amino acid changes at various positions in an antibody sequence based on knowledge of the initial antibody sequence. (Yang, WP et al., 1995, J. Mol. Biol., 254, 392-403; Rader, C. et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915; , TJ et al., 1998, Nature Biotechnology, 16, 535-539).

  [76] In these studies, methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or E. coli mutator strains are used. Using, the first antibody variants were generated by altering the heavy and light chain gene sequences in the CDR1, CDR2, CDR3, or framework regions (Vaughan, TJ et al., 1998, Nature). Biotechnology, 16, 535-539; Adey, NB et al., 1996, Chapter 16, pp. 277-291, “Page Display of Peptides and Proteins”, supervised by Kay, BK et al., Academ. c Press). These methods of altering the sequence of the first antibody have resulted in a second antibody with improved affinity (Gram, H. et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 3576- Boder, ET et al., 2000, Proc. Natl. Acad. Sci. USA, 97, 10701-10705; Davies, J. and Riechmann, L., 1996, Immunotechnology, 2, 169-1ps; J. et al., 1996, J. Mol. Biol., 256, 77-88; Short, M.K. et al., 2002, J. Biol.Chem., 277, 16365-16370; 001, J. Biol. Chem., 276, 27622-27628).

  [77] Creating an anti-IGF-I receptor antibody with improved function using an antibody sequence according to the present invention by a similar directed strategy of changing one or more amino acid residues of an antibody Is also possible.

  [78] Conjugates of the invention include the antibodies, fragments, and analogs thereof disclosed herein linked to a cytotoxic agent. Preferred cytotoxic agents are maytansinoids, taxanes and CC-1065 analogs. It is also possible to prepare conjugates by in vitro methods. In order to link the cytotoxic agent to the antibody, a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photosensitive groups, peptidase labile groups and esterase labile groups. Preferred linking groups are disulfide groups and thioether groups. For example, conjugates can be constructed using a disulfide exchange reaction or by forming a thioether bond between an antibody and a cytotoxic agent.

  [79] Among the preferred cytotoxic agents are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogues. Suitable maytansinoids are US Pat. Nos. 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; 322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499 and 5,846,545.

[80] Taxanes are also preferred cytotoxic agents. Taxanes suitable for use in the present invention are disclosed in US Pat. Nos. 6,372,738 and 6,340,701.
[81] CC-1065 and its analogs are also preferred cytotoxic agents for use in the present invention. CC-1065 and its analogs are disclosed in US Pat. Nos. 6,372,738; 6,340,701; 5,846,545 and 5,585,499.

  [82] An attractive candidate for the preparation of such cytotoxic conjugates is CC-1065, a potent antitumor antibiotic isolated from a culture broth of Streptomyces zelensis It is. CC-1065 is about 1000 times more potent in vitro than commonly used anticancer drugs such as doxorubicin, methotrexate and vincristine (BK Bhuyan et al., Cancer Res., 42, 3532-3537 (1982). )).

  [83] Cytotoxic agents such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and calicheamicin are also suitable for the preparation of the conjugates of the invention and the drug molecule It is also possible to link the antibody molecule through an intermediate carrier molecule such as serum albumin.

[84] For diagnostic applications, the antibodies of the invention will typically be labeled with a detectable moiety. The detectable moiety can be anything capable of producing a detectable signal, either directly or indirectly. For example, the detectable moiety can be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹³¹ I; a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin; or alkaline phosphatase, It can also be an enzyme such as beta-galactosidase or horseradish peroxidase.

  [85] Any method known in the art can be used to conjugate the antibody to the detectable moiety, including Hunter et al., Nature 144: 945 (1962); David et al. , Biochemistry 13: 1014 (1974); Pain et al., J. Biol. Immunol. Meth. 40: 219 (1981); and Nygren, J. et al. Histochem. and Cytochem. 30: 407 (1982).

  [86] The antibodies of the present invention can be used in any known assay method, such as 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)).

  [87] The antibodies of the present invention are also useful for in vivo imaging, in which the subject is preferably treated with an antibody labeled with a detectable moiety such as a radio-opaque agent or a radioisotope. Is administered into the blood stream and assayed for the presence and location of the labeled antibody in the host. This imaging technique is useful for staging and treatment of malignant tumors. The antibody can be labeled with any moiety that is detectable in the host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

  [88] The antibodies of the present invention are also useful as affinity purification agents. In this process, the antibody is immobilized on a suitable support, such as Sephadex resin or filter paper, using methods well known in the art.

[89] The antibodies of the present invention are also useful as reagents in biological research based on inhibiting the function of IGF-I receptors in cells.
[90] For therapeutic application, an antibody or conjugate of the invention is administered to a subject in a pharmaceutically acceptable dosage form. They can be administered intravenously as a bolus or by continuous infusion over a period of time, intramuscular, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes Can be administered. It is also possible to exert local and systemic therapeutic effects by administering antibodies by intratumoral, peritumoral, intralesional, or peripathic routes. Suitable pharmaceutically acceptable carriers, diluents and excipients are well known and can be determined by one of ordinary skill in the art to assure a clinical situation. Examples of suitable carriers, diluents and / or excipients include: (1) Dulbecco's phosphate buffered saline containing about 1 mg / ml to 25 mg / ml human serum albumin, pH about 7.4, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose. It is also possible to carry out the method of the invention in vitro, in vivo, or ex vivo.

  [91] In other therapeutic treatments, one or more additional therapeutic agents and the antibody, antibody fragment or conjugate of the invention can be co-administered or administered sequentially. Suitable therapeutic agents include, but are not limited to, cytotoxic agents or cytostatic agents. Taxol is also a cytotoxic agent and a preferred therapeutic agent.

  [92] Cancer therapeutic agents are agents that seek to kill or limit the growth of cancer cells while minimizing damage to the host. Thus, these agents can take advantage of any difference in cancer cell properties from healthy host cells (eg, metabolism, angiogenesis or cell surface antigen presentation). Differences in tumor morphogenesis are potential sites of intervention: for example, a second therapeutic agent is useful in delaying angiogenesis within a solid tumor and thereby slowing its growth rate. It is also possible to use an antibody such as Other therapeutic agents include, but are not limited to, adjuncts such as granisetron HCl, androgen inhibitors such as leuprolide acetate, antibiotics such as doxorubicin, antiestrogens such as tamoxifen, interferon alpha- Antimetabolites such as 2a, cytotoxic agents such as taxol, enzyme inhibitors such as ras farnesyl transferase inhibitor, immunomodulators such as aldesleukin, and nitrogen mustard derivatives such as melphalan HCl.

  [93] Therapeutic agents that can also be combined with EM164 to improve anticancer efficacy include a variety of agents used in oncology treatment (Reference: Cancer, Principles & Practice of Oncology, DeVita, V T., Hellman, S., Rosenberg, SA, 6th edition, Lippincott-Raven, Philadelphia, 2001), for example docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Herceptin), , Tamoxifen, Toremifene, Letrozole, Anastrozole, Fulvestrant, Exemestane, Goserelin, Oxaliplatin, Carboplatin, Cisplata Dexamethasone, antidone, bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronate, streptozocin, rituximacin, rituximabine , Busulfan, chlorambucil, fludarabine, imatinib, cytarabine, ibritumomab (zevalin), tositumomab (bexal), interferon alfa-2b, melphalan, bortezomib (velcade), altretamine, asparaginase, gefitinib (irresa), erlotib EGF receptor antibody (cetuximab, Abx-EGF), epothilone, and cytotoxicity There are conjugates of antibodies against drugs, and cell surface receptors. Preferred therapeutic agents are platinum agents (carboplatin, oxaliplatin, cisplatin, etc.), taxanes (paclitaxel, docetaxel, etc.), gemcitabine, or camptothecin.

  [94] One or more additional therapeutic agents can be administered before, simultaneously with, or after the antibody, antibody fragment or conjugate of the invention. One skilled in the art will appreciate that for each therapeutic agent, there may be advantages in a particular order of administration. Similarly, one of skill in the art will understand that for each therapeutic agent, the length of time between administration of the agent and the antibody, antibody fragment or conjugate of the invention will vary.

  [95] The skilled artisan will understand that the dosage of each therapeutic agent will depend on the uniqueness of the agent, but a preferred dosage is from about 10 mg / square meter to about 2000 mg / square meter, more preferably about 50 mg. It can also be in the range of from / square meter to about 1000 mg / square meter. For preferred agents such as platinum agents (carboplatin, oxaliplatin, cisplatin), the preferred dosage is about 10 mg / square meter to about 400 mg / square meter, and for taxanes (paclitaxel, docetaxel), the preferred dosage is about 20 mg. For gemcitabine, the preferred dosage is about 100 mg / square meter to about 2000 mg / square meter, and for camptothecin, the preferred dosage is about 50 mg / square meter to about 350 mg / square meter. It is. The dosage of these and other therapeutic agents may depend on whether the antibody, antibody fragment or conjugate of the invention is administered simultaneously or sequentially with the therapeutic agent.

  [96] Administration of an antibody, antibody fragment or conjugate of the invention, and one or more additional therapeutic agents, whether co-administered or sequentially administered, as described above for therapeutic applications It is also possible to do this. It will be appreciated by those skilled in the art that pharmaceutically acceptable carriers, diluents, and excipients suitable for co-administration will depend on the uniqueness of the particular therapeutic agent being co-administered.

  [97] When present in an aqueous dosage form rather than a lyophilized form, the antibody will typically be formulated at a concentration of about 0.1 mg / ml to 100 mg / ml, but outside these ranges Wide variations in are acceptable. For disease treatment, the appropriate dosage of the antibody or conjugate will be the type of disease being treated, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, as defined above. Will depend on the course of previous therapy, the patient's medical history and response to antibodies, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

  [98] Depending on the type and severity of the disease, preferably from about 1 mg / square meter to about 2000 mg / square meter of antibody, whether by one or more separate administrations or by continuous infusion The first candidate dosage is more preferably about 10 mg / square meter to about 1000 mg / square meter of antibody by either one or more separate administration or continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other medications may be useful and are not excluded.

  [99] The invention also includes kits that include one or more of the elements described herein and instructions for using these elements. In a preferred embodiment, the kit of the present invention includes the antibody, antibody fragment or conjugate of the present invention, and a therapeutic agent. Instructions for use of this preferred embodiment include instructions for inhibiting the growth of cancer cells using the antibodies, antibody fragments or conjugates, and therapeutic agents of the invention, and / or antibodies, antibody fragments of the invention. Or includes instructions for methods of treating patients with cancer using conjugates and therapeutic agents.

  [100] Preferably, the antibody used in the kit has the same amino acid sequence as murine antibody EM164 produced in murine hybridoma EM164 (ATCC deposit no. PTA-4457), or the antibody is an epitope-binding fragment, Both antibodies and fragments specifically bind to insulin-like growth factor I receptor. The antibodies and antibody fragments used in the kits are also a surface reconstituted version of the EM164 antibody, a humanized version of the EM164 antibody, or a modified version of the EM164 antibody having at least one nucleotide mutation, deletion or insertion. It is also possible. These three versions of antibodies and antibody fragments retain the same binding specificity as the EM164 antibody.

  [101] Preferably, the therapeutic agent used in the kit is docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane , Goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, valetrone Zoledronate, streptozocin, rituximab (Rituxan), idarubicin, busulfan, chloram Sill, fludarabine, imatinib, cytarabine, ibritumomab (zevalin), tositumomab (bexal), interferon alfa-2b, melphalan, bortezomib (velcade), altretamine, asparaginase, gefitinib (irresa), erlonitib (anti-TGF receptor) Selected from the group consisting of an antibody (cetuximab, Abx-EGF) and epothilone. More preferably, the therapeutic agent is a platinum agent (carboplatin, oxaliplatin, cisplatin, etc.), a taxane (paclitaxel, docetaxel, etc.), gemcitabine, or camptothecin.

  [102] The elements of the kit of the invention are in a form suitable for the kit, such as a solution or a lyophilized powder. One skilled in the art will appreciate that the concentration or amount of elements of the kit will vary depending on the identity of each element of the kit and the intended use.

  [103] Cancers mentioned in the kit instructions and cells derived therefrom include breast cancer, colon cancer, ovarian cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial carcinoma, pancreatic cancer, melanoma , Multiple myeloma, neuroblastoma, and rhabdomyosarcoma.

EXAMPLES [104] The present invention will now be described by reference to the following examples, which are illustrative only and not intended to limit the invention.

Example 1
Murine EM164 antibody [105] In this first example, the complete primary amino acid structure and cDNA sequence of the murine antibody of the invention, as well as its binding properties and means for its expression in recombinant form are disclosed. Accordingly, a complete disclosure of the antibodies of the present invention and methods for their preparation is provided so that one of ordinary skill in the immunological arts can prepare such antibodies without undue experimentation.

A. Generation of Anti-IGF-I Receptor Monoclonal Antibody Hybridoma [106] A cell line expressing the human IGF-I receptor with the Y1251F mutation to express a number of IGF-I receptors (10 ⁷ per cell) Used for immunization. Although the Y1251F mutation in the cytoplasmic domain of the IGF-I receptor resulted in a lack of transformation and anti-apoptotic signaling, it has no effect on IGF-I binding and mitogenic signaling 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 of this example bound to the extracellular domain of the IGF-I receptor, which was identical in both the Y1251F mutant and the wild type receptor, this mutation was otherwise The production was not affected.

  [107] Y1251F mutant human IGF-containing a Y1251F mutation from 3T3-like cells of IGF-I receptor-deficient mice by transfecting a puromycin resistance gene with a human IGF-I receptor gene. Cell lines expressing the I receptor were generated and selected using puromycin (2.5 microgram / ml) and by FACS sorting for high IGF-I receptor expression (Miura, M. et al. , 1995, J. Biol. Chem., 270, 22639-22644). Cell lines with high levels of IGF-I receptor expression were further selected using high concentrations of puromycin, such as 25 microgram / ml, which are toxic to most cells. Surviving colonies were picked and selected to show high levels of IGF-I receptor expression.

[108] Y1251F mutant human IGF-I receptor overexpressing cells (5 × 10 ⁵ cells, suspended in 0.2 ml PBS) on day 0, 6 month old CAF1 / J female mice by intraperitoneal route I was immunized. Animals were boosted with 0.2 ml cell suspension as follows: day 2, 1 × 10 ⁶ cells; day 5, 2 × 10 ⁶ cells; day 7, day 9, day 12, and Day 23, 1 × 10 ⁷ cells. On day 26, the mice were sacrificed and the spleen was removed.

[109] The spleen was ground between two ground glass slides to obtain a single cell suspension that was washed with serum-free RPMI medium (SFM) containing penicillin and streptomycin. The spleen cell pellet was resuspended in 0.8 ml of 0.83% (w / v) ammonium chloride solution in water for 10 minutes on ice to lyse the red blood cells and then washed with serum free medium (SFM). . Spleen cells (1.2 × 10 ⁸ ) were collected in vitro with myeloma cells (4 × 10 ⁷ ) from the nonsecretory mouse myeloma cell line P3X63Ag8.653 (ATCC, Rockville, Md .; catalog number CRL1580). And washed with serum free RPMI-1640 medium (SFM). The supernatant was removed and the cell pellet was resuspended in the remaining medium. Place the test tube in a beaker with water at 37 ° C. and add 1.5 ml of polyethylene glycol solution (75 mM HEPES, pH 8 in 50% PEG (w / v), average molecular weight 1500) dropwise with gentle shaking. Slowly added at a rate of 0.5 ml / min. After waiting for 1 minute, 10 ml of SFM was added as follows: 1 ml over the first minute, 2 ml over the next minute, and 7 ml over the next minute. Then another 10 ml was slowly added over 1 minute. Cells are pelleted by centrifugation, washed with SFM, and 5% fetal bovine serum (FBS), hypoxanthine / aminopterin / thymidine (HAT), penicillin, streptomycin, and 10% hybridoma cloning supernatant (HCS). Resuspended in supplemented RPMI-1640 growth medium. Cells were seeded into 96 well flat bottom tissue culture plates with 2 × 10 ⁵ spleen cells in 200 μl per well. After 5-7 days, 100 μl per well was removed and replaced with growth medium supplemented with hypoxanthine / thymidine (HT) and 5% FBS. General conditions used for immunization and hybridoma production are described in J. Am. Langone and H.C. Vunakis (supervised, Methods in Enzymology, Vol. 121, “Immunochemical Technologies, Part I”; 1986; Academic Press, Florida). Harlow and D.H. Lane (“Antibodies: A Laboratory Manual”; 1988; Cold Spring Harbor Laboratory Press, New York). Other techniques of immunization and hybridoma production can also be used, as is well known to those skilled in the art.

  [110] As described below, for binding to purified human IGF-I receptor by ELISA, and for specific binding to cells overexpressing human IGF-I receptor by ELISA and FACS screening, and human • Culture supernatants from hybridoma clones were screened for lack of binding to cells overexpressing insulin receptor. Clones with higher binding affinity were grown and subcloned into cells overexpressing the human IGF-I receptor than to cells overexpressing the human insulin receptor. Subclonal culture supernatants were further screened by the binding assay. By this method, subclone 3F1-C8-D7 (EM164) was selected and the heavy and light chain genes were cloned and sequenced as described below.

[111] The human IGF-I receptor was isolated for use in screening supernatants from hybridoma clones for binding to the IGF-I receptor by the following method. Prepare biotinylated IGF-I by modifying recombinant IGF-I with a biotinylation reagent such as sulfo-NHS-LC-biotin, sulfo-NHS-SS-biotin, or NHS-PEO ₄ -biotin did. Biotinylated IGF-I was absorbed onto streptavidin-agarose beads and incubated with lysates from cells that overexpress human wild type or Y1251F mutant IGFR. The beads were washed and eluted with a buffer containing a surfactant such as 2-4 M urea and Triton X-100 or octyl-β-glucoside. The eluted IGF-I receptor is dialyzed against PBS and analyzed for purity by SDS-PAGE under reducing conditions, and the alpha and beta chain bands of the IGF-I receptor with molecular weights of about 135 kDa and 95 kDa, respectively. It has been shown.

[112] Immulon-4HB ELISA plate (Dynatech) was purified from purified human IGF-I receptor sample (urea / octyl-β-glucoside elution of affinity purified sample) to check for binding of hybridoma supernatant to purified IGF-I receptor Prepared by dialysis from) and diluted with 50 mM CHES buffer, pH 9.5 (100 μl; 4 ° C., overnight). Block wells with 200 μl 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 derived from hybridoma clones Supernatant (100 μl; diluted with blocking buffer) for about 1-12 hours, washed with TBS-T buffer, and goat anti-mouse IgG Fc antibody-horseradish peroxidase (HRP) conjugate (100 μl ; in blocking buffer, 0.8μg / ml; Jackson ImmunoResearch Laboratories ) and incubated, then washed and detected at 405nm using ABTS _/ H 2 _{O 2} substrate (0.1M citric San'yuru Liquid, medium pH4.2, 0.5mg / ml ABTS, 0.03 % H 2 O 2). Typically, the supernatant from the 3F1 hybridoma subclone produces a signal of about 1.2 absorbance units within 3 minutes of color development, which is obtained for supernatants from several other hybridoma clones. In contrast to the 0.0 value. General conditions for this ELISA are as follows: Harlow and D.H. Standard ELISA conditions for antibody binding and detection as described in Lane (“Using Antibodies: A Laboratory Manual”; 1999, Cold Spring Harbor Laboratory Press, NY), which conditions can also be used It is.

[113] Specific for human IGF-I receptor using ELISA for cell lines overexpressing human Y1251F-IGF-I receptor and for cell lines overexpressing human insulin receptor The hybridoma supernatants that bind specifically and do not specifically bind to the human insulin receptor were screened. Both cell lines were generated from 3T3-like cells of IGF-I receptor-deficient mice. By rapid trypsin / EDTA treatment, IGF-I receptor overexpressing cells and insulin receptor overexpressing cells are collected separately from tissue culture flasks, suspended in growth medium containing 10% FBS, and pelleted by centrifugation. And washed with PBS. To the wells of an Immulon-2HB plate coated with phytohemagglutinin (100 μl of 20 μg / ml PHA), washed cells (100 μl of about 1-3 × 10 ⁶ cells / ml) were added, centrifuged, And it was made to adhere to the well coated with PHA for 10 minutes. The plate containing the cells was tapped to remove the PBS and then dried overnight at 37 ° C. The wells were blocked with 5 mg / ml BSA solution in PBS for 1 hour at 37 ° C. and then gently washed with PBS. An aliquot of supernatant from the hybridoma clone (100 μl; diluted in blocking buffer) is then added to the well containing the IGF-I receptor overexpressing cells and the well containing the insulin receptor overexpressing cells; And incubated for 1 hour at ambient temperature. Wells were washed with PBS, incubated with goat anti-mouse IgG Fc antibody-horseradish peroxidase (HRP) conjugate (100 μl; 0.8 μg / ml in blocking buffer), then washed, and then Binding was detected using ABTS / H ₂ O ₂ substrate. Typically, upon incubation with cells overexpressing the IGF-I receptor, the supernatant from the 3F1 hybridoma subclone produces a signal of 0.88 absorbance units within 12 minutes of color development, which is human. Contrast with the 0.22 extinction unit value obtained upon incubation with cells overexpressing insulin receptor.

[114] Hybridomas were grown in Integra CL350 flasks (Integra Biosciences, MD) according to manufacturer's specifications to provide purified EM164 antibody. Based on quantification by ELISA using antibody standards and by SDS-PAGE / Coomassie blue staining, yields of approximately 0.5-1 mg / ml of antibody were obtained in supernatants collected from Integra flasks. Affinity on a protein A-agarose bead column under standard purification conditions, loaded and washed with 100 mM Tris buffer containing 3 M NaCl, pH 8.9, and then eluted with 100 mM acetic acid solution containing 150 mM NaCl. • The antibody was purified by chromatography. The elution fraction containing the antibody was neutralized with cold 2M K ₂ HPO ₄ solution and dialyzed at 4 ° C. in PBS. Antibody concentration was determined by measuring absorbance at 280 nm (quenching coefficient = 1.4 mg ⁻¹ ml cm ⁻¹ ). Analysis of the purified antibody samples by SDS-PAGE and Coomassie blue staining under reducing conditions showed only about 55 kDa and 25 kDa antibody heavy and light chain bands, respectively. The isotype of the purified antibody was IgG ₁ with kappa light chain.

B. Binding properties of EM164 antibody [115] Purified EM164 by fluorescence activated cell sorting (FACS) using cells overexpressing the human IGF-I receptor and cells overexpressing the human insulin receptor. Specific binding of the antibody was demonstrated (Figure 1). 100 μl cold FACS buffer (Dulbecco's MEM medium) using cells overexpressing IGF-I receptor and cells overexpressing insulin receptor (2 × 10 ⁵ cells / ml) in round bottom 96 well plates. EM164 antibody (50-100 nM) in 1 mg / ml BSA) for 1 hour. Cells were pelleted by centrifugation and washed with cold FACS buffer by tapping and then incubated with goat anti-mouse IgG antibody-FITC conjugate (100 μl; 10 μg / ml in FACS buffer) on ice for 1 hour. . Cells were pelleted, washed and resuspended in 120 μl of 1% formaldehyde solution in PBS. Plates were analyzed using a FACSCalibur reader (BD Biosciences).

  [116] Incubation of cells overexpressing the IGF-I receptor with EM164 antibody resulted in a strong fluorescence shift, which was not a significant shift when cells overexpressing the insulin receptor were incubated with EM164 antibody. (FIG. 1), which demonstrated that the EM164 antibody is selective in binding to the IGF-I receptor and does not bind to the insulin receptor. Control antibody, anti-IGF-I receptor antibody 1H7 (Santa Cruz Biotechnology) and anti-insulin receptor alpha antibody (BD Pharmingen Laboratories) overexpress IGF-I receptor and insulin receptor, respectively Upon incubation with the cells, a fluorescence shift occurred (Figure 1). A strong fluorescence shift was also observed by FACS assay using EM164 antibody and human breast cancer MCF-7 cells expressing IGF-I receptor (Dufourny, B. et al., 1997, J. Biol. Chem., 272). , 31163-31171), which showed that the EM164 antibody binds to the human IGF-I receptor on the surface of human tumor cells.

[117] Using either directly coated IGF-I receptor (affinity purified using biotinylated IGF-I as described above) or indirectly captured biotinylated IGF-I receptor The dissociation constant (K _d ) for binding of human IGF-I receptor to EM164 antibody was determined by ELISA titrating antibody binding at several concentrations. Biotinylated IGF-I by biotinylating lysates from cells overexpressing IGF-I receptor, solubilized with detergent, using PEO-maleimide-biotin reagent (Pierce, Molecular Biosciences). The receptor was prepared, affinity purified using anti-IGF-I receptor beta chain antibody immobilized on NHS-agarose beads, and 2-4 M in buffer containing NP-40 detergent. Eluted with urea and dialyzed in PBS.

[118] Biotinylated IGF-I receptor and EM164 by coating Immulon-2HB plates with 100 μl of 1 μg / ml streptavidin in carbonate buffer (150 mM sodium carbonate, 350 mM sodium bicarbonate) overnight at 4 ° C. The K _d for antibody binding was determined. Wells coated with streptavidin are blocked with 200 μl blocking buffer (10 mg / ml BSA in TBS-T buffer), washed with TBS-T buffer, and biotinylated IGF-I receptor (10-10 100 ng) and ambient temperature for 4 hours. The wells containing the indirectly captured biotinylated IGF-I receptor were then washed and at several concentrations (5.1 × 10 ⁻¹³ M-200 nM) EM164 antibody and ambient temperature in blocking buffer. Incubated for 2 hours and then incubated overnight at 4 ° C. The wells are then washed with TBS-T buffer and incubated with goat anti-mouse IgG _{H + L} antibody-horseradish peroxidase conjugate (100 μl; 0.5 μg / ml in blocking buffer) followed by washing. And at 405 nm with ABTS / H ₂ O ₂ substrate. For single site binding, K _d values were estimated by non-linear regression.

  [119] E.E. Harlow and D.H. Similar binding titrations using Fab fragments of the EM164 antibody prepared by papain digestion of the antibody as described in Lane ("Using Antibodies: A Laboratory Manual"; 1999, Cold Spring Harbor Laboratory Press, NY). Went.

[120] The binding titration curve for binding of EM164 antibody to biotinylated human IGF-I receptor yielded a _Kd value of 0.1 nM (FIG. 2). The Fab fragment of the EM164 antibody also binds very tightly to the human IGF-I receptor with a _Kd value of 0.3 nM, which also causes monomeric binding of the EM164 antibody to the IGF-I receptor. It was also shown to be very strong.

[121] This extremely low dissociation constant for the binding of the IGF-I receptor by the EM164 antibody is that after washing the antibody bound to the immobilized IGF-I receptor for a long period of 1-2 days, This was partly because the k _off rate was very slow, as confirmed by the observation of a strong binding signal.

  [122] As demonstrated by incubating lysates of human breast cancer MCF-7 cells solubilized with detergent with EM164 antibody immobilized on protein G-agarose beads (Pierce Chemical Company), The EM164 antibody can also be used for immunoprecipitation of the IGF-I receptor. Rabbit polyclonal anti-IGF-I receptor beta chain (C-terminal) antibody (Santa Cruz Biotechnology) and goat anti-rabbit IgG antibody-horseradish peroxidase conjugate were then washed and enhanced chemiluminescence (ECL) detection Performed to detect Western blots of EM164 antibody immunoprecipitates. Western blot of EM164 immunoprecipitates from MCF-7 cells showed a band corresponding to the beta chain of about 95 kDa IGF-I receptor and about 220 kDa pro-IGF-I receptor. For other cell types, similar immunoprecipitation was performed to check the species specificity of EM164 antibody binding, and it still binds to the IGF-I receptor from cos-7 cells (African green monkey). However, it did not bind to the IGF-I receptor of 3T3 cells (mouse), CHO cells (Chinese hamsters) or goat fibroblasts (goats). EM164 antibody does not detect SDS-denatured human IGF-I receptor in Western blots of lysates from MCF-7 cells, so that EM164 antibody conforms to native non-denatured human IGF-I receptor. It was shown to bind to the epitope.

  [123] Contains a cysteine-rich domain (residues 1-468) adjacent to the L1 and L2 domains, fused to it with a 16-mer C-terminal piece (residues 704-719), and with a C-terminal epitope tag The final, truncated alpha chain construct was used to further characterize the binding domain of the EM164 antibody. This smaller IGF-I receptor lacking residues 469-703 has been reported to bind to IGF-I, although less tightly compared to the native 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 partially excised IGF-I receptor alpha chain construct was prepared comprising residues 1-468, fused to the C-terminal piece of residues 704-719, and flanked by the C-terminal myc epitope tag. . A stable cell line expressing this construct and also a cell line transiently expressing the construct in human embryonic kidney 293T cells was constructed. EM164 antibody was observed to bind strongly to this partially excised IGF-I receptor alpha chain construct. Of the two antibodies tested, IR3 (Calbiochem) also bound to this partially excised alpha chain, but 1H7 antibody (Santa Cruz Biotechnology) did not bind, which caused the epitope of EM164 antibody to be that of 1H7 antibody. It was shown to be clearly distinct from the epitope.

C. Inhibition of IGF-1 binding to MCF-7 cells by EM164 antibody [124] IGF-I binding to human breast cancer MCF-7 cells was inhibited by EM164 antibody (FIG. 3). MCF-7 cells were incubated for 2 hours in serum-free medium with or without 5 μg / ml EM164 antibody, followed by incubation with 50 ng / ml biotinylated IGF-I at 37 ° C. for 20 minutes. The cells were then washed twice with serum free medium to remove unbound biotin-IGF-I and then lysed in 50 mM HEPES, pH 7.4 containing 1% NP-40 and protease inhibitors. An Immulon-2HB ELISA plate was coated with a mouse monoclonal anti-IGF-I receptor beta chain antibody and was used to capture IGF-I receptor and bound biotin-IGF-I from the lysate. Binding of the coated antibody to the cytoplasmic C-terminal domain of the beta chain of the IGF-I receptor did not interfere with the binding of biotin-IGF-I to the extracellular domain of the IGF-I receptor. Wells were washed, incubated with streptavidin-horseradish peroxidase conjugate, washed again and then detected with ABTS / H ₂ O ₂ substrate. Inhibition of IGF-I binding to MCF-7 cells by 5 μg / ml EM164 antibody is quantitative in nature and is approximately that of the ELISA background obtained using a control lacking biotin-IGF-I. It was equivalent.

  [125] In addition to the assay described above for inhibition of IGF-I binding to MCF-7 cells by EM164 antibody, EM164 antibody displaces bound IGF-I from MCF-7 cells by the following assay ( has proved to be very effective in disposing), and under physiological conditions an antagonistic anti-IGF-I receptor antibody is bound by an endogenous physiological ligand (such as IGF-I or IGF-II). It is suitable that it is desirable to replace In this IGF-I displacement assay, MCF-7 cells grown in 12-well plates are serum depleted and then biotinylated IGF-I (20-50 ng / ml) and 37 ° C. (or in serum-free medium) (4 ° C.) for 1-2 hours. Cells with bound biotinylated IGF-I were then treated with EM164 antibody or control antibody (10-100 μg / ml) at 37 ° C. (or 4 ° C.) for 30 minutes to 4 hours. Cells were then washed with PBS and lysed at 4 ° C. in lysis buffer containing 1% NP-40. An ELISA was performed as described above to capture the IGF-I receptor from the lysate and then a streptavidin-horseradish peroxidase conjugate was used to detect biotinylated IGF-I bound to the receptor. . By this ELISA, the EM164 antibody removed previously bound biotinylated IGF-I from the cells almost completely at 37 ° C. (90% within 30 minutes and ˜100% within 4 hours) and 4 ° C. It was proved that about 2% substitution was possible at 2 hours. In another experiment, incubation of NCI-H838 lung cancer cells with biotin-IGF-I, followed by washing and incubation with EM164 antibody for 2 hours at 4 ° C. resulted in an 80% reduction in bound biotin-IGF-I. Thus, the EM164 antibody is very effective in replacing previously bound IGF-I from cancer cells, which replaces IGF-I receptor by replacing the bound endogenous physiological ligand. It may be therapeutically important to antagonize the body.

  [126] Western blot using anti-IGF-I receptor beta chain antibody (Santa Cruz Biotechnology; sc-713) after incubation of EM164 antibody and MCF-7 cells at 4 ° C for 2 hours (or 37 ° C for 30 minutes) Based on the analysis, no significant downregulation of IGF-I receptor occurred, but incubation with EM164 antibody for a longer time at 37 ° C. resulted in 25% downregulation of IGF-I receptor. . Thus, in this short experiment, at both 4 ° C. and 37 ° C., the EM164 antibody inhibited IGF-I binding and the displacement of the bound IGF-I was due to the binding of the EM164 antibody binding. It cannot be explained by the downward control. The mechanism by which EM164 antibody strongly inhibits the binding of IGF-I to the IGF-I receptor and replaces the previously bound IGF-I is either through sharing of the binding site or through steric closure. It may be competing for binding either through (steric occlusion) or through an allosteric effect.

D. Inhibition of IGF-I receptor-mediated cell signaling by EM164 antibody [127] Treatment of breast cancer MCF-7 cells and osteosarcoma SaOS-2 cells with EM164 antibody results in inhibition of IGF-I receptor autophosphorylation. And intracellular IGF-I receptor signaling was almost completely inhibited, as shown by inhibition of phosphorylation of downstream effectors such as insulin receptor substrate 1 (IRS-1), Akt and Erk1 / 2 ( 4-6).

[128] In FIG. 4, MCF-7 cells were grown in 12-well plates for 3 days in standard medium and then 20 μg / ml EM164 antibody (or anti-B4 control antibody) in serum-free medium. And then stimulated with 50 ng / ml IGF-I for 20 minutes at 37 ° C. Then, an ice-cold lysis buffer containing a protease inhibitor and a phosphatase inhibitor (50 mM HEPES buffer, pH 7.4, 1% NP-40, 1 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 The cells were lysed in 5 mM EDTA, 10 μM leupeptin, 5 μM pepstatin, 1 mM PMSF, 5 mM benzamidine, and 5 μg / ml aprotinin). ELISA plates were pre-coated with anti-IGF-I receptor beta chain C-terminal monoclonal antibody TC123 and incubated with lysate samples at ambient temperature for 5 hours to capture IGF-I receptor. The wells containing the captured IGF-I receptor were then washed and incubated with a biotinylated anti-phosphotyrosine antibody (PY20; 0.25 μg / ml; BD Transaction Laboratories) for 30 minutes, then washed and streptavidin- Incubated with horseradish peroxidase conjugate (0.8 μg / ml) for 30 minutes. Wells were washed and detected with ABTS / H ₂ O ₂ substrate. Using a control anti-B4 antibody showed no inhibition of IGF-I-stimulated IGF-I receptor autophosphorylation. In contrast, treatment with EM164 antibody completely inhibited IGF-I-stimulated IGF-I receptor autophosphorylation (FIG. 4).

[129] To demonstrate inhibition of phosphorylation of insulin receptor substrate 1 (IRS-1), an ELISA was used to capture IRS-1 from the lysate using immobilized anti-IRS-1 antibody, and then phosphorylated IRS The associated p85 subunit of phosphatidylinositol-3-kinase (PI-3 kinase) binding to -1 was measured (Jackson, JG et al., 1998, J. Biol. Chem., 273, 9994-10003. ). In FIG. 5, MCF-7 cells were treated with 5 μg / ml antibody (EM164 or IR3) in serum-free medium for 2 hours and then stimulated with 50 ng / ml IGF-I at 37 ° C. for 10 minutes. Indirect capture of anti-IRS-1 antibody (rabbit polyclonal; Upstate Biotechnology) by incubating with coated goat anti-rabbit IgG antibody on an ELISA plate, which is then incubated overnight at 4 ° C. Was used to capture IRS-1 from cell lysate samples. The 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. The wells were then washed and detected with ABTS / H ₂ O ₂ substrate (FIG. 5). As shown in FIG. 5, EM164 antibody is more effective at inhibiting IGF-I-stimulated IRS-1 phosphorylation than IR3 antibody, and in the absence of IGF-I, EM164 antibody is When incubated with EM164 antibody did not show any agonist activity on IRS-1 phosphorylation.

[130] Activation of other downstream effectors such as Akt and Erk1 / 2 was also performed by Western blot of lysates and phosphorylation specific antibodies (rabbit polyclonal anti-phospho Ser ⁴⁷³ Akt polyclonal antibody and anti-phospho-ERK1 / 2 antibody). ; Inhibited by EM164 antibody in a dose-dependent manner in SaOS-2 cells (FIG. 6) and in MCF-7 cells as demonstrated using Cell Signaling Technology). The pan-ERK antibody demonstrated that all lanes were loaded with equal amounts of protein (FIG. 6). Treatment of SaOS-2 cells with EM164 antibody did not inhibit EGF-stimulated phosphorylation of Erk1 / 2, thus demonstrating that inhibition of the IGF receptor signaling pathway by EM164 antibody was specific.

E. Inhibition of IGF-I, IGF-II and serum-stimulated human tumor cell growth and survival by EM164 antibody [131] With respect to proliferation and survival responses to IGF-I, several humans under serum-free conditions Tumor cell lines were tested. These cell lines were treated with EM164 antibody in the presence of IGF-I, IGF-II, or serum, and after 2-4 days, proliferative and survival responses were measured using the MTT assay. Approximately 1500 cells were plated in 96-well plates in standard medium with serum and the following day the medium was replaced with serum-free medium (serum-free RPMI medium supplemented with transferrin and BSA, or Dufourny, B. et al., 1997, J. Biol. Chem., 272, 31163-31171 as specified in phenol red). After 1 day growth in serum-free medium, cells are incubated with about 75 μl of 10 μg / ml antibody for 30 minutes to 3 hours, after which 25 μl of IGF-I (or IGF-II or serum) solution is added. To a final concentration of 10 ng / ml IGF-I, or 20 ng / ml IGF-II, or 0.04-10% serum. In some experiments, cells were first stimulated with IGF-I for 15 minutes or both IGF-I and EM164 antibody were added together before adding EM164 antibody. The cells were then grown for an additional 2-3 days. MTT (3- (4,5) -dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; 5 mg / ml solution in 25 μl PBS) was then added and the cells were allowed for 2-3 hours. Returned to incubator. The medium was then removed and replaced with 100 μl DMSO, mixed, and the absorbance of the plate was measured at 545 nm. Some human tumor cell lines show a proliferative and survival response when IGF-I or IGF-II or serum is added, which is significantly inhibited by the EM164 antibody, which includes antibodies against IGF-I. It was irrelevant whether IGF-I was added before the antibody, or IGF-I was added before the antibody, or IGF-I and the antibody were added together (Table 1).

Table 1. Inhibition of IGF-I stimulated tumor cell proliferation and survival by EM164 antibody

^a MTT assay of 3-4 days growth / survival of cells in response to 10 ng / ml IGF-I in serum free medium containing 5-10 μg / ml EM164 antibody.
^b Cells in 1.25-10% serum in the presence of 5-10 μg / ml EM164 antibody by MTT assay or colony formation assay based on comparison with control (with serum but no antibody) Inhibition of proliferation; MCF-7 cells based on controls (no serum but no antibodies and no serum but no antibodies) to account for autocrine / paracrine IGF stimulation by cells, The degree of inhibition was quantitatively measured for NCI-H838 cells and SK-N-SH cells. ND indicates that the data is inferior due to lack of data or difficulty in staining.

[132] EM164 antibody strongly inhibited the growth and survival of breast cancer MCF-7 cells stimulated by IGF-I or serum (FIGS. 7 and 8). In another experiment, EM164 antibody strongly inhibited IGF-II stimulated proliferation and survival of MCF-7 cells. Previous reports with commercially available antibodies such as IR3 antibodies have shown that these antibodies only weakly inhibit the growth and survival of serum-stimulated MCF-7 cells, The IR3 and 1H7 antibodies are as confirmed in FIG. 7 (Cullen, KJ et al., 1990, Cancer Res., 50, 48-53). In contrast, EM164 antibody was a potent inhibitor of MCF-7 cells stimulated by serum or IGF. As shown in FIG. 8, EM164 antibody is equally effective in inhibiting the growth and survival of MCF-7 cells over a wide range of serum concentrations (0.04-10% serum).

[133] Growth inhibition of MCF-7 cells by EM164 antibody was measured by counting cells. Therefore, approximately 7500 cells were plated in RPMI medium containing 10% FBS in the presence or absence of 10 μg / ml EM164 antibody in 12 well plates. After 5 days of growth, the number of cells in the untreated control sample was 20.5 × 10 ⁴ cells, in contrast, only 1.7 × 10 ⁴ cells in the sample treated with the EM164 antibody. Treatment with EM164 antibody inhibited MCF-7 cell proliferation by about 12-fold in 5 days. This inhibition by the EM164 antibody was significantly higher than the 2.5-fold inhibition reported with the IR3 antibody in the 6-day assay of MCF-7 cells (Rohlik, QT et al., 1987, Biochem. Biophys.Res.Commun., 149, 276-281).

  [134] Proliferation and survival of IGF-I and serum stimulated non-small cell lung cancer line NCI-H838 was also strongly inhibited by EM164 antibody compared to control anti-B4 antibody (FIG. 9). Treatment with EM164 antibody in serum-free medium resulted in a smaller signal for both NCI-H838 and MCF-7 cells than the untreated sample, which is likely due to the EM164 antibody's autocrine and parasite secretion of these cells. It may be because secreted IGF-I and IGF-II stimulation was also inhibited (FIGS. 7 and 9). Treatment with EM164 antibody also greatly reduced the colony size of HT29 colon cancer cells.

  [135] The EM164 antibody is therefore effective in inhibiting serum-stimulated proliferation of tumor cells such as MCF-7 cells and NCI-H838 cells by more than 80%. It is unique among IGF-I receptor antibodies.

  [136] The EM164 antibody caused cell growth arrest at the G0 / G1 phase of the cell cycle and abrogated the mitogenic effect of IGF-I. For cell cycle analysis, MCF-7 cells were treated with IGF-I (20 ng / ml) for 1 day in the presence or absence of EM164 (20 μg / ml) and then propidium iodide staining and flow cytometry. Analyzed by. As shown in FIG. 25, cycling of cells in response to IGF-I stimulation in the absence of EM164 (41% of cells were in S phase and 50% were in G0 / G1 phase) was treated with EM164 treatment. Suppressed with cells (only 9% of cells were in S phase and 77% were in G0 / G1 phase).

  [137] In addition to inhibiting cell proliferation, EM164 antibody treatment resulted in cell apoptosis. For apoptosis measurements, caspase-mediated cleavage of cytokeratin CK18 protein was measured in NCI-H838 lung cancer cells incubated with IGF-I or serum for 1 day in the presence or absence of EM164 (FIG. 26). When IGF-I or serum is added in the absence of EM164, the signal of caspase-cleaved CK18 is lower compared to controls without IGF-I, and IGF-I and serum prevent caspase activation It was shown that. Treatment with EM164 suppressed the anti-apoptotic effects of IGF-I and serum, as shown by the higher levels of cleaved CK18 obtained in the presence of EM164 than in the absence of EM164.

F. Synergistic inhibition of human tumor cell growth and survival by EM164 antibody in combination with other cytotoxic and cytostatic agents [138] Taxol and EM164 antibody are administered in combination with non-small cell lung cancer rather than taxol alone Significantly more inhibitory to Calu6 cell proliferation and survival. Similarly, the combined use of camptothecin and EM164 antibody was significantly more inhibitory than camptothecin alone on the growth and survival of colon cancer HT29 cells. Since the EM164 antibody alone is not expected to be as toxic to cells as an organic chemical toxic agent, the synergy between the mitogenic effect of the EM164 antibody and the cytotoxic effect of the chemical toxic agent is: It can be very effective in combination cancer therapy in a clinical setting.

  [139] The combined effect of EM164 antibody and anti-EGF receptor antibody (KS77) is on the growth and survival of several tumor cell lines such as HT-3 cells, RD cells, MCF-7 cells, and A431 cells , Significantly more inhibitory than either EM164 antibody or KS77 antibody alone. Thus, the synergistic effect of combining two growth factor receptor neutralizing antibodies such as IGF-I receptor and EGF receptor may also be useful for clinical cancer treatment.

  [140] Based on the efficacy of EM164 antibody as a single agent in inhibiting the growth and survival of various human cancer cell lines as shown in Table 1, combining EM164 antibody with other anticancer therapeutic agents Further efficacy studies were conducted. In these studies on a variety of cancer cell lines, the combined treatment of EM164 antibody and other anticancer therapeutic agents resulted in higher anticancer efficacy than either EM164 or other therapeutic agents alone. Thus, these combinations of EM164 and other therapeutic agents are very effective in inhibiting cancer cell growth and survival. Therapeutic agents that can be used in combination with EM164 to improve anticancer efficacy include a variety of agents used in oncology treatment (Reference: Cancer, Principles & Practice of Oncology, DeVita, VT, Hellman. , S., Rosenberg, SA, 6th edition, Lippincott-Raven, Philadelphia, 2001), for example, docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Herceptin), capecitabine, taxene, Letrozole, anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone Antid, bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronate, streptozocin, rituximab, rituximab, rituximab, rituximab Fludarabine, imatinib, cytarabine, ibritumomab (zevalin), tositumomab (bexal), interferon alfa-2b, melphalan, bortezomib (velcade), altretamine, asparaginase, gefitinib (Iressa), erlonitib (anti-TGF receptor) (Cetuximab, Abx-EGF), epothilone, and cytotoxic agents and cell surfaces There are conjugates of antibodies to receptor.

  [141] For these combination therapy, alkylating agent, platinum agent, hormone therapy, antimetabolite, topoisomerase inhibitor, anti-microtubule agent, differentiation agent, anti-angiogenic or anti-angiogenic therapy, radiation therapy, corpus luteum Agonists and antagonists of forming hormone releasing hormone (LHRH) or gonadotropin releasing hormone (GnRH), inhibitory antibodies or small molecule inhibitors to cell surface receptors, and other chemotherapeutic agents (Reference: Cancer, Principles & Practice of Oncology, DeVita, VT, Hellman, S., Rosenberg, SA, 6th edition, Lippincott-Raven, Philadelphia, 2001) A combination of EM164. In one example, the LHRH antagonist antid (0.1-10 micromolar) and EM164 antibody (0.1-10 nanomolar) were used in combination over either EM164 or antid alone. , MCF-7 breast cancer cell proliferation was significantly inhibited. In the example of combination therapy with platinum agents, combination treatment with EM164 antibody (10 microgram / ml) and cisplatin (0.1-60 microgram / ml) compared to inhibition by either EM164 antibody or cisplatin alone. Thus, the proliferation and survival of MCF-7 breast cancer cells were more inhibited.

  [142] These combinations of EM164 antibody with other therapeutic agents include breast cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, cervical cancer, ovarian cancer, melanoma, multiple myeloma, neuroblastoma, It is effective against several types of cancer, including rhabdomyosarcoma and osteosarcoma. The EM164 antibody and the therapeutic agent can be administered for cancer therapy simultaneously or sequentially.

  [143] Conjugates of EM164 antibody and cytotoxic agents are also useful in targeting delivery of cytotoxic agents to tumors that overexpress the IGF-I receptor. It is also possible to use conjugates of EM164 antibody and radiolabels or other labels for the treatment and imaging of tumors that overexpress the IGF-I receptor.

G. Effect of EM164 treatment as a single agent or in combination with anticancer agents in human cancer xenografts in immunodeficient mice [144] By injection of 1 × 10 ⁷ Calu-6 cells subcutaneously in immunodeficient mice A cell lung cancer Calu-6 xenograft was established. As shown in FIG. 10, using 5 mice per treatment group, these mice containing the established 100 mm ³ Calu-6 xenograft were treated with EM164 antibody alone (0.8 mg / mouse 2 weeks / week). 6 times, intravenous injection), or taxol alone (5 times taxol every 2 days, intraperitoneal injection; 15 mg / kg), or in combination with taxol and EM164 antibody treatment, or PBS alone (200 μl / Mice were treated twice a week 6 times intravenously). Compared to the PBS control, EM164 antibody treatment significantly delayed tumor growth. Based on mouse body weight measurements, no EM164 antibody toxicity was observed. Taxol alone treatment was effective until day 14, after which the tumor began to grow again. However, tumor growth was significantly delayed in the taxol and EM164 antibody combination treatment group compared to the taxol alone treatment group.

[145] Human pancreatic cancer xenografts were established in 5 week old female SCID / ICR mice (Taconic) by subcutaneous injection of 10 ⁷ BxPC-3 cells in PBS (day 0). Then, using 5 mice in each of the 5 treatment groups, mice bearing established 80 mm ³ tumors were isolated from EM164 alone (Day 12, Day 16, Day 19, Day 23, Day 26 days, 29 days, 36 days, 43 days, 50 days, 54 days, 58 days, 61 days and 64 days, 13 mg of 0.8 mg / mouse intravenously into the lateral tail vein Internal injection), gemcitabine alone (on days 12 and 19, 150 mg / kg / mouse twice, intraperitoneal injection), gemcitabine and EM164 combined according to the above schedule, PBS alone, and control antibody alone (According to the same schedule as EM164). As shown in FIG. 27, tumor xenografts in EM164 alone or in combination with gemcitabine resulted in 4 out of 5 animals in the EM164 treated group and all 5 animals in the combined treatment group. A complete regression of first occurred. Measurable tumor regrowth was seen in more than one animal only at day 43 in the EM164 group and day 68 in the combination treatment group, and significantly smaller compared to the control treatment group on day 74 Average tumor volumes were produced (P = 0.029 and 0.002 respectively; two-tailed T test; FIG. 27). In another study, EM164 antibody treatment (alone or in combination with anti-EGF receptor antibody; intraperitoneal injection) inhibited the growth of BxPC-3 xenografts established in mice.

  [146] Murine EM164 and humanized EM164 antibodies have demonstrated equivalent inhibition of BxPC-3 xenograft growth established in mice, thus the potency of humanized EM164 is in vivo and that of murine EM164 It was proved to be equivalent. When comparing the different modes of administration of the EM164 antibody, both intraperitoneal and intravenous administration of the EM164 antibody showed equivalent inhibition of established BxPC-3 xenograft growth in mice. In another xenograft study, treatment of an A-673 human rhabdomyosarcoma / Ewing sarcoma xenograft established in mice with EM164 antibody significantly delayed proliferation.

H. Cloning and sequencing of EM164 antibody light and heavy chains [147] Total RNA was purified from EM164 hybridoma cells. Reverse transcriptase reactions were performed using 4-5 μg of total RNA and either oligo dT primers or random hexamer primers.

[148] Using the RACE method described in Co et al. (J. Immunol., 148, 1149-1154 (1992)) and in Wang et al. (J. Immunol. Methods, 233, 167-177 (2000)). PCR reactions were performed using degenerate primers as described. The RACE PCR method requires an intermediate step of adding a poly G tail to the 3 ′ end of the first strand cDNA. The RT reaction was purified on a Qianeasy (Qiagen) column and eluted in 50 μl of 1 × NEB buffer 4. The eluate was subjected to a dG tail addition reaction with 0.25 mM CoCl ₂ , 1 mM dGTP, and 5 units of terminal transferase (NEB) in 1 × NEB buffer 4. The mixture was incubated at 37 ° C. for 30 minutes and then 1/5 (10 μl) of the reaction was added directly to the PCR reaction and utilized as template DNA.

  [149] RACE and degenerate PCR reactions were identical except that the primers and template were different. A terminal transferase reaction was used directly for the RACE PCR template, while an RT reaction mixture was used directly for the degenerate PCR reaction.

  [150] Same 3 'light chain primer in both RACE and degenerate PCR reactions:

And 3 'heavy chain primer:

Was used.
[151] In RACE PCR, one poly C 5 ′ primer:

Is used for both heavy and light chains, while degenerate 5'end PCR primers are:

And for the heavy chain:

Of the same amount.

  [152] In the primer sequence, the mixed base is 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 .

  [153] The PCR reaction was carried out using the following program: 1) 94 ° C for 3 minutes, 2) 94 ° C for 15 seconds, 3) 45 ° C for 1 minute, 4) 72 ° C for 2 minutes, 5) Return to step 2 29 cycles, 6) End at the final extension step at 72 ° C. for 10 minutes.

[154] The PCR product was cloned into pBluescript II SK + (Stratagene) using restriction enzymes generated by PCR primers.
[155] By conventional means, several individual light and heavy chain clones were sequenced to identify and avoid sequence errors that the polymerase could generate (Figures 12 and 13). Using the Chothia canonical taxonomy definition, three light and heavy chain CDRs were identified (FIGS. 12-14).

  [156] When searching the NCBI IgBlast database, the anti-IGF-I receptor antibody light chain variable region is probably derived from the mouse IgVk Cr1 germline gene, while the heavy chain variable region is presumably IgVh J558. cDerived from germline genes (FIG. 15).

  [157] Protein sequencing of the murine EM164 antibody was performed to confirm the sequences shown in FIGS. The purified EM164 antibody heavy and light chain protein bands were transferred from a gel (SDS-PAGE, reducing conditions) to a PVDF membrane, excised from the PVDF membrane, and analyzed by protein sequencing. The N-terminal sequence of the light chain was determined by Edman sequencing to be: DVLMTQTPLS (SEQ ID NO: 20), which matched the N-terminal sequence of the cloned light chain gene obtained from the EM164 hybridoma.

  [158] The N-terminus of the heavy chain was found to be blocked and not capable of Edman protein sequencing. Via post-source digestion (PSD), the trypsin digested peptide fragment of the heavy chain of mass 1129.5 (M + H +, monoisotopic) was fragmented and its sequence was determined to be GRPDYYGSSK (SEQ ID NO: 21). It was. Via post-source degradation (PSD), another trypsin digested peptide fragment of the heavy chain of mass 2664.2 (M + H +, monoisotopic) is also fragmented and its sequence is: SSSTAYMQLSLSTSEDSAVYYFAR (SEQ ID NO: 22) Was identified. Both of these sequences perfectly match those of CDR3 and framework 3 (FR3) of the cloned heavy chain gene obtained from EM164 hybridoma.

I. Recombinant expression of EM164 antibody [159] The light and heavy chain pair sequences were cloned into a single mammalian expression vector (Figure 16). The human variable sequence PCR primer generates a restriction site that allows the human signal sequence to be attached while in the pBluescript II cloning vector and for the light or heavy chain, respectively, an EcoRI site. The variable sequence was cloned into a mammalian expression plasmid using and BsiWI or HindIII and ApaI sites (FIG. 16). The light chain variable sequence was cloned in frame onto the human IgK constant region and the heavy chain variable sequence was cloned into the human Ig gamma 1 constant region sequence. In the final expression plasmid, the human CMV promoter driven the expression of both light and heavy chain cDNA sequences. Expression and purification of the recombinant mouse EM164 antibody proceeded according to methods well known in the art.

(Example 2)
Humanized version of EM164 antibody [160] In order to provide a humanized version suitable as a therapeutic or diagnostic agent, surface reconstruction of EM164 antibody is generally described in US Pat. No. 5,639,641 Proceed according to the method and as follows.

A. Surface prediction [161] Solvent accessibility of variable region residues of antibody sets with elucidated structures was used to predict surface residues of murine anti-IGF-I receptor antibody (EM164) variable regions. The amino acid solvent accessibility (Table 2) of 127 unique antibody structure file sets was calculated with the MC software package (Pedersen et al., 1994, J. Mol. Biol., 235, 959-973). From this set of 127 structures, the 10 most similar light and heavy chain amino acid sequences were determined by sequence alignment. For each variable region residue, the average solvent accessibility was calculated and an average accessibility position greater than 30% was considered a surface residue. Only for structures with two identical adjacent residues, positions with an average accessibility between 25% and 35% were further investigated by calculating the accessibility of individual residues.

Table 2-127 antibody structures from the Brookhaven database used to predict the surface of anti-IGF-I receptor antibody (EM164)

B. Molecular modeling:
[162] A molecular model of murine EM164 was generated using the Oxford Molecular Software Package AbM. The antibody framework was constructed using the structure file of the antibody with the most similar amino acid sequence, 2jel of the light chain and 1nqb of the heavy chain. Non-canonical CDRs were constructed by searching a C-α structure database containing non-redundant elucidated structures. Residues located within 5 Å of the CDRs were determined.

C. Human Ab selection [163] The surface position of murine EM164 was compared to the corresponding position of the human antibody sequence in the Kabat database (Johnson, G. and Wu, TT (2001) Nucleic Acids Research, 29: 205-). 206). Antibody surface management software SR (Searle 1998) was used to extract and juxtapose antibody surface residues from natural heavy and light chain human antibody pairs. The human antibody surface with the most identical surface residues was chosen to specifically replace the murine anti-IGF-I receptor antibody surface residues, especially considering the position within 5 cm of the CDR.

D. PCR mutagenesis [164] Murine EM164 cDNA clones (above) were subjected to PCR mutagenesis to construct surface reconstituted human EM164 (here huEM164). A primer set was designed to create the 8 amino acid changes required for all tested versions of huEM164, and an additional primer was designed to create two separate 5Å residue changes (Table 3). . PCR reaction was performed using the following program: 1) 94 ° C. for 1 minute, 2) 94 ° C. for 15 seconds, 3) 55 ° C. for 1 minute, 4) 72 ° C. for 1 minute, 5) Return to step 2 for 29 cycles 6) Finished at the final extension step at 72 ° C. for 4 minutes. The PCR product was digested with the corresponding restriction enzymes and cloned into the pBluescript cloning vector as described above. Clones were sequenced to confirm the desired amino acid changes.

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

E. Variable region surface residues [165] Antibody surface reconstitution described in Pedersen et al. (J. Mol. Biol., 235, 959-973, 1994) and Roguska et al. (Protein Eng., 9, 895-904, 1996). The technique begins with predicting surface residues of murine antibody variable sequences. Surface residues are defined as amino acids in which at least 30% of the total surface area is accessible to water molecules.

  [166] Among the 127 antibody structure file sets, the 10 most homologous antibodies were identified (FIGS. 17 and 18). For these parallel sequences, the solvent accessibility at each Kabat position was averaged, and the distribution of relative accessibility of each residue is shown in FIG. Both light and heavy chains have 26 residues with an average relative accessibility of at least 30% (FIG. 19): thus these residues were the predicted surface residues of EM164 . Some residues have an average accessibility between 25% and 35%, and these are further increased by averaging only those antibodies in which the two adjacent residues on either side of the residue are identical. It investigated (Table 4 and Table 5). After this further analysis, the original set of surface residues identified above remained unchanged.

Table 4-Surface residues and average accessibility of light and heavy chain variable sequences of EM164 antibody

Table 5

  Residues with an average accessibility between 25% and 35% were further analyzed by averaging antibody subsets where the two adjacent residues on either side of the residue under consideration are identical. Provides these borderline surface positions and its new average accessibility. NA refers to residues that do not have identical adjacent residues in the ten most similar antibodies.

F. Molecular modeling to determine which residues are within 5 cm of the CDRs [167] Analyzing the above molecular model generated with the AbM software package to determine which EM164 surface residues are within 5 cm of the CDRs Were determined. In order to reconstitute the surface of the murine EM164 antibody, all surface residues outside the CDRs must be changed to their human counterparts, but residues within 5 cm of the CDRs can also contribute to antigen specificity, Treated with special care. Therefore, these latter residues must be identified and carefully considered throughout the humanization process. The CDR definitions used for surface reconstruction combine the AbM definition for heavy chain CDR2 and the Kabat definition for the remaining 5 CDRs (FIG. 14). Table 6 shows residues that were within 5 か ら of either CDR residue in either the light chain or heavy chain sequence of the EM164 model.

Table 6 EM164 antibody framework surface residues within 5 cm of CDR
EM164 surface residue within 5 cm of CDR

G. Identification of the most homologous human surface [168] Candidate humans that resurface EM164 in the Kabat antibody sequence database using SR software that arranges the search for only specified residue positions against the antibody database The antibody surface was identified. In order to preserve natural pairing, both light and heavy chain surface residues were compared together. The most homologous human surfaces in the Kabat database were aligned in order of sequence identity. The top five surfaces are provided in Table 7. These surfaces were then compared to identify which of them required minimal changes within 5 cm of the CDR. The leukemia B cell antibody, CLL 1.69, required a minimal number of surface residue changes (total 10) and only two of these residues were present within 5 cm of the CDRs.

  [169] The full-length variable region sequence of EM164 was also juxtaposed against the Kabat human antibody database, and again CLL 1.69 was identified as the most similar human variable region sequence. Taken together, these sequence comparisons identified the human leukemia B cell antibody CLL 1.69 as a preferred choice for the human surface of EM164.

Table 7-Top 5 human sequences extracted from Kabat database

  Parallel was generated by SR (Pedersen 1993). EM164 surface residues within 5 cm of the CDR are underlined.

H. Construction of the humanized EM164 gene [170] Ten surface residue changes of EM164 (Table 7) were generated using PCR mutagenesis techniques as described above. Since 8 of the surface residues of CLL 1.69 are not within 5 cm of the CDRs, these versions were changed from murine to human in all versions of humanized EM164 (Tables 8 and 9). . Two light chain surface residues within 5 ３ of the CDRs (Kabat positions 3 and 45) were changed to human or left murine. Taken together, these options generate four humanized versions of the constructed EM 164 (FIGS. 22 and 23).

  [171] Of the four humanized versions, version 1.0 has all 10 human surface residues. With regard to changes in the vicinity of the CDR, the most conservative version is version 1.1, which retained both murine surface residues that were within 5 cm of the CDR. Four humanized EM164 antibody genes were cloned into antibody expression plasmids for use in transient transfection and stable transfection (FIG. 16).

Table 8-Residue changes of humanized EM164 antibody version 1.0-1.3

I. Comparison of affinity of murine EM164 antibody and humanized EM164 antibody version for binding to full length IGF-I receptor and to partially excised IGF-I receptor alpha chain [172] Biotinylated full length as described above Through binding competition assays using human IGF-I receptor or myc-epitope tagged partially excised IGF-I receptor alpha chain, the affinity of humanized EM164 antibody version 1.0-1.3 was determined using murine EM164 antibody. Compared with the ones. In human embryonic kidney 293T cells, humanized EM164 antibody samples were obtained by transient transfection of the appropriate expression vector, and antibody concentrations were determined by ELISA using purified humanized antibody standards. For ELISA binding competition measurements, a mixture of humanized antibody samples and various concentrations of murine EM164 antibody was used to indirectly capture biotinylated full length IGF-I receptor or myc-epitope tagged partially excised IGF-I receptor. Incubated with body alpha chain. After equilibration, a goat anti-human Fab _'2 antibody - with horseradish peroxidase conjugate, to detect bound human antibody. Theoretically yields a straight line with slope = (K _{d humanized Ab} / K _{d murine Ab} ), ([bound murine Ab] / [bound humanized Ab]) versus ([murine Ab] / [humanized]. Ab]) plot was used to determine the relative affinities of the humanized and murine antibodies.

[173] A typical competition assay is shown in FIG. Immulon-2HB ELISA plates were coated with 5 μg / ml streptavidin in 100 μl carbonate buffer per well for 7 hours at ambient temperature. Wells coated with streptavidin are blocked with 200 μl blocking buffer (10 mg / ml BSA in TBS-T buffer) for 1 hour, washed with TBS-T buffer, and biotinylated IGF-I receptor ( 5 ng per well) and overnight at 4 ° C. The wells containing indirectly captured biotinylated IGF-I receptor were then washed and humanized EM164 antibody (15.5 ng) and murine antibody (0 ng, or 16.35 ng, in 100 μl blocking buffer, Or 32.7 ng, or 65.4 ng, or 163.5 ng) of the mixture for 2 hours at ambient temperature and then overnight at 4 ° C. Then, the wells were washed with TBS-T buffer and goat anti-human Fab _'2 antibody - horseradish peroxidase conjugate (100 [mu] l; in blocking buffer, 1 [mu] g / ml) and incubated for 1 hour, then washed And at 405 nm with ABTS / H ₂ O ₂ substrate.

The plot of [174] ([bound murine Ab] / [bound humanized Ab]) versus ([murine Ab] / [humanized Ab]) shows a slope of 0.52 (= K _{d humanized Ab} / K _d yielded a straight line (r ² = 0.996) with _{murine Ab} ). Thus, humanized antibody version 1.0 bound to the IGF-I receptor more closely than the murine EM164 antibody. Murine EM164 and humanized EM164 antibody versions 1.0, 1.1, 1.2 and 1.3 for binding to full length IGF-I receptor or to partially excised IGF-I receptor alpha chain Since similar values of slopes ranging from about 0.5 to 0.8 were obtained for all competition, all humanized versions of EM164 antibody had similar affinity, all of which were the parent murine EM164. It was shown to be superior to that of the antibody. The chimeric version of the EM164 antibody with the 92F → C mutation in the heavy chain showed a slope of about 3 in similar binding competition with the murine EM164 antibody, indicating that the 92F → C mutant of EM164 is an IGF It was shown to have a 3-fold lower affinity for binding to the -I receptor than the murine EM164 antibody. The humanized EM164 v1.0 antibody showed a similar inhibition of IGF-I stimulated MCF-7 cell proliferation and survival as the murine EM164 antibody (FIG. 24). Inhibition of serum-stimulated MCF-7 cell proliferation and survival by humanized EM164 v1.0 antibody was similar to inhibition by murine EM164 antibody.

Table 9

  The Kabat numbering system is used for light chain and heavy chain variable region polypeptides of different versions of EM164Ab. Amino acid residues are grouped into framework regions (FR) and complementarity determining regions (CDRs) according to their position in the polypeptide chain.

  Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, 1991, NIH Publication No. Adopted from 91-3242.

J. et al. Process of providing improved anti-IGF-I receptor antibody starting from murine antibody sequences and humanized antibody sequences described herein [175] of anti-IGF-I receptor antibody EM164 and humanized variants thereof Amino acid and nucleic acid sequences were used to create other antibodies that have improved properties and are also within the scope of the present invention. Such improved properties include increased affinity for the IGF-I receptor. Several studies have investigated the effects on properties such as binding and expression levels of introducing one or more amino acid changes at various positions in an antibody sequence based on knowledge of the original antibody sequence. (Yang, WP, et al., 1995, J. Mol. Biol., 254, 392-403; Rader, C., et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915; TJ et al., 1998, Nature Biotechnology, 16, 535-539).

  [176] In these studies, methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or E. coli mutator strains are used, and CDR1, CDR2, CDR3, or frame By altering the heavy and light chain gene sequences in the work region, the first antibody variants were generated (Vaughan, TJ et al., 1998, Nature Biotechnology, 16, 535-539; Adey, NB et al., 1996, Chapter 16, pp. 277-291, “Page Display of Peptides and Proteins”, supervised by Kay, BK et al., Academic Press). These methods of altering the sequence of the initial antibody have resulted in a second antibody with improved affinity through the use of standard screening techniques (Gram, H. et al., 1992, Proc. Natl. Acad. USA, 89, 3576-3580; Boder, ET et al., 2000, Proc. Natl. Acad. Sci. USA, 97, 10701-10705, Davies, J. and Riechmann, L., nte, 1996, Imm. Thompson, J. et al., 1996, J. Mol. Biol., 256, 77-88; Short, M.K. et al., 2002, J. Biol.Chem., 277, 16365-1 6370; Furukawa, K. et al., 2001, J. Biol. Chem., 276, 27622-27628).

  [177] It is also possible to generate anti-IGF-I receptor antibodies with improved function using the antibody sequences described in the present invention by a similar designated strategy that changes one or more amino acid residues of an antibody. There are antibodies with suitable groups, such as free amino or thiol groups at convenient attachment points for covalent modifications, for example for the attachment of therapeutic agents.

K. Alternative expression systems for murine, chimeric and other anti-IGF-I receptor antibodies [178] From mammalian plasmids similar to those used to express humanized antibodies (above), murine anti-IGF- I receptor antibody was also expressed. Expression plasmids are known to have a murine constant region containing a light chain kappa sequence and a heavy chain gamma-1 sequence (McLean et al., 2000, Mol Immunol., 37, 837-845). These plasmids were designed to accept any antibody variable region, such as a murine anti-IGF-I receptor antibody, by simple restriction digests and cloning. Usually, further PCR of anti-IGF-I receptor antibodies was required to generate restriction sites that were compatible with those in the expression plasmid.

  [179] Another approach to expressing full length murine anti-IGF-I receptor antibody was to replace the human constant region of the chimeric anti-IGF-I receptor antibody expression plasmid. A chimeric expression plasmid (FIG. 16) was constructed using variable region cassettes and both light and heavy chain constant region cassettes. By restriction digestion, the antibody variable sequence was cloned in either of the constant region sequences using another restriction digest just as it was cloned into this expression plasmid. For example, for cloning the anti-IGF-I antibody variable region, kappa light chain cDNA and gamma-1 heavy chain cDNA were cloned from murine hybridoma RNA, such as the RNA described herein. Similarly, appropriate primers were designed from sequences available in the Kabat database (see Table 10). For example, RT-PCR was used to clone the constant region sequences and create the restriction sites necessary to clone these fragments into a chimeric anti-IGF-I receptor antibody expression plasmid. This plasmid was then used to express a full length murine anti-IGF-I receptor antibody in a standard mammalian expression system such as a CHO cell line.

Table 10-Primers designed to clone murine gamma-1 constant region and murine kappa constant region, respectively.

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

Description of Deposit [180] The hybridoma producing the murine EM164 antibody was deposited on June 14, 2002 in the American Type Culture Collection, PO Box 1549, Manassas, VA 20108, under the terms of the Budapest Treaty. And assigned the ATCC deposit number PTA-4457.

[181] Specific patents and printed publications are referred to in this disclosure, each of which is fully incorporated herein by reference.
[182] While this invention has been described in detail and with reference to specific embodiments, those skilled in the art can make various changes and modifications without departing from the spirit and scope of this invention. It will be clear that

[35] FIG. 1 shows a fluorescence activated cell sorting (FACS) analysis of specific binding of purified EM164 antibody to cells overexpressing human Y1251F IGF-I receptor or human insulin receptor. [36] FIG. 2 shows a binding titration curve for binding of EM164 antibody to biotinylated human IGF-I receptor. [37] FIG. 3 shows inhibition of biotinylated IGF-I binding to human breast cancer MCF-7 cells by EM164 antibody. [38] FIG. 4 shows inhibition by EM164 antibody of IGF-I-stimulated autophosphorylation of IGF-I in MCF-7 cells. [39] FIG. 5 shows inhibition of IRS-1 phosphorylation stimulated by IGF-I in MCF-7 cells by EM164 antibody. [40] FIG. 6 shows inhibition by EM164 antibody of IGF-I-stimulated signaling in SaOS-2 cells. [41] FIG. 7 shows the effect of EM164 antibody on the growth and survival of MCF-7 cells under different growth conditions as assessed by MTT assay. [42] FIG. 8 shows the effect of EM164 antibody on the growth and survival of MCF-7 cells in the presence of various concentrations of serum. [43] FIG. 9 shows inhibition by EM164 antibody of IGF-I stimulated and serum stimulated NCI-H838 cell proliferation and survival. [44] FIG. 10 shows the effect of treatment with EM164 antibody, taxol, or a combination of EM164 antibody and taxol on the growth of Calu-6 lung cancer xenografts in mice. [45] FIG. 11 shows the competition between the binding of humanized EM164 antibody (v.1.0) and murine EM164 antibody. [46] FIG. 12 shows the cDNA sequence (SEQ ID NO: 49) and amino acid sequence (SEQ ID NO: 50) of the leader region and variable region of the light chain of murine anti-IGF-I receptor antibody EM164. The arrow marks the beginning of framework 1. Three CDR sequences according to Kabat are underlined. [47] FIG. 13 shows the cDNA sequence (SEQ ID NO: 51) and amino acid sequence (SEQ ID NO: 52) of the heavy chain leader region and variable region of murine anti-IGF-I receptor antibody EM164. The arrow marks the beginning of framework 1. Three CDR sequences according to Kabat are underlined. [48] FIG. 14 shows the CDR amino acid sequences of the light and heavy chains of antibody EM164 as determined by the Chothia canonical class definition. Also shown is the AbM modeling software definition of the heavy 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, and 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. [49] FIG. 15 shows Cr1 (SEQ ID NO: 56) and J588. c (SEQ ID NO: 57) shows the light and heavy chain amino acid sequences of anti-IGF-I receptor antibody EM164, juxtaposed with the germline sequence of the gene. A dash symbol (-) indicates sequence identity. [50] FIG. 16 shows plasmids used to construct and express recombinant EM164 chimeric and recombinant humanized EM164 antibodies. A) light chain cloning plasmid, B) heavy chain cloning plasmid, C) mammalian antibody expression plasmid. [51] FIG. 17 shows the 10 most homologous light chain amino acid sequences screened from 127 antibodies in the structure file set used to predict the surface residues of EM164. eml64 LC (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). [52] FIG. 18 shows the 10 most homologous heavy chain amino acid sequences screened from 127 antibodies in the structure file set used to predict the surface residues of EM164. em164 HC (SEQ ID NO: 70), 1 nqb (SEQ ID NO: 71), 1 ngp (SEQ ID NO: 72), 1fbi (SEQ ID NO: 73), 1afv (SEQ ID NO: 74), 1 yuh (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). [53] Figure 19 shows the average accessibility of each of the variable region residues of (A) light chain and (B) heavy chain from the 10 most homologous structures. Numbers indicate Kabat antibody sequence position numbers. [54] FIG. 20 shows the light chain variable region amino acid sequences of murine EM164 (muEM164) and humanized 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). [55] FIG. 21 shows the heavy chain variable region amino acid sequences of murine (muEM164, SEQ ID NO: 87) and humanized EM164 antibody (huEM164, SEQ ID NO: 88). [56] FIG. 22 shows huEM164 v1.0 variable region DNA and amino acids of both 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) Indicates the sequence. [57] FIG. 23 shows humanized 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 v1.3 (DNA, The light chain variable region DNA and amino acid sequence of SEQ ID NO: 97; amino acid, SEQ ID NO: 98) are shown. [57] FIG. 23 shows humanized 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 v1.3 (DNA, The light chain variable region DNA and amino acid sequence of SEQ ID NO: 97; amino acid, SEQ ID NO: 98) are shown. [58] FIG. 24 shows inhibition of IGF-I stimulated MCF-7 cell proliferation and survival by humanized EM164 v1.0 and murine EM164 antibodies. [59] FIG. 25 shows that EM164 suppresses cycling of MCF-7 cells stimulated by IGF-I. [60] FIG. 26 shows that EM164 suppresses the anti-apoptotic effects of IGF-1 and serum. Treatment with EM164 results in apoptotic cell death, as evidenced by increased levels of CK18 protein cleavage. [61] FIG. 27 shows the effect of treatment with EM164 antibody, gemcitabine, or a combination of EM164 antibody and gemcitabine on the growth of human BxPC-3 pancreatic cancer xenografts in immunodeficient mice.

Claims (31)

(A) a first therapeutic agent, wherein said first therapeutic agent is an antibody or an epitope-binding fragment thereof, and said antibody or said fragment specifically binds to insulin-like growth factor I receptor. :
(I) an antibody having the same amino acid sequence as murine antibody EM164 produced by mouse hybridoma EM164 (ATCC Deposit No. PTA-4457), or an epitope-binding fragment thereof,
(Ii) a resurfaced antibody having the same binding specificity as murine antibody EM164, or an epitope-binding fragment thereof,
(Iii) a human or humanized antibody having the same binding specificity as murine antibody EM164, or an epitope-binding fragment thereof,
(Iv) a functional equivalent of an antibody having the same binding specificity as murine antibody EM164, or an epitope-binding fragment thereof,
(V) a variant of murine antibody EM164 having at least one nucleotide mutation, deletion or insertion compared to murine antibody EM164 and having the same binding specificity as murine antibody EM164, or an epitope-binding fragment thereof, And (vi) said first therapeutic agent selected from the group consisting of murine antibody EM164 produced by murine hybridoma EM164 (ATCC Deposit No. PTA-4457), or an epitope-binding fragment thereof, and (b) A composition comprising a second therapeutic agent.   The second therapeutic agent is docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin , Carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, simalezoto, zoledrozole Rituximab (Rituxan), idarubicin, busulfan, chlorambucil, fludarabine, imati , Cytarabine, ibritumomab (zevalin), tositumomab (bexal), interferon alfa-2b, melphalan, bortezomib (velcade), altretamine, asparaginase, gefitinib (Iressa), erlonitib (Tarceva), anti-EGF receptor antibody (cetuximab) The composition of claim 1, selected from the group consisting of (Abx-EGF) and epothilone.   The composition of claim 1, wherein the second therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, and camptothecin.   A first therapeutic agent is administered to the patient at a dosage of about 1 mg / square meter to about 2000 mg / square meter, and a second therapeutic agent is administered at a dosage of about 10 mg / square meter to about 2000 mg / square meter; The composition of claim 1.   A first therapeutic agent is administered to the patient at a dosage of about 10 mg / square meter to about 1000 mg / square meter, and a second therapeutic agent is administered at a dosage of about 50 mg / square meter to about 1000 mg / square meter; The composition of claim 1.   A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier or diluent. (A) The first therapeutic agent:

A composition comprising the first therapeutic agent as described above, which is an antibody or antibody fragment comprising at least one complementarity determining region having an amino acid sequence selected from the group consisting of: and (b) a second therapeutic agent . (A) a first therapeutic agent, wherein the first therapeutic agent is an antibody or antibody fragment comprising at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable The regions are SEQ ID NOS: 1-3, respectively:

Including three consecutive complementarity determining regions having the amino acid sequences shown in FIG.

A composition comprising the first therapeutic agent as described above, comprising three consecutive complementarity determining regions having the amino acid sequence shown in (b), and (b) a second therapeutic agent. (A) a first therapeutic agent, wherein the first therapeutic agent is an antibody or a fragment thereof, and the antibody is SEQ ID NO: 7:

A composition comprising a first therapeutic agent as described above, comprising a heavy chain variable region having at least 90% sequence identity in the amino acid sequence shown in (2), and (b) a second therapeutic agent.   10. The composition of claim 9, wherein the heavy chain variable region has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.   The composition of claim 9, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 7. (A) a first therapeutic agent, wherein the first therapeutic agent is an antibody or a fragment thereof, and the antibody is SEQ ID NO: 8:

A composition comprising the first therapeutic agent as described above, comprising a light chain variable region having at least 90% sequence identity in the amino acid sequence shown in (b), and (b) a second therapeutic agent.   13. The composition of claim 12, wherein the light chain variable region has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8.   13. The composition of claim 12, wherein the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 8. (A) The first therapeutic agent:

A composition comprising the first therapeutic agent as described above, which is an antibody or fragment thereof, comprising a light chain variable region having a sequence selected from the group consisting of: and (b) a second therapeutic agent. (A) a first therapeutic agent, SEQ ID NO: 13:

A composition comprising the first therapeutic agent, which is an antibody or fragment thereof, comprising a heavy chain variable region having the sequence shown in (b), and (b) a second therapeutic agent.   The second therapeutic agent is docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin , Carboplatin, cisplatin, dexamethasone, antid, bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronic acid, zoledronic acid ), Idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine Ibritumomab (zevalin), tositumomab (bexal), interferon alfa-2b, melphalan, bortezomib (velcade), altretamine, asparaginase, gefitinib (Iressa), erlonitib (Tarceva), anti-EGF receptor antibody (cetuximab), Abx-E And the composition of any one of claims 7 to 16 selected from the group consisting of epothilone.   17. The composition of any one of claims 7-16, wherein the second therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, and camptothecin.   A method of inhibiting the growth of cancer cells, comprising contacting the cells with the composition of claim 1.   A method of treating a patient having cancer comprising administering to said patient an effective amount of the composition of claim 1.   A method of treating a patient having cancer comprising administering to said patient an effective amount of the pharmaceutical composition of claim 6.   The cancer is from breast cancer, colon cancer, ovarian carcinoma, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial carcinoma, pancreatic cancer, melanoma, multiple myeloma, neuroblastoma, and rhabdomyosarcoma The method according to any one of claims 19 to 21, wherein the cancer is selected from the group consisting of: (A) a first therapeutic agent, wherein the first therapeutic agent is an antibody having the same amino acid sequence as murine antibody EM164 produced in murine hybridoma EM164 (ATCC deposit number PTA-4457), or The first therapeutic agent, which is an epitope-binding fragment, wherein the antibody or the fragment specifically binds to an insulin-like growth factor I receptor;
(B) a second therapeutic agent; and (c) a kit comprising instructions for use. A method of inhibiting the growth of cancer cells comprising:
(A) a first therapeutic agent, wherein the first therapeutic agent is an antibody having the same amino acid sequence as murine antibody EM164 produced in murine hybridoma EM164 (ATCC deposit number PTA-4457), or An epitope-binding fragment, wherein the antibody or the fragment specifically binds to an insulin-like growth factor I receptor, and (b) is contacted with a second therapeutic agent. Including said method. A method of treating a patient having cancer, wherein the patient has an effective amount:
(A) a first therapeutic agent, wherein the first therapeutic agent is an antibody having the same amino acid sequence as murine antibody EM164 produced in murine hybridoma EM164 (ATCC deposit number PTA-4457), or Administering an epitope binding fragment, said first therapeutic agent, wherein said antibody or said fragment specifically binds to insulin-like growth factor I receptor, and (b) a second therapeutic agent Including said method.   25. The method of claim 24, wherein the cells are contacted simultaneously with the first therapeutic agent and the second therapeutic agent.   25. The method of claim 24, wherein the cells are contacted with the first therapeutic agent and the second therapeutic agent sequentially and in any order.   26. The method of claim 25, wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously.   26. The method of claim 25, wherein the first therapeutic agent and the second therapeutic agent are administered sequentially and in any order.   The second therapeutic agent is docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin , Carboplatin, cisplatin, dexamethasone, antid, bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronic acid, zoledronic acid ), Idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine Ibritumomab (zevalin), tositumomab (bexal), interferon alfa-2b, melphalan, bortezomib (velcade), altretamine, asparaginase, gefitinib (Iressa), erlonitib (Tarceva), anti-EGF receptor antibody (cetuximab), Abx-E 26. The method of claim 24 or 25, selected from the group consisting of: and epothilone.   26. The method of claim 24 or 25, wherein the second therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, and camptothecin.

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