EA009807B1 - Anti-igf-i receptor antibody - Google Patents

Abstract

Antibodies, humanized antibodies, resurfaced antibodies antibody fragments, derivatized antibodies, and conjugates of same with cytotoxic agents, which specifically bind to, and inhibit, insulin-like growth factor-I receptor, antagonize the effects of IGF-I, IGF-II and serum on the growth and survival of tumor cells, and which are substantially devoid of agonist activity. The antibodies and fragments thereof may be used, optionally in conjunction with other therapeutic agents, in the treatment of tumors that express elevated levels of IGF-I receptor, such as breast cancer, colon cancer, lung cancer, ovarian carcinoma, synovial sarcoma, prostate cancer and pancreatic cancer and the derivatized antibodies may be used in the diagnosis and imaging of tumors that express elevated levels of IGF-I receptor.

Description

FIELD OF THE INVENTION

This invention relates to antibodies that bind to an insulin-like factor-1 receptor (UEP receptor). More specifically, this invention relates to antibodies against the UE receptor, which inhibit the cellular functions of the UB-1 receptor. Even more specifically, this invention relates to antibodies against the UE-1 receptor, which are antagonists of the effects of 1CB-1, UE-ΙΙ and serum on the growth and survival of tumor cells and which are essentially devoid of agonistic activity.

This invention also relates to fragments of these antibodies, humanized and altered versions of these antibodies, conjugates of these antibodies, derivatives of antibodies and their use in diagnostic, research and therapeutic applications. Further, the present invention relates to improved antibodies or fragments thereof, which are derived from the above antibodies and fragments thereof. In another aspect, the invention relates to a polynucleotide encoding these antibodies or fragments thereof, and to vectors containing these polynucleotides.

State of the art

The receptor for the insulin-like factor-Ι (Ι CE-1 receptor) is a transmembrane heterotetrameric protein that has two extracellular alpha chains and two beta chain extending across the membrane in the disulfide-linked β-α-α-β configuration. The binding of ligands, which are insulin-like growth factor I (SE-Ι) and insulin-like growth factor II (SE-ΙΙ), the extracellular domain of the 1CE-1 receptor activates its intracellular tyrosine kinase domain, leading to autophosphorylation of this receptor and phosphorylation of the substrate . The UEP receptor is homologous to the insulin receptor, having a high sequence similarity of 84% in the tyrosine kinase domain of the beta chain and low sequence similarity of 48% in the cysteine-rich extracellular domain (I1isy., A. e! A1., 1986, EMBO, 5, 2503- 2512; Yeschia-Uatejds, Υ. E! A1., 1986, 1. ΙοΙ. Siet., 261, 1672716731; LeKoy, Ό. E! A1., 1995, Eppospe Reu1e \\ b. 16, 143-163). JU-Ereceptor and its ligands (JU-Ι and JU-ΙΙ) play important roles in numerous physiological processes, including growth and development during embryogenesis, metabolism, cell proliferation and cell differentiation in adults (LeKoy, Ό., 2000, Epboschlodu, 141, 1287-1288; LéKoy, Ό., 1997, етеетеете Е апдд1 1. 1. 1. 1. Mob., 336, 633-640).

JU-Ι and ICE-II function both as endocrine hormones in the blood, where they are predominantly present in complexes with JU-binding proteins, and as paracrine and autocrine growth factors that are produced locally (NTsie1, K.E., 1990 , Eig. 1. Vusjet., 190, 445-462; CoSysk, ν.8. Appb C1ttopz, Ό.Κ. 1993, Appi. Keu. Ryuzyu1. 55, 131-153).

It was assumed that the UE-receptor is involved in stimulating the growth, transformation and survival of tumor cells (Always, K. e! A1., 1997, Vusyet. Vyuriz. Ac! A, 1332, E105-E126; B1akezueu, UAA e! A1., 1997, 1o11 ok Epbosgto1odu, 152, 339-344; Ka1eko, M., Kiyeg, ν.1., apb MSHeg, A.E. 1990, Mo1. Ce11. Vu1., 10, 464-473). Thus, it is known that several types of tumors express higher than normal levels of the UEP receptor, including breast cancer, colon cancer, ovarian cancer, synovial sarcoma, and pancreatic cancer (Kyapb ^ a1a, N.M. e ! a1, 2000, Epbospe Keu1e ^ 8, 21, 215-244; \ Ve ^ ne ^. N. apb BeKoy, Ό., 1995, Abu. Sapseg Kez. 68, 183-223; Narregk1e1b, L.S. e! a1., 1997, 1. Ra! yo.., 183, 412-417; Epeg, 8. e! a1, 1999, C !, 44, 704-708; un Pat, R.A. e! a1. , 1994, 1. Syp. Ra! Yo1., 47, 914-919; X1e, Υ. E! A1., 1999, Sapseg Kez., 59, 3588-3591; Vegdtapp, I.e! A1., 1995, Sapseg Kez., 55, 20072011). Ιπ νίϋΌ, it was found that UE-Ι and ICE-II are strong mitogens for several human cell lines, such as lung cancer, breast cancer, colon cancer, osteosarcoma, and cervical cancer (Apkgarr, P.P. apb Νеνаπ, Ό.Κ, 1993, Сапсег Кез., 53, 33993404; Сi11ep, К.1., 1990, Сапсег Кез., 50, 48-53; Negtap! O, I., 2000, Се11 Сго \\ 1st & P1kkegep! 1a1yup, 11, 655664; Sio, Υ.8. E! A1., 1995, 1. At. Co11. 8igd., 181, 145-154; Carre1, S.S. e! A1., 1994, Sapseg Kez., 54, 28032807; 8! E11eg, M.A. e! A1, 1996, Sapseg Kez. 56, 1761-1765). Several of these tumors and tumor cell lines also express UE-Ι or UE-ΙΙ, which can stimulate their growth in an autocrine or paracrine manner. Yuschpp, K.A. e! A1., 1996, 1. Vu1. Siet., 271, 11477-11483).

Epidemiological studies have shown a correlation between elevated plasma levels of UE-Ι (and lower levels of UE-binding protein-3) with an increased risk of prostate cancer, colon cancer, lung cancer and breast cancer (Syap, 1.M. e! A1 ., 1998, 8s1epse, 279, 563566; No. o1k, A. e! A1, 1998, 1. Yab. Sapseg Sz!., 90, 911-915; Ma, 1. e! A1, 1999, 1. Ya! 1. Sapseg] pz !, 91, 620-625; Υϋ, N. e! A1., 1999, 1. Yab. Sapsegs yz!, 91, 151-156; Napkshzop, 8.E. e! A1., 1998, Lapse !, 351, 1393-1396). Strategies for lowering plasma UE-Ι levels or inhibiting UEP receptor function have been proposed to prevent cancer (νυ, Υ. Е! А1., 2002, Sapseg Kez., 62, 1030-1035; Spjögd, A apb Soyep R., 2000 , 1. Ce11. Ryuzyu1., 183, 1-9).

The JUP receptor protects tumor cells from apoptosis caused by growth factor deprivation, linkage independence, or treatment with cytotoxic drugs (ΝηνηπΌ, M. apb. Always, K., 2001, Epboschodu 142, 1073-1081; Always, K. e! A1. , 1997, Vusyet. Vyryuz. Ac! A, 1332, E105-E126). The domains of the UEb receptor were identified by mutation analysis, which are crucial for its mitogenic, transforming, and antiapoptotic activities.

- 1 009807

For example, the tyrosine residue of 1251 SE-1 receptor was identified as critical for anti-apoptotic and transforming activities, but not for its mitogenic activity (O'Soppog, B. e! A1., 1997, Mo1. Ce11. ΒίοΙ., 17, 427 -435; Msta, M. e! A1, 1995, 1. ΒίοΙ. SEet. 270, 22639-22644). The intracellular signal transmission pathway of the ligand-activated UE-1 receptor includes the phosphorylation of tyrosine residues of insulin receptor substrates (1B8-1 and 1B8-2), which recruit phosphatylinositol-3-kinase (ΡΙ-3-kinase) to the membrane. The membrane-bound phospholipid products of β-3 kinase activates the serine / threonine kinase Ak !, the substrates of which include the pro-apoptotic protein β, which phosphorylates to an inactive state (EaNa. Z.V., Vgipe !, A. apb Sgiepbegd, M.E., 1999, Sepek & Oeue1ortep !, 13, 2905-2927; Ki11k, 6., K11rre1, A. apb Aebet, M.1., 1997, Mo1. Ce11. 1ο1. 17, 1595-1606). Mitogenic signal transmission of the 1CE-1 receptor in human breast cancer MCE-7 cells requires ΡΙ-3 kinase and is independent of the mitogen-activated protein kinase, while the transmission of the survival signal in differentiated PC12 rat pheochromocytoma cells requires ΡΙ-3 kinase , and signal transmission pathways of mitogen-activated protein kinase (Όιι Goigpu, B. e! a1., 1997, 1. Βώ! CEet., 272, 31163-31171; Ρа ^^^ ζак, M., 3аШе1, А.В. Apb LeBoe, Ό., 1997, 1. ΒΦΕ CEet., 272, 154-161).

It has been shown that downregulation of the level of the 1CE-1 receptor by antisense strategies reduces the oncogenicity of several tumor cell lines, such as melanoma, lung cancer, ovarian cancer, glioblastoma, neuroblastoma, and rhabdomyosarcoma (VekssoGG, M. e! A1. , 1994, Sapseg Vek., 54, 4848-4850; Bee, S.-T. e! A1, 1996, Sapseg Vek., 56, 3038-3041; Mi11eg, M. e! A1., 1998, 1p !. 1. Sapseg, 77, 567-571; Tto_) ap, 1. e! A1., 1993, Zc1epse, 259, 94-97; Wow, X. e! A1., 1998, Sapseg Vek., 58, 5432-5438; 3Ear1to, Ό.Ν. e! A1, 1994, 1. C1. 1 batch!., 94, 1235-1242). Similarly, it was reported that the dominant negative mutant of the 1CE-1 receptor reduces the oncogenicity of w1uo and the growth of six transformed Ba! -1 cells overexpressing the 1CE-1 receptor; e! A1, 1994 Bgos. Νη11. Asab. Zsk IZA, 91, 2181-2185).

Tumor cells expressing antisense against the 1CE-1 mRNA receptor undergo extensive apoptosis when injected into animals in bio-diffusion chambers. This observation makes the 1CE-1 receptor an attractive therapeutic target based on the hypothesis that tumor cells are more sensitive than normal cells to apoptosis caused by inhibition of the 1CE-1 receptor (VekshsoGG, M. e! A1., 1995, Sapseg Vek ., 55, 2463-2469; Βα ^ ι ^ α, V., 1995, Sapseg Vek., 55, 249-252).

Another strategy for inhibiting the function of the 1CE-1 receptor in tumor cells has been to use antibodies against the 1CE-1 receptor, which bind to the extracellular domain of the 1CE-1 receptor and inhibit its activation. Several attempts have been reported to develop murine monoclonal antibodies against the 1CE-1 receptor, of which two inhibitory antibodies, 1B3 and ΙΗ7, are available and their use has been reported in several studies of the UEP receptor.

Antibody ΙΒ3 was developed using a partially purified placental insulin receptor preparation to immunize the mice that formed the antibody, GW1, which was selective for binding to the insulin receptor, and two antibodies, ΙΒ2 and GW3, were obtained that showed preferential immunoprecipitation of the UEP receptor (receptor somatomethine-C), but only weak immunoprecipitation of the insulin receptor (Ki11, E.S. e! a1., 1983, 1. ΒώΕ CEet., 258, 6561-6566).

The 1H7 antibody was obtained by immunizing mice with a purified placental preparation of the Ι6Ε-Ι receptor, which yielded an 1H7 inhibitory antibody, along with three stimulatory antibodies (L1, δ.-b. E! A1., 1993, Β ^ ΐκιιι. Β ^ οреук. Century. Sottip. ., 196, 92-98; Hupp, L. E! A1., 1992 ΡΐΌα Να11. Asab. 8sk IZA, 89, 5356-5360).

In another report, a panel of murine monoclonal antibodies specific for the human ΕΙ6ΕΙ receptor was obtained by immunizing mice with transfected 3T3 cells expressing high levels of the UEb receptor, which were classified into seven groups using competitive binding studies and for their inhibition or stimulation of Ι6Ε- binding Ι with transfected 3T3 cells (8 ° C, M.A. e! A1., 1992, 1. ΒώΕ CEet., 267, 12955-12963).

Thus, although the ΙΒ3 antibody is the most commonly used inhibitory antibody for studies of the UEP receptor, it has the drawback that it exhibits agonistic activity in transfected 3T3 and CHO cells expressing the human Ι6ΕΙ receptor (Ka! Е. E! A1., 1993, 1. ΒώΕ SEet., 268, 2655-2661; Z ^ ek-BeAtk, S. apb Vo! E, V.A., 1990, Β ^ ΐκιη. Β ^ ёрЕук. Век Sottip., 171, 1244-1251). Similarly, among this panel of antibodies developed by Zooc e! A1., most of the inhibitory antibodies 24-57 and 24-60 also showed agonistic activity in transfected 3T3 cells (Zooc, MA e! a1., 1992, 1. ΒώΕ CEet., 267, 1295512963). Although the ΙΒ3 antibody has been reported to inhibit the binding of Ι6Ε-Ι (but not Ι6Ε-ΙΙ) to expressed receptors in intact cells and after solubilization, it has been shown to inhibit the ability of Ι6Ε-Ι and Ι6Ε-ΙΙ to stimulate DNA synthesis in cells completely Apb Vo! E, V.A.,

1990, Β ^ ΐκιιι. Β ^ οрЕук. Century. Sottip., 171, 1244-1251). It was concluded on the basis of the chimeric constructs of the insulin-UEP receptor that the binding epitope of the antibody! B3 is region 223-274

- 2 009807

SE-1 receptor (SiyyaRkop, T.A. apb Ki !! er, XV.!. 1990, 1. ΒίοΙ. Sb., 265, 18663-18667; 8ook, M.A. e! A1., 1992, 1. Βίο1. Sb., 267, 12955-12963).

The MCP-7 human breast cancer cell line is usually used as a model cell line to demonstrate the growth reaction of UR-1 and UR-11 ίη νίίτο (OiRoitu, B. e! A1., 1997, 1. Βίο1. Sb., 272, 31163-31171). In MCP-7 cells, 1K3 antibody completely blocks the stimulating effect of exogenously added Ju-1 and Ju-ΙΙ under serum-free conditions by approximately 80%. Antibody ΙΚ3 also slightly inhibits (by less than 25%) the growth of MCP-7 cells in 10% serum (Cu11ep, K.1. E! A1., 1990, Sapseg Kek., 50, 48-53). This weak inhibition of serum-stimulated MCP-7 cell growth by antibody ΙΚ3 ш3ygo may be related to the results of the study of νίνΌ, in which treatment with antibody не3 did not significantly inhibit the growth of MCP-7 xenograft in nude mice (Ajeada, C.E.! A1., 1989, 1. Cl. 1 ^ ek!., 84, 1418-1423).

Due to the weak agonistic activities of ΙΚ3 and other described antibodies and their inability to significantly inhibit the growth of tumor cells, such as MCP-7 cells, under more physiological conditions of stimulation with serum (instead of stimulation with exogenously added UR-Ι or UR-ΙΙ under serum-free conditions), there is a need for new antibodies against JRL receptors, which significantly inhibit the growth of tumor cells stimulated by serum, but which themselves do not show significant agonistic activity.

SUMMARY OF THE INVENTION

Thus, the aim of the present invention is to provide antibodies, fragments of antibodies and derivatives of antibodies that specifically bind to an insulin-like growth factor Ι and inhibit the cellular activity of this receptor by antagonistic effects on this receptor, and are also essentially devoid of agonistic activity against this receptor.

Thus, in the first embodiment, a murine antibody EM164 is provided, which is fully characterized here with respect to the amino acid sequences of the variable regions of both its light chain and its heavy chain, cDNA sequences of genes for the variable regions of the light and heavy chain, identification of its SEC (determining complementarity of regions), identification of its surface amino acids and methods for its expression in recombinant form.

In a second embodiment, a remodeled or humanized version of the EM164 antibody is provided, where surface residues of this antibody or fragments thereof are replaced in both light and heavy chains for closer resemblance to known human antibody surfaces. Such humanized antibodies may have increased utility compared to murine EM164, as therapeutic or diagnostic agents. Humanized versions of the EM164 antibody are also fully characterized here with respect to their respective amino acid sequences of the variable regions of both light and heavy chains, gene sequences for the variable regions of the light and heavy chains, identification of SECs, identification of their surface amino acids, and disclosure of methods for their expression in recombinant form .

In a third embodiment, an antibody is provided that is capable of inhibiting the growth of cancer cells by more than about 80% in the presence of a growth promoter such as, for example, serum, insulin-like growth factor Ι and insulin-like growth factor ΙΙ.

In a fourth embodiment, an antibody or antibody fragment is provided having a heavy chain including SECs having the amino acid sequences represented by 8E0 GO N0: 1-3, respectively:

8ΥΑ.ΜΗ (8Ε9ΙΟΝΟ: 1),

ΕΙΝΡ8ΝΟΚΤΝΥΝΕΚΡΚΚ (8Е0 GO N0: 2),

ΟΚΡΏΥΥΟδδΚλνΥΕΰν (8Eph GO N0: 3);

and having a light chain that contains SEC, having the amino acid sequences represented by 8E0 GO N0: 4-6:

Κ8808ΐνΗ8ΝνΝΤΥΕΕ (8Е () ГО N0: 4);

ΚνΞΝΚΕδ (8ERGOZH): 5);

GDSZNURRT (8Ef GO N0: 6).

In a fifth embodiment, antibodies are provided having a heavy chain that has an amino acid sequence that has at least 90% sequence identity with the amino acid sequence represented by 8E0 GO N0: 7:

ΟνφΕφΟδΟΑΕΕνΚΡΟΑδνΚΕδΟΚΑβΟΥΤΕΤΞΥΑ'ΜΗΑΎΚφΚΡΟφΩΕΕλΊΟΕΙ ΝΡ8ΝσΚΤΝΥΗΕΚΓΚΚΚΑΤΕΤνθΚ888ΤΑΥΜ () Ε88ΕΤ8ΕΟ8ΑνΥΥΓΑΚ <3ΚΡΟΥ Υ688ΚΨΥΕΈ) νΐνθΑΟΤΤντν88 (8Eph GO N0: 7)

Similarly, antibodies are provided having a light chain that has an amino acid

- 3 009807 sequence, which has at least 90% sequence identity with the amino acid sequence represented by 8EO GO N0: 8:

OUMT0TR8ERU8ESO0A818SK88081UN81F / HT ¥ ^ LE UE0KRO08RKSHU KU8KKR8OUROKR8S8OZOTSRTEK18E.UEAEOES1UUSR0O8NURRTROOOTK EE1KK (8Ef GO N0: 8).

In a sixth embodiment, antibodies are provided having a humanized or surface-rearranged variable region of the light chain having an amino acid sequence corresponding to one of 8E0 GO N0: 9-12:

ΠΨΜΤ0ΤΡΕ8ΕΡν8ΕΟΟΡΑ8Ι80Κ8808ΐνΗ8ΝνΝΤΥΕΕΨΥΕ0ΚΡΟ98ΡΚΕΕΙΥ ΚνδΝΚΡδΟνΡϋΚΡδΟδΟΑΟΤΟΡΤΕΚΙδΚνΕΑΕΏΕΟΙΥΥΟΡΟαδΗνΡΡΤΡΟΟσΤ ΚΕΕΙΚΚ (8Eph GO N0: 9);

ОУМТ0ТРЬ8ЕРУ8ЬПРА818СК88081УН8 ПТАУУЕУАТ0КРСг (28РКШ ¥ Κν8ΝΚΡ86νΡϋΚΡ8Ο8ΟΑΟΤΟΡΤΕΚΙ8ΚνΕΑΕϋΕΟΙΥΥΟΡ0Ο8ΗνΡΡΤΡ (3ΟΟΤ КЬГКК (8Еф Г ΝΟ: 10);

θνΕΜΤ0ΤΡΕ8ΕΡν8ΕΟΟΡΑ8Ι8ΟΚ3808ΐνΗ8ΝνΝΤΥΕΕΨΥΕςΚΡθς8ΡΚΕΕΙΥ KU8PKR8OUROKR8OZSAOOTORTEK18KUEAEOES1UUSR0O8NURRTROOOOT ΚΤ.ΕΙΚΚ (8Ef GO N0: 11); or

Ο \ νΜΤρΤΡΕ8ΕΡν8ΕΟΟΡΑ5Ι8ΟΒ.85ζ) 8ΐνΗ8ΝνΝΤΥΕΕΑνΥΕ (2ΚΡΟ08ΡΚΕΕΙΥ КУ8МКР8СУРОКР8О8ОА0ТПРТЕК18КУЕАЕЕЕ1УУСР0О8НУРРТР0О0Т ΚΕΕΙΚΒ. (8Εφ ΕΟ N0: 12)

Similarly, antibodies are provided having a humanized or rearranged heavy chain variable region having an amino acid sequence corresponding to 8E0 GO N0: 13:

0νζ) Ενζ) 8ΟΑΕννΚΡΟΆ8νΚΕ80ΚΑ8ϋΥΤΕΤ8Υν / ΜΗ \ ννΚ0ΕΡ60ΟΕΕ \ νΐΟΕΙ ΝΡ8ΝΟΚΤΝΥΝ0ΚΡ0ΟΚΑΤΕΊΎΟΚ833ΤΑΥΜ0Ε88ΕΤ8ΕΟ5ΑνΥΥΡΑΚ0ΚΡΟΥ ΥΟ38ΚλνΥΡϋνΨΟ0 <} ΤΤντν88 (Zef GO N0: 13).

In a seventh embodiment, antibodies or antibody fragments of the invention are provided that have improved properties. For example, antibodies or antibody fragments having improved affinity for the SE-1 receptor are prepared by maturing the affinity of an antibody or fragment of the invention.

The present invention further provides conjugates of these antibodies, wherein the cytotoxic agent is covalently attached, either directly or via a cleavable or non-cleavable linker, to an antibody or epitope-binding fragment of an antibody of the invention. In preferred embodiments, the cytotoxic agent is taxol, maytansinoid, CC-1065 or an analogue of CC-1065.

The invention further provides antibodies or fragments thereof that are further labeled for use in research or diagnostic applications. In preferred embodiments, the implementation of this label is a radioactive label, fluorophore, chromophore, visiographic agent or metal ion.

A diagnostic method is also provided in which said labeled antibodies or fragments are administered to a subject who is suspected of having cancer, and the distribution of this label in the body of the subject is measured or monitored.

In an eighth embodiment, the invention provides methods for treating a subject having cancer by administering an antibody, antibody fragment, or antibody conjugate of the invention, alone or in combination with other cytotoxic or therapeutic agents. This cancer may be, for example, one or more of breast cancer, colon cancer, ovarian cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial cancer, pancreatic cancer, or another cancer that needs to be determined in which the levels of the ju-1 receptor are elevated.

In a ninth embodiment, the invention provides methods of treating a subject having cancer by administering an antibody, antibody fragment, or antibody conjugate of the invention, alone or in combination with other cytotoxic or therapeutic agents. In particular, preferred cytotoxic and therapeutic agents include docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Neheeri), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestin-9-state, 4-state carboxylate, tin, dexamethasone, antide, bevacizumab (ΛνηδΙίη). 5-fluorouracil, leucovarin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronate, streptozocin, rituximab (Kyihap), idarubicin, busulfin liburabinulobimiblubloimblobin, chlorambibutinumlobloimblobin, chlorambinblubinibloidinum tositumomab (Wehhag), interferon alfa-2b, melpholam, bortezomib (Be1sabe), altretamine, asparaginase, gefitinib (1ge§8a), erlonitib (Tagseua), anti-ECE receptor antibody (Ce1ih1tE, Abot-eon-ep. More preferably, this therapeutic agent is a platinum-containing agent (such as carboplatin, oxaliplatin, cisplatin), taxane (such as paclitaxel, docetaxel), gemcitabine or camptothecin.

The cancer may be, for example, one or more of the cancers, such as breast cancer, colon cancer, ovarian cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial cancer, pancreatic cancer, melanoma, multiple myeloma , neuroblastoma and rhabdomyosarcoma, or another cancer that still needs to be determined, in which the levels of the UE-1 receptor are elevated.

In a tenth embodiment, the invention provides kits comprising one or more of the elements described herein and instructions for using these elements. In a preferred embodiment, a kit of the invention includes an antibody, antibody fragment or conjugate of the antibody of the invention and a therapeutic agent. Instructions for this preferred embodiment include instructions for inhibiting the growth of cancer cells using an antibody, fragment or conjugate of an antibody of the invention and a therapeutic agent and / or instructions for a method of treating a patient having cancer using an antibody, fragment or conjugate of an antibody of the present invention and therapeutic agent.

Brief Description of the Figures

FIG. 1 shows an analysis using cell sorting with excitation of fluorescence (EAC8) of specific binding of purified EM 164 antibody to cells overexpressing the UE-1 receptor Υ1251Ε or human insulin receptor;

FIG. 2 is a binding titration curve for binding of an EM164 antibody to a human biotinylated SE-1 receptor.

FIG. 3 - inhibition of the binding of biotinylated human 1CE-1 receptor to human breast cancer MCE-7 cells with antibody EM 164.

FIG. 4 - inhibition of UE-1-stimulated autophosphorylation of the UE-1 receptor in MCE-7 cells with EM164 antibody.

FIG. 5 - inhibition of UE-1-stimulated ΙΚδ-1-phosphorylation in MCE-7 cells with EM164 antibody.

FIG. 6 - inhibition of JU-1 receptor-stimulated signal transduction in 8a08-2 cells with EM164 antibody.

FIG. 7 shows the effect of EM164 antibody on the growth and survival of MCE-7 cells under various growth conditions, as assessed by MTT analysis.

FIG. 8 shows the effect of the EM 164 antibody on the growth and survival of MCE-7 cells in the presence of various serum concentrations.

FIG. 9 - inhibition of the growth and survival of IS1-H838 cells stimulated by UE-1 serum and antibody EM164.

FIG. 10 shows the effect of treatment with an EM164 antibody, taxol, or a combination of an EM164 antibody and taxol on the growth of a Ca1i-6 lung cancer xenograft in mice.

FIG. 11 - competition between the binding of a humanized antibody EM164 (ν.1.0) and mouse antibodies EM164.

FIG. 12 - cDNA- (8Ef ΙΌ N0: 49) and amino acid (8Ef ΙΌ N0: 50) sequences of the leader region and the light chain variable region of the mouse antibody against the EM 161 receptor EM 164. The arrow indicates the beginning of frame 1. Three SIC sequences in according to KaLa! underlined.

FIG. 13 - cDNA- (8Ef ΙΌ N0: 51) and amino acid (8Ef ΙΌ N0: 52) sequences of the leader region and the variable region of the heavy chain of the mouse anti-EM-1 receptor EM 164. The arrow indicates the beginning of frame 1. Three SIC sequences in according to KaLa! underlined.

FIG. 14 shows the amino acid sequences of the SICs of the light and heavy chains of the EM164 antibody, determined based on the definitions of the canonical class C1u11ia. The definitions of the ABM program for heavy chain SICs are also shown. Light chain: SIC1 is 8Eph GO N0: 4, SIC2 is 8Eph GO N0: 5 and SIC 3 is 8Eph GO N0: 6. The heavy chain: SIK1 is 8Ef GO N0: 1, SIK2 is 8Ef GO N0: 2 and SIK3 is 8Ef GO N0: 3. The heavy chain ALM: SIK1 is 8Ef GO N0: 53, SIK2 is 8ES) GO N0: 54 and SIK3 is 8ES) GO N0: 55.

FIG. 15 shows the amino acid sequences of the light chain and heavy chain for the anti-YEL receptor antibody EM164 aligned with the germline sequences for the Cr1 genes

- 5 009807 (8EC ΙΌ N0: 56) and 1558.S (8Ε0 ΙΌ N0: 57). The dotted line (-) indicates sequence identity.

FIG. 16 shows plasmids used for the construction and expression of recombinant chimeric and humanized antibodies EM 164. A) a cloning plasmid for a light chain, B) a cloning plasmid for a heavy chain, C) an expression plasmid for a mammalian antibody.

FIG. 17 shows the 10 most homologous amino acid sequences of light chains obtained by screening of 127 antibodies in a set of structural files used to predict surface residues of EM164. et164 bC (8E0 ΙΌ N0: 58), 2_) e1 (8E0 ΙΌ N0: 59), 2rer (8E0 ΙΌ N0: 60), 1npb (8E0 ΙΌ N0: 61), 1ke1 (8E0 ΙΌ N0: 62), 1Juh (8Е0 ΙΌ N0: 63), 1ί§Τ (8Е0 ΙΌ N0: 64), 1! Е! e (8E0 ΙΌ N0: 65), 1sk (8E0 ΙΌ N0: 66), 1b1n (8E0 ΙΌ N0: 67, 1s1y (8E0 ΙΌ N0: 68), Consensus (8E0 ΙΌ N0: 69).

FIG. 18 shows the 10 most homologous amino acid sequences of light chains obtained by screening of 127 antibodies in a set of structure files used to predict surface residues of EM164. et164 NS (8E0 ΙΌ N0: 70), 1pd (8E0 ΙΌ N0: 71), 1pd (8E0 ΙΌ N0: 72), 11Y (8E0 ΙΌ N0: 73), 1a1y (8E0 ΙΌ N0: 74), 1uy (8E0 ΙΌ N0: 75), 1p1d (8E0 ΙΌ N0: 76), 165b (8E0 ΙΌ N0: 77), 1ae6 (8E0 ΙΌ N0: 78), 1A8 (8E0 ΙΌ N0: 79), ZIL (8E0 ΙΌ N0: 80), Consensus (8Е0 ΙΌ N0: 81).

FIG. 19 - the average availability for each of the residues of the variable region of the light (A) and heavy (B) chains of the 10 most homologous structures. The numbers represent the position numbers of the antibody sequence according to KaLa !.

FIG. 20 - amino acid sequences of the variable region of the light chain for mouse EM164 (thiEM164) and humanized EM164 (IIEM164) antibodies. TiEM164 (8Е0 ΙΌ N0: 82), ИИЕМ164 У1.0 (8Е0 ΙΌ N0: 83), ИИЕМ164 νΙ.1 (8Е0 ΙΌ N0: 84), ИИЕМ164 У1.2 (8Е0 ΙΌ N0: 85), ИИЕМ164 У1.3 ( 8E0 ΙΌ N0: 86).

FIG. 21 - amino acid sequences of the variable region of the heavy chain for murine EM164 (thiEM164, 8E0 ΙΌ N0: 87) and humanized EM164 (IIEM164, 8E0 ΙΌ N0: 88) antibodies.

FIG. 22 - DNA sequence and amino acid sequence of the variable region 111.1EM164 ν1.0 for both light (DNA, 8Е0 ΙΌ N0: 89, amino acid 8Е0 ΙΌ N0: 90) and heavy (DNA, 8Е0 ΙΌ N0: 91, amino acid 8Е0 ΙΌ N0: 92).

FIG. 23 - DNA sequence and amino acid sequence of the variable region of the light chain for the humanized EM164 ν1.1 (DNA, 8Е0 ΙΌ N0: 93, amino acid 8Е0 ΙΌ N0: 94), ν1.2 (DNA, 8Е0 ΙΌ N0: 95, amino acid 8Е0 ΙΌ N0: 96) and ν1.3 (DNA, 8Е0 ΙΌ N0: 97, amino acid 8Е0 ΙΌ N0: 98).

FIG. 24 - inhibition of ΙΟΕ-Ι-stimulated growth and survival of MCE-7 cells with the humanized antibody EM164 ν1.0 and mouse antibody: EM164.

FIG. 25 shows that EM164 inhibits the ΙΟΕ-Ι-stimulated passage of the cell cycle of MCE-7 cells.

FIG. 26 shows that EM 164 inhibits the anti-apoptotic effect of ΙΟΕ-Ι and serum. Treatment with EM164 results in apoptotic cell death, as indicated by increased levels of the cleaved SK18 protein.

FIG. 27 shows the effect of treatment with an EM164 antibody, gemcitabine, or a combination of an EM164 antibody and gemcitabine on the growth of human pancreatic cancer BxPC-3 xenografts in immunodeficient mice.

Information confirming the possibility of carrying out the invention

The present invention has discovered and improved new antibodies that specifically bind to the human insulin-like growth factor Ι (ΙΟΕ-ΙΒ) receptor on the cell surface. These antibodies and fragments have a unique ability to inhibit the cellular functions of this receptor without the ability to activate the receptor itself. Thus, while previously known antibodies that specifically bind and inhibit ΙΟΕ-ΙΒ also activate the receptor even in the absence of ΙΟΕ-ΙΒ ligands, the antibodies or fragments of the present invention are ΙΟΕ-ΙΒ antagonists, but are essentially devoid of agonistic activity . In addition, antibodies and antibody fragments of the present invention inhibit the growth of human tumor cells, such as MCE-7 cells, in the presence of serum by more than 80%, which is a higher degree of inhibition than the degree of inhibition obtained using previously known antibodies against ΙΟΕ-Ι receptor.

The present invention begins with a murine anti-ΙΟΕ-тора receptor antibody, here EM164, which is fully characterized with respect to the amino acid sequences of both light and heavy chains, identification of CMEA, identification of surface amino acids and a method for its expression in recombinant form.

Germ line sequences are shown in FIG. 15 aligned with the EM164 sequence. This comparison identifies possible somatic mutations in EM164, including one of each mutations in CMEA 1 in the light chain and CMEA 2 in the heavy chain.

The primary amino acid sequence and DNA sequence of the light and heavy chains of the EM164 antibody and humanized versions are described herein. However, the scope of the invention is not limited to antibodies and fragments containing these sequences. Instead, all antibodies and fragments that specifically bind to an insulin-like factor receptor

- 6 009807 growth I and counteract the biological activity of this receptor, but which are essentially devoid of agonistic activity, fall within the scope of this invention. Thus, antibodies and antibody fragments may differ from the EM 164 antibody or humanized derivatives in the amino acid sequence of their framework, SEC light chain and heavy chain, but still fall within the scope of this invention.

SOY antibodies EM 164 identified by modeling and their molecular structures were predicted. Again, although these SOYs are important for the recognition of epitopes, they are not indispensable for antibodies and fragments of the present invention. Thus, antibodies and fragments are provided that have improved properties formed, for example, by the maturation of the affinity of an antibody of the invention.

A variety of antibodies and antibody fragments, as well as antibody mimetics can be easily obtained by mutation, deletion and / or insertion in sequences of variable and constant regions that flank a particular set of SOY. So, for example, various classes of L for a particular set of SOY are possible, by replacing various heavy chains, whereby, for example, types and isotypes of antibodies 1dO1-4, 1dM, 1d A1 -2, 1dE can be obtained. 1de. Similarly, artificial antibodies can be made within the scope of this invention by placing a particular set of SOY in a fully synthetic framework. The term "variable" is used here to describe certain parts of the variable domains, which differ in their sequence among antibodies and are used in the binding and specificity of each specific antibody in relation to its antigen. However, this variability is usually not evenly distributed across all the variable domains of these antibodies. Usually it is concentrated in three segments, called complementarity determining regions (SOY) or hypervariable regions in the variable domains of both light and heavy chains. The more highly conserved parts of the variable domains are called the framework (EF). The variable domains of the heavy and light chains, each, contain four frame regions that take on a significant configuration of beta folds connected by three CEJs that form loops connecting the beta-fold structure and, in some cases, form part of the beta-fold structure. SOY in each chain are held together in close proximity by EJ-regions and, with SOY from the other chain, contribute to the formation of an antigen-binding site of antibodies (E.A. KaLa! C1 a1. , ΝΙΗ). The constant domains do not directly participate in the binding of an antibody to an antigen, but exhibit various effector functions, such as the participation of this antibody in antibody-dependent cellular toxicity.

Humanized antibodies or antibodies adapted for non-rejection by other mammals can be obtained using several technologies, such as surface reconstruction of antibodies and transplantation of SOY. In surface remodeling technology, molecular modeling, statistical analysis, and mutagenesis are combined to bring the non-CEA surfaces of the variable regions to resemble the surfaces of known target host antibodies. Strategies and methods for altering antibody surfaces and other methods for reducing the immunogenicity of antibodies in a different host are described in US Pat. No. 5,639,641, which is incorporated herein by reference in its entirety. In the technology of transplantation of CEA, SEA of mouse heavy and light chains are transplanted into a fully human framework sequence.

The invention also includes functional equivalents of the antibodies described herein. Functional equivalents have binding characteristics that are comparable to the binding characteristics of these antibodies, and include, for example, chimeric, humanized, and single chain antibodies, as well as fragments thereof. Methods for obtaining such functional equivalents are described in PCT Application AO 93/21319, European Patent Application No. 239400; PCT Application AO 89/09622; European patent application 338745 and European patent application 332424, which are incorporated in their entirety by reference.

Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of an antibody of the invention. Essentially the same as applied to the amino acid sequence is defined here as a sequence with at least about 90% and more preferably at least about 95% sequence identity with respect to another amino acid sequence determined using the EAZTA search method in accordance with Reagco apb Yrtai, Prgos . Ν; ι11. Asab. Zsg IZA 85, 2444-2448 (1988).

Chimeric antibodies preferably have constant regions produced essentially or exclusively from constant regions of a human antibody, and variable regions produced essentially or exclusively from a sequence of variable regions from a mammal other than a human. Humanized forms of these antibodies are obtained by substituting complementarity determining regions, for example, a mouse antibody, into a human framework domain, for example, see PCT Publication No. AO92 / 22653. Humanitariannet antibodies preferably have constant regions and variable regions other than the complementarity determining regions (SEA), semi

- 7 009807 derived, essentially, or exclusively from the corresponding regions of human antibodies, and SER. obtained essentially or exclusively from a mammal other than human.

Functional equivalents also include single chain antibody fragments, also known as single chain antibodies (xEu). These fragments contain at least one fragment of the variable amino acid sequence of the antibody heavy chain (V _n ) attached to at least one fragment of the variable light chain sequence of the antibody (V _b ) with one or more linkers connecting them. Such a linker may be a short, flexible peptide selected to ensure that the correct three-dimensional folding of the domains (V _b ) and (V _n ) is formed upon their binding, so that the binding specificity of the target molecule of the whole antibody from which this single-chain fragment is derived antibodies. Typically, the carboxyl terminus of the sequence (Y _b ) or (Y _n ) can be covalently linked by such a peptide linker to the amino terminus of the complementary sequence (Y _b ) and (Y _n ). Single chain antibody fragments can be generated by molecular cloning using a phage antibody display library or similar methods. These proteins can be produced either in eukaryotic cells or in prokaryotic cells, including bacteria.

Single chain antibody fragments contain amino acid sequences having at least one of the variable or complementarity determining regions (SECs) of the whole antibodies described in this description, but lack some or all of the constant regions of these antibodies. These constant regions are not required for antigen binding, but form the bulk of the structure of whole antibodies. Single chain antibody fragments can therefore overcome some of the problems associated with the use of antibodies containing part of the constant region or the entire constant region. For example, single chain antibody fragments tend to be free of unwanted interactions between biological molecules and the constant region of the heavy chain or other undesirable biological activity. In addition, single chain antibody fragments are significantly smaller than whole antibodies and can therefore have higher capillary permeability than whole antibodies, allowing single chain fragments to more efficiently locate and bind to antigen-binding target sites. Antibody fragments can be produced on a relatively large scale in prokaryotic cells, which facilitates their production. In addition, the relatively small size of single-stranded fragments leads to the fact that they are less likely to induce an immune response in the recipient than whole antibodies.

Functional equivalents further include antibody fragments that have the same or comparable binding characteristics as the binding characteristics of the whole antibody. Such fragments may contain one or both of an Eb fragment or an E (ab ') ₂ fragment. Preferably, these antibody fragments contain all six complementarity determining regions of the whole antibody, although fragments containing less than all such regions, for example, three, four or five COP, are also functional. Further functional equivalents can be members or can be combined with members of the following classes of immunoglobulins: 1dO, 1dM, 1dA, 1dO or 1dE and their subclasses.

Awareness of the amino acid and nucleic acid sequences for antibodies against the EM-164 receptor for EM 164 and its humanized variants, which are described herein, can be used to develop other antibodies that also bind to human human immunogymic receptors and inhibit the cellular functions of this human -1 receptor. Some studies have studied the effect of introducing one or more amino acid substitutions at different positions in an antibody sequence, based on knowledge of the primary sequence of an antibody, on its properties such as binding and expression level (Wapd, A.P. e! A1., 1995, 1. Mo1. ΒίοΙ., 254, 392-403; Kabeg, S. e! A1., Proc. Νη11. Asab. 8c1. I8A, 95, 8910-8915; Waidyap, T.1. E! A1., 1998, Ya1ige Vu! Esipoodu 16, 535-539).

In these studies, primary antibody variants were generated by changing the sequences of the heavy and light chain genes in the SECE SOK2, SEC3 or frame regions using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, PCR error-prone, DNA shuffling, or mutator strains E. soi (Waidai, T. 1. E! A1., 1998, Ya! Igu Vu! Esipododu, 16, 535-539; Abeu, Ν.Β. e! A1., 1996, Syar! Eg 16, pp. 277-291, ίη Ryade Όίκр1ау о £ Rurbbek apb Rgo! Eshk, Ebk. Kau, V.K. e! A1., Asabetyu Rgekk). These methods of changing the sequence of the primary antibody have led to improved affinities of secondary antibodies (Ogat, N. e! A1., 1992, Propos. Asab. 8ο. I8A, 89, 3576-3580; Vobeg, E.T. e! A1., 2000 , Proc. N311. Assab. Δα. I8A, 97, 10701-10705; Pau1ek, 1. apb K1saytapp, b., 1996, 1 type1, 2, 169-179; Tyotrkop, 1. e! A1., 1996, 1. Mo1. Vu1., 256, 77-88; 8Boy, M.K. e! A1., 2002, 1. Vu1. Siet., 277, 16365-16370; Eigskata, K. e! A1, 2001, 1. Vu1 Siet., 276, 27622-27628).

Using a similar directed strategy for changing one or more amino acid residues of an antibody, the antibody sequences described in this invention can be used to develop antibodies against the UE-1 receptor with improved functions.

The conjugates of this invention contain an antibody, fragments and their analogs described herein,

- 8 009807 associated with a cytotoxic agent. Preferred cytotoxic agents are maytansinoids, taxanes, and CC-1065 analogs. These conjugates can be prepared by ίη νίΐτο methods. A binding group is used to bind the cytotoxic agent to the antibody. Suitable linking groups are well known in the art and include disulfide groups, simple thioether groups, acid unstable groups, photolabile 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 simple thioether bond between an antibody and a cytotoxic agent.

Maytansinoids and maytansinoid analogues are among the preferred cytotoxic agents. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Suitable maytansinoids are described in US Pat. Nos. 4,424,219; 4,256,746; 4,294,767; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,316,650; 4,362,663; 4,364,866; 4,450,254; 4322348; 4,371,533; 6333410; 5,457,092; 5585499 and 5846545.

Taxanes are also preferred cytotoxic agents. Taxanes suitable for use in this invention are described in US Pat. Nos. 6,372,738 and 6,340,701.

CC-1065 and its analogues are also preferred cytotoxic drugs for use in this invention. CC-1065 and its analogs are described in US Pat. Nos. 6372738; 6,340,701; 5846545 and 5585499.

An attractive candidate for such cytotoxic conjugates is CC-1065, which is a strong antitumor antibiotic isolated from the 81ger1 xc1sp515 culture broth. CC-1065 is approximately 1,000 times stronger than commonly used anti-cancer drugs, such as doxorubicin, methotrexate and vincristine (V.K. Wijuap e! A1., Sapseg Vek., 42, 3532-3537 (1982)).

Cytotoxic drugs, such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and calicheamicin, are also suitable for the preparation of the conjugates of this invention, and these drug molecules can also be coupled to antibody molecules via an intermediate molecule - a carrier such as serum albumin.

For diagnostic applications, the antibodies of the invention are typically labeled with a detectable molecule. A detectable molecule can be any molecule that is capable of producing, directly or indirectly, a detectable signal. For example, the detected molecule may be a radio3 14 32 35 131 isotope, such as H, C, P, 8 or I; a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine or luciferin; or an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art can be used to conjugate an antibody to a detectable molecule, including the methods described by Hepl, e! A1., No. 1 Shge 144: 945 (1962); Όανίά, e! A1., B1set1k1gu 13: 1014 (1974); Rush, e! A1, I. 1ttypo1. Me111. 40: 219 (1981) and Yudgep, I. NYUSNet. apb suiset. 30: 407 (1982).

The antibodies of this invention can be used in any known assay method, such as competitive binding assays, direct or indirect sandwich assays, and immunoprecipitation assays (Ζο1η. Mopos1op1 Apbobob1ek: A Mapia1 o £ Tes1shk | Ik. Pp. 47-158 (SHS Rgekk, 1ps., 1987)).

The antibodies of the invention are also useful for visiography of w νίνΌ, where an antibody labeled with a detectable molecule, such as an opaque radiation agent or a radioactive isotope, is administered to a subject, preferably into the bloodstream, and the presence or location of this labeled antibody in the host is analyzed. This method of visiography is applicable in determining the stage and treatment of malignancy. The antibody can be labeled with any molecule that is detectable in the host, using nuclear magnetic resonance, radiology or other means of detection known in this field.

The antibodies of the invention are also useful as affinity purification agents. In this method, antibodies are immobilized on a suitable carrier, such as Sephadex resin or filter paper, using methods well known in the art.

The antibodies of the invention are also useful as reagents in biological studies based on their inhibition of the ΙΟΕ-Ι receptor function in cells.

For therapeutic applications, the antibodies and conjugates of this invention are 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, by intramuscular, subcutaneous, intraarticular, intrasinovial, intrathecal, oral, topical or inhalation routes. An antibody can also be administered by intratumoral, near-tumor methods, by the route of introduction into or near the lesion, for the manifestation of local as well as systemic therapeutic actions. Suitable pharmaceutically acceptable carriers, diluents and

- 9 009807 excipients are well known and can be determined by specialists in this field, as may be required by the clinical situation. Examples of suitable carriers, diluents and / or excipients include: (1) Dulbecco's phosphate buffered saline, pH approximately 7.4, containing about 1 mg / ml to 25 mg / ml human serum albumin, (2) 0.9% saline (0.9% w / v NaCl) and (3) 5% (w / v) dextrose. The method of the invention may be practiced ίη νίίτο, ίη νίνο or ex νίνο.

In other therapeutic treatments, the antibodies, antibody fragments or conjugates of the invention are co-administered or sequentially administered with one or more additional therapeutic agents. Suitable therapeutic agents include, but are not limited to, cytotoxic or cytostatic agents. Taxol is a preferred therapeutic agent, which is also a cytotoxic agent.

Anticancer therapeutic agents are those that seek to kill or limit the growth of cancer cells, causing only minimal damage to the host. Thus, such agents can exploit any difference in the properties of cancer cells (e.g., metabolism, vascularization or presentation of cell surface antigens) from the properties of healthy host cells. Differences in tumor morphology are potential sites for intervention: for example, the second therapeutic substance may be an antibody such as an anti-UVP antibody that is useful in slowing the vascularization of the interior of a solid tumor, which slows down the growth rate of the tumor. Other therapeutic agents include, but are not limited to, additional agents such as granisterone, HC1, androgen inhibitors such as leuprolide acetate, antibiotics such as doxorubicin, antiestrogens such as tamoxifen, antimetabolites such as interferon alpha-2a, cytotoxic agents such as taxol, enzyme inhibitors such as a farnesyl transferase inhibitor, immunomodulators such as aldesleukin and derivatives of nitrogen analogues of mustard gas (mustard gas) such as melphalan, HC1 and the like.

Therapeutic agents that can be combined with EM164 for improved anticancer efficacy include various agents used in oncology practice (Ref: Sapseg, Przep1p1e8 & PrzepNeo ίί Opso1odu, OeUuja, U.T., Neitap, 8., Vokepzegd, 8 .a., 6 ebyyup ^4b, YrrepsoIRauep. RY1abe1rYa, 2001), such as docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Negseryp), capecitabine, tamoxifen, toremifene, letrozole, aiastrozol, fulvestrant, exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Ανа8ί ^ η), 5-fluorouracil, leucovarin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronate, streptozocin, ruludicamidulibrucimidibrudicimidibrucidrucimidrucimidrucimidibrucidrucimidrucimidrucimidrucidumibrucidrucimidrucimidrucimidrucimidrucimidrucimidrucimidrucimidrucimidrucidumibrucidrucimidrucimbucidibrucidrucidumibrucidrucimudicu bicidrucibiculidrucibiculidrucimidrucidumibrucidrucimudicubimcidrucibu imatinib, cytarabine, ibritumomab (^ etaHn), tositumomab (Wehhag), interferon alfa-2b, melpholam, bortezomib (Ve1sabe), altretamine, asparaginase, gefitinib (1ge88a), erlonitib (Ta ^ anti-receptor) (Ceschrhtta, ABx-ECP) and epothilones and conjugates of cytotoxic drugs and Ithel against cell surface receptors. Preferred therapeutic agents are platinum-containing agents (such as carboplatin, oxaliplatin, cisplatin), taxanes (such as paclitaxel, docetaxel), gemcitabine or camptothecin.

One or more additional therapeutic agents may be administered before, simultaneously with or after the antibody, fragment or conjugate of the antibody of the present invention. One skilled in the art will recognize that for each therapeutic agent there may be advantages with respect to a particular administration route. Similarly, one skilled in the art will understand that for each therapeutic agent, the length of the intervals at which each agent and antibody, antibody fragment or conjugate of the invention will be administered will vary.

Although one skilled in the art will recognize that the dose of each therapeutic agent will depend on the particular agent, preferred doses may range from about 10 to about 2000 mg / sq., More preferably from about 50 to about 1000 mg / sq. . m. For preferred agents, such as platinum-containing agents (carboplatin, oxaliplatin, cisplatin), the preferred dose is about 10 to about 400 mg / sq. m, for taxanes (paclitaxel, docetaxel), the dose is preferably about 20 to about 150 mg / sqm, for gemcitabine, the preferred dose is about 100 to about 2000 mg / sqm, and for camptothecin, the preferred dose is about 60 mg / sqm - approximately 350 mg / sq.m. The dose of these and other therapeutic agents may depend on whether the antibody, fragment or conjugate of an antibody of the invention is administered simultaneously or sequentially with the therapeutic agent.

Administration of an antibody, antibody fragment or conjugate of the invention and one or more additional therapeutic agents, whether administered together or sequentially, can be performed as described above for therapeutic applications. Suitable pharmaceutically acceptable carriers, diluents and excipients for co-administration will be known to those skilled in the art and will depend on the identity of the particular

- 10 009807 therapeutic agent, which is administered together.

When an antibody is present in an aqueous dosage form, rather than in a lyophilized form, this antibody is usually prepared at a concentration of about 0.1-100 mg / ml, although wide variation outside these ranges is allowed. For the treatment of a disease, the appropriate dose of an antibody or conjugate will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether these antibodies are administered for prophylactic or therapeutic purposes, the course of previous therapy, the patient’s clinical history and response antibody and at the discretion of the attending physician. Preferably, this antibody is administered to a patient once or over a series of administrations.

Depending on the type and severity of the disease, preferably the starting dose for administration to a patient is a dose of from about 1 to about 2000 mg / m2 of antibody, more preferably from about 10 to about 1000 mg / m2 of antibody, for example, by one or more single administrations or continuous infusion. For repeated administrations over several days or longer, depending on the condition, the administration is repeated until the desired suppression of disease symptoms is observed. However, other dosage regimens may be used and are not excluded.

The invention also includes kits containing one or more of the elements described herein and instructions for using these elements. In a preferred embodiment, a kit of the invention includes an antibody, antibody fragment or conjugate of the antibody of the invention and a therapeutic agent. Instructions for this preferred embodiment include instructions for inhibiting the growth of cancer cells using an antibody, fragment or conjugate of an antibody of the invention and a therapeutic agent and / or instructions for a method of treating a patient having cancer using an antibody, fragment or conjugate of an antibody of the present invention, and a therapeutic agent.

Preferably, the antibody used in this kit has the same amino acid sequence as the murine EM164 antibody produced by the mouse hybridoma EM164 (ATCC assortment and PTA-4457), or the antibody is an epitope-containing fragment thereof, both the antibody and this fragment specifically bind to the receptor of the insulin-like growth factor I. The antibody and antibody fragment used in this kit may also be an EM164 version of the antibody modified at the antibody surface that has at least at least one nucleotide mutation, deletion or insertion. Antibodies and antibody fragments of each of these three versions retain the same binding specificity as the EM164 antibody.

Preferably, the therapeutic agent used in this kit is selected from the group consisting of therapeutic agents such as docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Negserip), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, ezestanest, fulvest goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Auazyp), 5-fluorouracil, leucovarin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbin, estramustrone, strontium, mitroxanthus, mitrostane ptozocin, rituximab (Kyihap), idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine, ibritumomab (2uea1sh), tositumomab (Wehhag), interferon alfa-2b, melpholum, bortezemiebin (aspezeminum), asparafinum (aspezeminum), asparafinum (aspezeminum), asparafinum (aspezemin), erlonitib (Tateehua), anti-EOE receptor antibody (Ce1x1utb, ABx-EOE) and epothilone. More preferably, this therapeutic agent is a platinum-containing agent (such as carboplatin, oxaliplatin, cisplatin), taxane (such as paclitaxel, docetaxel), gemcitabine or camptothecin.

The elements of the kits of the present invention are in a suitable kit form, for example, in the form of a solution or lyophilized powder. It will be understood by a person skilled in the art that the concentration or amount of these elements of the kits will vary depending on the identity and intended use of each element of the kits.

The types of cancer and cells from these types of cancer that are referenced in the kit instructions include breast cancer, colon cancer, ovarian cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial cancer, pancreatic cancer , melanoma, multiple myeloma, neuroblastoma, and rhabdomyosarcoma.

Examples

The invention is now described with reference to the following examples, which are illustrative only and are not intended to limit the invention.

Example 1. Mouse antibody EM164.

This first example describes the complete primary amino acid structure and cDNA sequence of the murine antibody of the invention, together with its binding properties and the method of its expression in recombinant form. Thus, a complete and complete description of the antibodies of the present invention and its preparation is provided, so that the average specialist in the field of immunology is able to obtain the specified antibody without undue experimentation.

- 11 009807

A. Generation of a hybridoma monoclonal antibody against the JH-1 receptor.

A cell line expressing a human JH-1 receptor with a mutation of Υ1251Γ was used for immunization, since it expressed a high number of JH-1 receptors (~ 10 ⁷ per cell). The Υ1251Γ mutation in the cytoplasmic domain of the JH-1 receptor led to a loss of transformation (transformation of cells into tumor cells) and antiapoptotic signal transmission, but did not affect the binding of JH-1 and JH-1-stimulated mitogenic signal transmission (O'Soppog, K. s1 a1., 1997, Mo1. Ce11. ΙοΙ., 17, 427-435; Msta, M. e1 a1, 1995, 1. Vu1. SN., 270, 22639-22644). This mutation did not otherwise affect the generation of antibodies, since the antibody of this example binds to the extracellular domain of the JR-1 receptor, which was identical for both the Υ125ΙΡ mutant and the wild-type receptor.

The cell line expressing the JR-1 receptor with the Υ1251Ρ mutation was generated from 3T3-like cells of a mouse deficient in the JR-1 receptor by transfection with the human JR-1 receptor gene together with the puromycin resistance gene and selected using puromycin (2.5 μg / ml) and RASZ-sorting for high expression of the UR-1 receptor (Mshta, M. e1 a1., 1995, ί. Βίο1. SN., 270, 22639-22644). A cell line having a high expression level of the UR-1 receptor was further selected using a high concentration of puromycin, for example 25 μg / ml, which was toxic to most of these cells. Surviving colonies were selected, and colonies showing a high expression level of the UR-1 receptor were selected.

Female CAP1 / 1 mice, 6 months old, were immunized intraperitoneally per day with 0 cells overexpressing the human Υ1251Ρ mutant UR-1 receptor (5x10 ⁶ cells suspended in 0.2 ml PBS). The animals were re-injected with 0.2 ml of cell suspension as follows: day 2, 5.2 x 10 ⁶ cells; day 5, 2x10 ⁶ cells; days 7, 9, 12 and 23, 1x10 ⁷ cells. On day 26, the mouse was sacrificed and its spleen removed.

The spleen was crushed between two frosted glass slides to obtain a suspension of individual cells, which was washed with serum-free medium KPM1 containing penicillin and streptomycin (8PM). The spleen cell pellet was resuspended in 10 ml of a 0.83% (w / v) solution of ammonium chloride in water for 10 min on ice to lyse red blood cells and then washed with serum-free medium (8PM). Spleen cells (1.2 × 10 ⁸ ) were combined with myeloma cells (4 × ^{10 7} ) from the non-secreting mouse myeloma cell line P3X63Ad8.653 (ATCC, Koskushe. ME; Ca! # SKB1580) in vitro and washed with serum-free KPM1 1640 medium (8РМ). The supernatant was removed and the cell pellet was resuspended in the rest of the medium. The tube was placed in a beaker with water at 37 ° C and slowly added at a drop speed of 0.5 ml / min. 1.5 ml of a solution of polyethylene glycol (50% PEG (w / v), average molecular weight of 1500 in 75 mm HEPE8, pH 8) with gentle shaking of the tube. After standing for one minute, 10 ml of 8PM was added as follows: 1 ml during the first minute, 2 ml during the second minute and 7 ml during the third minute. Then another 10 ml was added slowly over one minute. Cells were pelleted by centrifugation, washed in 8PM, and resuspended in KPM11640 growth medium supplemented with 5% fetal calf serum (FCS), hypoxanthine / aminopterin / thymidine (NAT), penicillin, streptomycin and 10% cloning hybridoma supplement (HC8). Cells were seeded in 96-well flat-bottomed tissue culture plates at 2 x 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 (NT) and 5% FCS. General conditions used for immunization and hybridoma production were as described ί. Lapdope apb N. Uipak1k (Ebk., Mebubk ίη Ep7ut1odu, Uo1. 121, 1ttypo1od1sa1 Tesishdiek, Paradise I, 1986; Asabeiis Rgekk, P1opba) and E. Nag1o u apb. Laphe (Apyoblek: A baobaFgu Mapia1, 1988; Co1b Zrppd Natot babaFgu Rgekk, No. \ ν Wogk). Other methods of immunizing and producing hybridomas may also be used, as is well known to those skilled in the art.

Cultural supernatants from hybridoma clones were screened for binding to a purified human UR-1 receptor and for the absence of specific binding to cells overexpressing the human insulin receptor using E18A and PAC8 screening, as described below. Clones exhibiting a higher binding affinity for cells overexpressing the human URS receptor than for cells overexpressing the human insulin receptor were propagated and subcloned. The culture supernatants of these subclones were further screened by the above binding assays. Using this procedure, a 3P1-C8-E7 subclone (EM164) was selected and the heavy and light chain genes were cloned and sequenced as described below.

The human UR-1 receptor was isolated for use in screening supernatants from hybridoma clones for their binding to the UR-1 receptor using the method described below. Biotinylated UR-Ι was obtained by modification of recombinant UR-Ι using biotinylation reagents such as sulfo-ННЗ-ЬС-biotin, sulfo-НН8-88-biotin or PN8-REO _4- biotin. Biotinylated JR-Ι was absorbed on streptavidin-agarose granules and incubated with a lysate from cells that overexpressed wild-type human RK or Υ1251P-mutant RK. These granules

- 12 009807 was washed and eluted with a buffer containing 2-4 M urea and a detergent, such as Triton X-100 or octyl-b-glucoside. The eluted YuB-1 receptor was dialyzed against PBS and analyzed for purity by SDS-PAGE under reducing conditions, which showed alpha and beta chains of the YuB-1 receptor with molecular weights of approximately 135 kDa and 95 kDa, respectively.

To check for binding of hybridomas supernatants to a purified YB-1 receptor, a 1TTI1OP-4HB EY8A plate was coated with a sample of a purified human YB-1 receptor (obtained by dialysis from elution with a urea / octyl-b-glucoside affinity purified sample) diluted in 50 mM CHE8 buffer at pH 9.5 (100 μl; 4 ° C, overnight). Wells were blocked with 200 μl blocking buffer (10 mg / ml BSA in TB8-T buffer containing 50 mM Tris, 150 mM NaCl, pH 7.5, and 0.1% Tween-20) and incubated with supernatants from hybridoma clones ( 100 μl; diluted in blocking buffer) for approximately 1-12 hours, washed with TB8-T buffer and incubated with a goat anti-mouse 1dC-Bs horseradish horseradish peroxidase (NKR) conjugate (100 μl; 0.8 μg / ml blocking buffer; Chaskcop 1tipoKe8eate EaotaFpec), followed by washing and detection using an ABT8 / H ₂ O ₂ substrate at 405 nm (0.5 mg / ml ABT8, 0.03% H ₂ O ₂ in 0.1 M citrate buffer, pH 4.2). Typically, the supernatant from the 3B1 hybridoma subclone gave a signal of approximately 1.2 units of optical density within 3 min of development, in contrast to the values of 0.0 obtained for supernatants from some other hybridoma clones. The general conditions for this EB18A were similar to the standard EB18A conditions for antibody binding and detection described by E. Nat1ote apb Ό. Lapé (Apboblée8: A Bálaga Lágu Mapia1, 1988; Cólb 8rppd Natíl Láboga logu Przekk, Youe Wogk), which can also be used.

Hybridoma supernatants were screened for specific binding to the human UB-1 receptor, but not to the human insulin receptor, using E18A on cell lines that overexpressed the human U1251B-UB-1 receptor, and on cell lines that overexpress the human insulin receptor. Both cell lines were obtained from 3T3-like cells of YBΙ receptor-deficient mice. Overexpressing UB-1 receptor cells and overexpressing insulin receptor cells were separately collected from tissue culture flasks by rapid trypsin / EDTA treatment, suspended in growth medium containing 10% FCS, precipitated by centrifugation and washed with PBS. The washed cells (100 μl of approximately 1-3 x 10 ⁶ cells / ml) were added to the wells of a 1Ti1op-2HB plate coated with phytohemagglutinin (100 μl of 20 μg / ml PHA), centrifuged and allowed to attach to the PHA-coated wells for 10 min . The cell plate was inverted with a tap to remove PBS and then dried overnight at 37 ° C. Wells were blocked with a 5 mg / ml solution of BMA in PBS for 1 h at 37 ° C and then washed carefully with PBS. Aliquots of supernatants from hybridoma clones (100 μl; diluted in blocking buffer) were added to wells containing overexpressing YB-1 receptor cells, and to wells containing overexpressing insulin receptor cells, and incubated at ambient temperature for 1 hour. The wells were washed with PBS, incubated with a goat anti-mouse 1dC-Bc horseradish conjugate - horseradish peroxidase (100 μl; 0.8 μg / ml in blocking buffer) for 1 h, followed by washing and then binding was detected using an ABT8 / H ₂ O substrate ₂ . A typical superiatant from 3B1 hybridoma subclone after incubation with cells overexpressing the YB-1 receptor gave a signal of 0.88 units of optical density within 12 minutes of development, in contrast to the value of 0.22 unit of optical density obtained with cells overexpressing the human insulin receptor .

This hybridoma was grown in 1p flasks (Una CI 350 (1p1eda Vyukaepsek, Magu1apb) according to the manufacturer's descriptions, to obtain purified EM164 antibody. Yield about 0.5-1 mg / ml of the antibody was obtained in collected supernatants from 1p1edt flasks, based on quantitative for determination of E8A and SDS-PAGE electrophoresis / Coomassie blue staining using antibody standards This antibody was purified by affinity chromatography on a column of Aagarose Protein granules under standard purification conditions for application and washing in 100 mM Trisbuffer, pH 8.9, containing 3 M NaCl, followed by elution in a 100 mM acetic acid solution containing 150 mm NaCl. The eluted fractions containing the antibody were neutralized with a cold 2 M solution of K ₂ HPO ₄ and dialyzed in PBS at 4 ° C. Antibody concentration was determined by measuring the optical density at 280 nm (extinction coefficient = 1.4 mg ^-1 ml cm ^-1 ). The purified antibody sample was analyzed by SDS-PAGE under reducing conditions and Coomassie blue stained, which showed only bands heavy and light chains of antibodies and at approximately 55 kDa and 25 kDa, respectively. The isotype of this purified antibody was 1dC ₄ with a kappa light chain.

B. Characterization of the binding of the antibody BM164.

The specific binding of the purified EM164 antibody was demonstrated by cell sorting with fluorescence stimulation (BAS8) using cells overexpressing the human UBΙ receptor and using cells overexpressing the human insulin receptor (FIG. 1). Incubation of antibody EM164 (50-100 nM) in 100 μl of cold BAS8 buffer (1 mg / ml BSA in

- 13 009807 MEM medium in the Dulbecco modification) was performed using cells overexpressing the human UR-1 receptor and using cells overexpressing the human insulin receptor (2 x 10 ⁵ cells / ml) in a round bottom 96-well plate for 1 h Cells were precipitated by centrifugation and washed with cold PAC8 buffer by careful shaking followed by incubation with a goat anti-mouse 1dC-Pc horseradish peroxidase conjugate antibody (100 μl; 10 μg / ml in PAC8 buffer) on ice for 1 h. Cells were precipitated washed and resusp ndirovali in 120 ul 1% formaldehyde solution in PBS. The tablet was analyzed using a PAC8Ca11b reader (VL VukOepsek).

A strong fluorescence shift was obtained after incubation of overexpressing UR receptor cells with the EM164 antibody, as opposed to a slight displacement after incubation of overexpressing insulin receptor cells with the EM164 antibody (Fig. 1), which showed that EM 164 antibody was selective in its binding to UR-1 -receptor and did not bind to the insulin receptor. The control antibodies, the anti-JR-1 receptor 1H7 antibody (8ap! And Cnx Vyslipodo) and the insulin anti-receptor antibody (VL PPrdschodep baLogaFpec), showed fluorescence shifts after incubation with cells overexpressing the receptor inr-1 receptor (Fig. 1). A strong fluorescence bias was also observed using PAC8 analysis using EM164 antibody and human breast cancer MCP-7 cells that expressed the UR-1 receptor (LiGoigpu, B. e! A1., 1997, Σ. Vu1. C1et., 272, 31171), which showed that the EM 164 antibody bound to the human UR-1 receptor on the surface of human tumor cells.

The dissociation constant (K _c ) for binding of the EM164 antibody to the human ju-1 receptor was determined by titration of the E8A binding antibody at several concentrations or directly by sensitizing the ju-1 receptor (affinity purified using biotinylated ju-1, as described above) , or with indirectly captured biotinylated UR receptor. The biotinylated LTP receptor was obtained by biotinylation of a solubilized detergent lysate from overexpressing LRL receptor cells using the ReOmaleimide-biotin reagent (P1egse, Mo1eci1ag Vu8x1epse8), which was affinity purified using an anti-H3L8 receptor granular antibody was eluted with 2-4 M urea in a buffer containing detergent no. 40 and dialyzed in PBS.

Determination of K _c for binding of the EM164 antibody to the biotinylated YRL receptor was carried out by sensitization of the IshtNop-LNB plate with 100 μl of 1 μg / ml streptavidin in carbonate buffer (150 mm sodium carbonate, 350 mm sodium bicarbonate) at 4 ° C overnight. The streptavidin-sensitized wells were blocked with 200 μl of blocking buffer (10 mg / ml BSA in TB8-T buffer), washed with TB8-T buffer and incubated with biotinylated URL-receptor (10-100 ng) for 4 hours at ambient temperature. Then, the wells containing the captured biotinylated YRL receptor were washed and incubated with EM164 antibody in blocking buffer at several concentrations (5.1 x 10 ^-13 M - 200 nM) for 2 hours at ambient temperature and then incubated overnight at 4 ° C. The wells were then washed with TB8-T buffer and incubated with a goat anti-mouse Ι§6 _{η +} ε - horseradish peroxidase conjugate conjugate (100 μl; 0.5 μg / ml in blocking buffer), followed by washing and detection using ABT8 / H substrate ₂ O ₂ at 405 nm. The value of K _{c was} determined by nonlinear regression for one-site binding.

A similar titration of binding was carried out using the PAb fragment of the EM164 antibody obtained by papain digestion of this antibody, as described by E. Nag1ote apb. Lape (Iktd Apyob1ek: A baobaFgu Mapia1, 1988; Sbb 8rpp§ Nagbog baobaogu Rgekk, Yout Watk).

Binding titration curve for binding of EM 164 antibody to biotinylated human YuRLretseptorom afforded value K _with 0.1 nM (FIG. 2). Fab fragment of EM164 antibody also binds human YURL receptor very tightly with _a K value of 0.3 nM, which indicated that the monomeric binding of the antibody to E164 YURL receptor was also very strong.

This extremely low value of the dissociation constant for binding of the YRL receptor to the EM 164 antibody was partly due to the very low rate of _GHG , confirmed by the strong binding signals observed after prolonged washing for 1-2 days of the antibody bound to the immobilized YRL receptor.

Antibody EM 164 can be used for immunoprecipitation of the LRL receptor, as shown by incubation of a human breast MCP-7 cell lysate detergent solubilized with a human antibody with EM164 antibody immobilized on protein Ο-agarose granules (Plxet Synetka Sotrapu). Western blot of the EM164 antibody immunoprecipitate was detected using a rabbit polyclonal antibody against the beta chain (C-terminus) of the YRL receptor (8ap! A Cnx Vuclipodu) and a goat antibody conjugate against mouse ΙβΟ - horseradish peroxidation followed by ) The EM164 immunoprecipitate western blot from MCP-7 cells showed bands corresponding to the beta chains of the YRL receptor at approximately 95 kDa and the proYURL receptor at approximately 220 kDa. Similar immunoprecipitations were carried out for other types of cells to check the species specificity of the binding of the EM164 antibody, which also bound to the ΙΟΕ-Ι receptor from CO8-7 cells (African green monkey), but did not bind to the Т-Ι receptor of 3T3 cells (mouse ), CHO cells (Chinese hamster) or goat fibroblast cells (goat). The EM164 antibody did not detect a denatured SDS human ΙΟΕ-Ι receptor in Western blot of lysates from MCE-7 cells, which indicated that it was associated with a conformational epitope of a native, non-denatured human ΙΟΕ-Ι receptor.

The binding domain of the EM 163 antibody was further characterized using a truncated alpha chain construct that contained a cysteine-rich domain flanked by the L1 and L2 domains (residues 1-468) fused to the 16-dimensional C-terminus (residues 704-719) and which ended with a C-terminal epitope tag. It was reported that this smaller ΙΟΕ-Ι receptor, which was devoid of residues 469-703, binds to ΙΟΕ-Ι, although it is less firmly compared to the native full-sized ΙΟΕ-Ι receptor (Mo1sha, b. C1 a1., 2000 , EEB8 Leyegck, 467, 226-230; Cpcrcssp. S. c1 a1., 1999, I. B1o1. Siesz., 274, 37251-37356). Thus, a shortened α-receptor alpha chain construct was obtained containing residues 1-468 fused to the C-terminal region, which contains residues 704719 and is flanked by the C-terminal epitope tag of the tus. A stable cell line was constructed that expressed this construct and which also expresses this construct transiently in 3T3 cells of a human embryonic kidney. Strong binding of the EM164 antibody to this truncated ΙΟΕ-ΙΟΕ receptor alpha chain was observed. Of the two antibodies tested, S.3 (Ca1O3set) also bound to this truncated alpha chain, but the 1H7 antibody (8ap1a Verse B1c111po1) did not bind, indicating that the epitope of the EM164 antibody was clearly different from the epitope of the 1H7 antibody.

C. Inhibition of ΙΟΕ-связыв binding to MCE-7 cells with EM164 antibody.

The binding of ΙΟΕ-Ι to human breast cancer cells was inhibited by antibody EM 164 (Fig. 3). MCE-7 cells were incubated in the presence or absence of 5 μg / ml of EM 164 antibody for 2 hours in serum-free medium, followed by incubation with 50 ng / ml of biotinylated ΙΟΕ-Ι for 20 min at 37 ° C. Then, these cells were washed twice with serum-free medium to remove unbound Biotin-ΙΟΕ-Ι and then lysed in 50 mM HEPE8, pH 7.4, containing 1% ΝΡ-40 and protease inhibitors. The Ttt1op-2HB plate was sensitized with a murine monoclonal antibody against the ΙΟΕ-Ι receptor beta chain and used to capture the ΙΟΕ-Ι receptor and the associated biotin-UE-Ι from the lysate. The binding of the sensitizing antibody to the cytoplasmic C-terminal domain of the ΙΟΕ-Ι receptor beta chain did not interfere with the binding of biotin-ΙΟΕ-Ι to the extracellular domain of the ΙΟΕ-Ι receptor. These wells were washed, incubated with horseradish streptavidin peroxidase conjugate, washed again, and then detected using ABT8 / H ₂ O ₂ substrate. The inhibition of ΙΟΕ-Ι binding to MCE-7 cells with EM 164 antibody (5 μg / ml) was essentially quantitative and was almost equivalent to the binding of the background EY8A obtained using a control without biotin-ΙΟΕ-Ι.

In addition to the above analysis of the inhibition of binding of ΙΟΕ-Ι to MCP-7 cells with EM164 antibody, the following analysis showed that the EM164 antibody was highly effective in displacing bound ΙΟΕ-Ι from MCP-7 cells, as is desirable under physiological conditions, so that the antagonistic antibody against the ог-Ι receptor, the bound endogenous physiological ligand (such as ΙΟΕ-Ι or ΙΟΕ-ΙΙ) displaced. In this assay, ΙΟΕ-Ι displacement MCP-7 cells grown in a 12-well plate were fasted in serum-free medium and then incubated with biotinylated ΙΟΕ-Ι (20-50 ng / ml) in serum-free medium at 37 ° C (or at 4 ° C) within 1-2 hours. Then these cells with bound biotinylated ΙΟΕ-Ι were treated with EM164 antibody or control antibody (10/100 μg / ml) at 37 ° C (or at 0 ° C) for 30 minutes - 1 hour Then, the cells were washed with PBS and lysed in lysis buffer containing 1% ΝΡ-40 at 4 ° C. EM8A was performed as described above to capture the ΙΟΕ-Ι receptor from the lysate and then detect the biotinylated ΙΟΕ-Ι associated with the receptor using the streptavidin-horseradish peroxidase conjugate. This EH8A showed that the EM164 antibody was able to displace pre-bound biotinylated ΙΟΕ-Ι from the cells almost completely (90% within 30 minutes and ~ 100% within 4 hours) at 37 ° C and approximately 50% in 2 hours at 4 ° C. In another experiment, рака-Η838 lung cancer cells were incubated with biotin-ΙΟΕ-Ι, then washed and incubated with EM164 antibody at 4 ° C for 2 h, which led to an 80% decrease in pre-bound biotin ΙΟΕ-Ι. Thus, the EM164 antibody was highly effective in displacing pre-bound biotin-ΙΟΕ-Ι from cancer cells, which would be important therapeutically for antagonism of the ΙΟΕ-Ι receptor by displacing the bound endogenous physiological ligand.

Incubation of MCΕ-7 cells with EM164 antibody at 4 ° C for 2 h (or at 37 ° C for 30 min) did not lead to significant downregulation of the ΙΟΕ-Ι receptor based on Western blot analysis using anti-beta antibodies ΙΟΕ-Ι receptor chains (8ap1a Sgi / Vu1ec11po1; §c-713), although longer incubation with the EM164 antibody at 37 ° C for 2 hours resulted in a 25% downregulation of the ΙΟΕ-Ι receptor. Thus, the inhibition of ΙΟΕ-ания binding and the displacement of ΙΟΕ-связ bound EM164 antibody both at 4 ° C and at 37 ° C in these experiments with a short period of time cannot be explained by downregulation of the receptor due to binding of the EM 164 antibody. inhibiting ΙΟΕ-Ι binding to the ΙΟΕ-Ι receptor and crowding out

- 15 009807 of the pre-bound ΙΟΡ-Ι antibody EM164 is, apparently, a competition for binding, either through the presence of a common binding site, or through steric occlusion, or through allosteric effects.

Ό. Inhibition of ΙΟΕ-Ι receptor-mediated antibody cell signal transduction

EM164.

The treatment of MCE-7 cancer cells and 8aO8-2 osteosarcoma cells with EM164 antibody almost completely inhibited the intracellular transmission of the ΙΟΡ-Ι receptor signal, as shown by the inhibition of autophosphorylation of the ΙΟΡ-Ι receptor and the inhibition of phosphorylation of its downstream effect transducers, such as substrate -1 insulin receptor (ΙΚ8-1), Ak1 and Egk1 / 2 (Fig. 4-6).

In FIG. 4, MCE-7 cells were grown in a 12-well plate in normal medium for 3 days and then they were treated with 20 μg / ml of EM164 antibody (or control anti-B4 antibody) in serum-free medium for 3 hours, followed by stimulation of 50 ng / ml ΙΟΡ-Ι for 20 min at 37 ° C. These cells were then lysed in ice-cold lysis buffer containing protease and phosphatase inhibitors (50 mM HEPE8 buffer, pH 7.4, 1% 40-40, 1 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 , 5 mM EDTA, 10 μM leupeptin, 5 μM pepstatin, 1 mM PMCE, 5 mM benzamide and 5 μg / ml aprotinin). The E8A plate was preliminarily sensitized with TC123 monoclonal antibody against the C-terminus of the ingredient beta chain and incubated with lysate samples for 5 hours at ambient temperature to capture the ΙΟΡ-Ι receptor. Then, the wells containing the captured ΙCE-1 receptor were washed and incubated with biotinylated antiphosphothyrosine antibody (ΡΥ20; 0.25 μg / ml; г Tgapkbisyop BabogaUpeD for 30 min, followed by washing and incubation with streptavidin-peroxidase conjugate (xidase 8 .mu.g / ml) for 30 min. The wells were washed and detected using substrate AVT8 / H ₂ O _2. The use of anti-V4antitela control showed no inhibition ΙΟΡ-Ι-stimulated autophosphorylation ΙΟΡ-Ιretseptora. in contrast, full ingibi sc ΙΟΡ-Ι-stimulated autophosphorylation ΙΟΡ-Ι-receptor was obtained after treatment with EM 164 antibody (Fig. 4).

To demonstrate the inhibition of phosphorylation of insulin receptor substrate-1 (ΙΚ8-1), EY8A was used using an immobilized anti-ΙΚ8-1 antibody to capture ΙΚ8-1 from lysates, followed by measurement of the associated p85 subunit of phosphatidylinositol-3-kinase (ΡΙ3-kinase), which binds with phosphorylated ΙΚ8-1 (Laskop, J. s1 a1., 1998, 1. ΒίοΙ. Siesh., 273, 9994-10003). In FIG. 5 MCE-7 cells were treated with 5 μg / ml antibody (EM164 or ΙΚ3) in serum-free medium for 2 hours, followed by stimulation with 50 ng / ml ΙΟΡ-Ι for 10 min at 37 ° C. The anti-δ-1 antibody (rabbit polyclonal; ir51a1e Vu1ec11po1odu) was directly captured by incubation with a goat anti-rabbit ΙβΟ antibody coated on an EY8A plate, which was then used to capture ΙΚ8-1 from cell lysate samples by incubation overnight at 4 ° C . Then, these wells were incubated with a murine monoclonal antibody against p85-ΡI-3 kinase (Ir51a1e Vu1ec11po1od) for 4 h followed by treatment with a goat anti-mouse ΙβΟ - NKR conjugate antibody for 30 min. Then the wells were washed and detected using an ABT8 / H ₂ O ₂ substrate (FIG. 5). As shown in FIG. 5, the EM164 antibody was more effective in inhibiting ΙΟΡ-Ι-stimulated ΙΚ8-1 phosphorylation when incubated with cells in the absence of ΙΟΡ-Ι.

Activation of other effectors downstream of the signal, such as Ak1 and Egk1 / 2, was also inhibited in a dose-dependent manner by the EM164 antibody in 8aO8-2 cells (Fig. 6) and in MCE-7 cells, as shown by Western blots lysates and antibodies specific for phosphorylation (rabbit polyclonal antibodies against phospho-8et ⁴⁷³ Ak1 and polyclonal antibodies against phospho-EKK1 / 2; Ce11 8shp1td Tesypododu). Antibody against total EKK showed the same protein load in all lanes (Fig. 6). Treatment of 8aO8-2 cells with EM164 antibody did not inhibit EOE-stimulated phosphorylation of Egk1 / 2, demonstrating the specificity of inhibiting the signal transduction pathway of the β-β receptor with EM 164 antibody.

E. Inhibition of stimulated ΙΟΡ-Ι, ΙΟΡ-ΙΙ and serum growth and survival of human tumor cells with EM164 antibody.

Several human tumor cell lines were tested under serum-free conditions for their growth and survival response to ΙΟΡ-Ι. These cell lines were treated with EM164 antibody in the presence of ΙΟΡ-Ι, ΙΟΡ-ΙΙ or serum, and their growth and survival reactions were measured using an MTT assay after 2-4 days. Approximately 1,500 cells were seeded in a 96-well plate in normal serum medium, which was replaced with serum-free medium the next day (either serum-free CPME supplemented with transferrin and BSA, or phenol-free red medium described by Iteoitu, B. e1 a1., 1997, I. Vu 1. Sies., 272, 31163-31171). After one for growth in serum-free medium, these cells were incubated with approximately 75 μl of 10 μg / ml antibody for 30 minutes 3 hours, followed by the addition of 25 μl of a ΙΟΡ-Ι solution (or ΙΟΡ-ΙΙ or serum) to obtain a final concentration of 10 ng / ml ΙΟΡ-Ι or 20 ng / ml ΙΟΡ-ΙΙ or 0.04-10% serum. In some experiments, these cells were first stimulated with ΙΟΡ-Ι for 15 minutes before adding EM164 antibody, or

- 16 009807 were added together as UE-Ι, and antibody EM164. Then the cells were allowed to grow over the next 2-3 days. Then a solution of MTT (3- (4,5) -dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide was added; 25 μl of a 5 mg / ml solution in PBS) and these cells were returned to the thermostat for 2-3 hours. Then the medium was removed and replaced with 100 μl of DMSO, mixed and the optical density of this plate was measured at 545 nm. Several human tumor cell lines showed a growth and survival response after addition of UE-Ι or UE-ΙΙ or serum that was significantly inhibited by EM164 antibody, regardless of whether the antibody was added before UE-Ι or UE-ляли was added before the antibody or as UE-Ι and the antibody was added together (table. 1).

Table 1. Inhibition of SE-L stimulated growth and survival of tumor cells with EM164 antibody

Type of tumor cells Growth reaction on UR-1 (MTT analysis: ratio of UR-1-treated cells to untreated cells in serum-free medium (a specified number of times) ^a % inhibition of antibody EM 164 UR-1 stimulated growth in serum-free medium Inhibition by antibody EM 164 cell growth / survival in 1.25-10% serum ⁵ MCP-7 (mammary gland) 1.7-2.8 one hundred% 85% NT-3 (cervix 2 70-90% ΝΌ Co1 205 (colon)) 2.3 fifty% Yes NT-29 1.5 60% Yes IS1-H838 (light) 3 one hundred% 85-90% Ca1i-6 1.6-1.8 85% Yes zk-y-1 1.4 one hundred% not N0144596 1.4 one hundred% Aeak1u A 549 1.2 80% N1) A 375 (melanoma) E6 90% not 8K-Me1-37 1.4 85% Νϋ

KO (rhabdomyosarcoma) 1.7 85-100% Yes 8aO8-2 (osteosarcoma) 2.5 one hundred% Yes A 431 (epidermoid) 2.2 85% Yes 8Κ-Ν-8Η (neuroblastoma) 2 85% 30-50%

^and MTT analysis of 3-4-day cell growth / survival in response to 10 ng / ml of SUE-Ι in serum-free medium containing 5-10 μg / ml of EM 164 antibody ^b Inhibition of cell growth in 1.25-10% serum in the presence of 5-10 μg / ml of EM164 antibody in accordance with an MTT assay or colony formation assay based on a comparison with a control (with serum but without antibody); the degree of inhibition was quantitatively measured for MCE-7, ΝΟΙ-Η838 and 8Κ-Ν-8Η cells per control (without serum, but with antibody and with serum but without antibody) to explain autocrine / paracrine JE stimulation by cells. ΝΏ indicates lack of data or bad data due to difficulties in staining.

Antibody EM 164 strongly inhibited the growth and survival of breast cancer cells MCE-7 stimulated by UE-Ι or serum (Figs. 7 and 8). In a separate experiment, EM 164 antibody strongly inhibited UE-ΙΙ-stimulated cell growth and survival of MCE-7 cells. Previous reports using commercially available antibodies, such as antibody No. 3, showed only weak inhibition of serum-stimulated growth and survival of MCE-7 cells, as confirmed in FIG. 7 for antibodies ΙΕ3 and 1H7 (Cu11ei, K.1. E! A1., 1990, Saiseg Yaek., 50, 48-53). In contrast, the EM 164 antibody was a potent inhibitor of serum-stimulated and UE-М cell growth of MCE-7. As shown in FIG. 8, the EM164 antibody was equally effective in inhibiting the growth and survival of MCE-7 cells in a wide range of serum concentrations (0.04-10% serum).

Cell growth inhibition of MCE-7 by EM164 antibody was measured by cell counting. So, at 12

- In a 17 009807 well plate, approximately 7500 cells were plated in KΡMI medium with 10% FCS, in the presence and absence of 10 μg / ml EM164 antibody. After 5 days of growth, the number of cells for the untreated control sample was 20.5 x 10 ⁴ cells, in contrast to the number of cells, only 1.7 x 10 ⁴ cells for the antibody-treated EM 164 sample. Antibody treatment with EM 164 inhibited the growth of MCE-7 cells by about 12 times in 5 days. This inhibition by EM164 antibody was significantly greater than the reported inhibition by 2.5 times using antibody No. 3 in a 6-day analysis for MCE-7 cells (KoY1k, F.T. e! A1., 1987, Vyusnet. Vyryuk. Kek. Sottip., 149, 276-281).

Stimulated by UE-Ι and serum growth and survival of the non-small cell lung cancer cell line N0-4838 were also strongly inhibited by the EM164 antibody compared to the B4 control antibody (Fig. 9). Treatment with the EM164 antibody in serum-free medium gave a smaller signal than the untreated sample for both HO-H838 cells and MCE-7 cells, presumably because the EM 164 antibody also inhibited the autocrine and paracrine stimulation of UE-Ι and UE-ΙΙ of these cells (Fig. 7 and 9). Colony size of HT29 colorectal cancer cells was also significantly reduced after treatment with antibody EM164.

Thus, the EM164 antibody is unique among all known anti-JUΙ receptor antibodies by its effectiveness in inhibiting serum-stimulated growth of tumor cells, such as MCE-7 and N0-4838 cells, by more than 80%.

Antibody EM 164 caused growth retardation in the С0 / С1 phase of the cell cycle and eliminated the mitogenic effect of UE-Ι. To analyze the cell cycle, MCE-7 cells were treated with UE-Ι (20 ng / ml) in the presence or absence of EM164 (20 μg / ml) for 1 day and then stained with propidium iodide and flow cytometry. As shown in FIG. 25, cell cycle passage in response to UE-стим stimulation in the absence of EM164 (with 41% of these cells in the 8th phase and 77% of these cells in the C0 / C1 phase) was inhibited in EM164-treated cells (with only 9% in 8- phase and 77% of these cells in the 00/01 phase).

In addition to inhibiting cell proliferation, treatment with EM164 antibody led to cell apoptosis. To measure apoptosis, caspase cytokeratin SK18 protein cleavage was measured in lung cancer cells N0-4 ^ 838 incubated with: UE-Ι or serum in the presence or absence of EM164, for day 1 (Fig. 26). In the absence of EM164, the addition of UE-Ι or serum resulted in a lower signal of caspase cleaved SK 18 in comparison with the control without UE-Ι, which indicated that UE-Ι and serum prevent caspase activation. Antibody treatment with EM 164 suppressed the anti-apoptotic effects of UE-Ι and serum, as indicated by higher levels of cleaved SK18 obtained in the presence of EM164 than in the absence of EM164 (Fig. 26).

E. Synergistic inhibition by antibody EM164 of the growth and survival of human tumor cells in combination with other cytotoxic and cytostatic agents.

The combined administration of EM164 antibody with taxol was significantly more inhibitory to the growth and survival of non-small cell lung cancer cells Ca1 and 6 than taxol alone. Similarly, the combination of EM164 antibody with camptothecin was significantly more inhibitory than camptothecin alone with respect to the growth and survival of HT29 colon cancer cells. Since the EM164 antibody alone was not expected to be as toxic to cells as organic chemotoxic drugs, the synergism between the predominantly cytotoxic effect of the EM164 antibody and the cytotoxic effect of the chemotoxic drug could be highly effective in combining cancer therapeutic agents in a clinical setting.

The combined effect of an EM164 antibody with an anti-SEE receptor antibody (K877) was significantly more inhibitory than one EM164 antibody or one K877 antibody on the growth and survival of several tumor cell lines, such as HT-3 cells, KI cells, MCE-7 cells and A431 cells. Thus, the synergistic effect of combining neutralizing antibodies for two growth factor receptors, such as the EU-L receptor and the ECE receptor, may also be applicable in the clinical treatment of cancer.

Based on the effectiveness of the EM164 antibody as the sole agent in inhibiting the proliferation and survival of various human cancer cell lines, shown in Table 1. 1, additional efficacy studies were conducted using combinations of the EM164 antibody with other anti-cancer therapeutic agents. In these studies on different cancer cell lines, the combined treatment with antibody EM164 and other anti-cancer therapeutic agents led to even higher anti-cancer efficacy than using either EM164 or another therapeutic agent alone. Thus, combinations of EM164 with other therapeutic agents are highly effective in inhibiting the proliferation and survival of cancer cells. Therapeutic agents that can be combined with EM164 for improved anticancer efficacy include a variety of agents used in oncology practice (Ref: Sapseg, Pr1ps1p1ez & Prgase o £ 0pso1od, Ieuya, U.T., Ne1tap, 8., Kokepzegd, 8 .A., 6 ' ¹ ' ebayup, ^^ рреηсой-Каνеη, РЫ1абе1рЫа, 2001), such as docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Negserip), capecitabine, tamoxifrostanol, torotrozolef, letrozolefromen, exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, d to

- 18 009807 sametazon, antide, bevacizumab (Auazyp), 5-fluorouracil, leykovarin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronate, streptozocin, rituximab (Kyihap), idarubicin, busulfan, chlorambucil , fludarabine, imatinib, cytarabine, ibritumomab (2еу11п), tositumomab (Веххаг), interferon alfa-2b, melpholam, bortezomib (Uecaye), altretamine, asparaginase, gefitinib (1Gezaa), anti-eloite , ABX-EOE), epothilones and conjugates of cytotoxic drugs ennyh drugs and antibodies against cell surface receptors.

For these combination therapies, EM164 is combined with one or more anticancer agents of various mechanisms of action, such as alkylating agents, platinum-containing agents, hormonal therapeutic agents, antimetabolites, topoisomerase inhibitors, antimicrobial agents, differentiation agents, antiangiogenic agents or anti-vascularization agents, radiation therapy, agonists and antagonists of the releasing factor of luteinizing hormone (SNA) or gonadotropin-releasing hormone (OPKN) and other chemistries therapeutic agents (Ref: Sapseg, Rgshs1r1ez & Rgasyse about £ Opso1odu, OeUya, WT, Ne1tap, 8, Kozepegd, 8.A. 6 eyyyup ^4b, Yrrepsoy-Kauep, RY1aye1rYa, 2001). In one embodiment, the combination of the LBH antide antagonist (0.1-10 micromolar) and the EM164 antibody (0.1-10 nanomolar) inhibited the proliferation of breast cancer cells MCE-7 significantly more than with either EM164 alone or one antide. In an example of combination therapy with a platinum-containing agent, this combination treatment with antibody EM164 (10 μg / ml) and cisplatin (0.1-60 μg / ml) led to a greater inhibition of the proliferation and survival of breast cancer cells MCE7 in comparison with the inhibition of either antibody EM164, or cisplatin alone.

These combinations of EM164 antibody with other therapeutic agents are effective against several types of cancers, including cancer of the breast, lung, colon, pancreas, cervix, ovary, melanoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma and osteosarcoma. The EM164 antibody and this therapeutic agent can be administered for cancer therapy either simultaneously or sequentially.

EM164 antibody conjugates with cytotoxic drugs are also valuable in targeted delivery of cytotoxic drugs to tumors overexpressing the UE-1 receptor. EM164 antibody conjugates with radioactive labels can be used in the treatment and visiography of tumors that overexpress the YE-1 receptor.

A. Effect of treatment with EM164, as the sole agent or in combination with anticancer agents, in human cancer xenografts in immunodeficient mice.

Xenografts of human non-small cell carcinoma Ca1i-6 were inoculated in immunologically deficient mice by subcutaneous injections of 1x10 ⁷ Ca1i-6 cells. As shown in figure 10, mice containing inoculated xenografts Ca1i-6 100 mm ³ were treated with one antibody EM 164 (6 injections 0.8 mg / mouse, ί.ν., twice a week) or one taxol (five injections of taxol , 1. R., every two days; 15 mg / kg) or a combination of treatments with Taxol and EM 164 antibody or one PBS (200 μg / mouse, 6 injections, twice a week, ί.ν.) using five mice per processing group. Tumor growth was significantly slowed by treatment with antibody EM 164 based on weight measurements of these mice. Although treatment with taxol alone was effective until day 14, then the tumor began to grow again. However, tumor growth was significantly delayed in the group that was treated with a combination of taxol and EM164 antibody, in comparison with the group that was treated with taxol alone.

Human pancreas xenografts were inoculated into 5-week-old female 8CGO / 1SK (Ta1cocs) mice by subcutaneous injection of 10 ⁷ BxRS-3 cells in PBS (day 0). Then, mice bearing inoculated tumors of 80 mm ³ were treated with one EM164 (13 injections of 0.8 mg / mouse, ί.ν., in the lateral tail vein, on days 12, 16, 19, 23, 26, 29, 36, 43 , 50, 54, 58, 61 and 64), one gemcitabine (two injections 150 mg / kg / mouse, 1. R. on days 12 and 19), a combination of gemcitabine and EM164 in accordance with the above administration schemes, one PBS and one control antibody (in accordance with the schemes shown for EM164) using five mice in each of the five treatment groups. As shown in FIG. 27, treatment with EM164 alone or in combination with gemcitabine led to a general regression of tumor xenografts in 4 of 5 animals in the EM164 treatment group and in all 5 animals in the combination treatment group. A measurable resumption of tumor growth was observed only in more than one animal, on day 43 in the EM164 group and on day 68 in the combination treatment group, resulting in significantly lower average tumor volumes on day 74 compared to control treatments (P = 0.029 and 0.002, respectively; two-sided T-test; Fig. 27). In another study, treatment with EM164 antibody (alone or in combination with an anti-UE-1 receptor antibody; intraperitoneal injection) inhibited the growth of grafted BxRS-3 xenografts in mice.

Murine EM164 and humanized EM164 antibodies showed equivalent inhibition of the growth of BxPC-3 grafted xenografts in mice, therefore demonstrating that the efficacy of humanized EM 164 is equivalent to that of mouse EM164 ίπ νί \ Ό. Compared to the different methods of administering the EM164 antibody, both intraperitoneal and intravenous administration of the EM164 antibody showed equivalent inhibition of the growth of xenograft BxRS-3 grafts in mice. In another xenograft study, treatment with the EM164 antibody showed a significant growth retardation in the grafted human A673 / Evang sarcoma grafted xenografts in mice.

H. Cloning and sequencing of the light and heavy chains of the EM164 antibody.

Total RNA was purified from EM164 hybridoma cells. Reverse transcriptase reactions were performed using 4-5 μg of total RNA and either oligo-bT or random hexamer primers.

PCR reactions were performed using the KACE method (rapid amplification of cDNA ends) described in Co e! a1. (1. Ittiη1., 148, 1149-1154 (1992)), and using degenerate primers described in Aapp e! a1. (1. Tttipo1. Meobob, 233, 167-177 (2000). The KACE PCR method required an intermediate step to add a poly C-tail at the 3'-ends of the first strand cDNA. The RT reaction mixtures were purified using O1apeaku columns (O1adep) and eluted in 50 μl of 1X # B buffer 4. The addition of the bC tail was performed on the eluate using 0.25 mM CoCl ₂ , 2 mM bSTP and 5 units of terminal transferase (ΝΉΒ) in 1 X ΝΉΒ buffer 4. This mixture incubated at 37 ° C for 30 min and then 1/5 of this reaction mixture (10 μl) was added directly to the PCR reaction, where it served as DNA the matrix.

CACE and degenerate PCR reactions were identical, with the exception of differences in primers and matrices. The terminal transferase reaction was used directly for the KACE-RSK matrix, while the RT reaction mixture was used directly for degenerate PCR reactions.

Both in KASE and in degenerate PCR reactions, the same 3'-primer of the light chain was used:

NTBK -! A! Adads! Saads !! dda! Dd! Dddaada! Dda! Asad !! dd! Ds (8E0 ΙΌ N0: 14) and the 3'-primer of the heavy chain: Β§12Ι ^ 1 - ddaada! S! AHadasada ! ddddd! d! sdaLddds (8Е0 ΙΌ N0: 15).

In CASE-PCR, one poly C-5'-primer was used for both the heavy and light chains:

ЕСОРо1уС - ТАТАТСТАСААТТСССССССССССССССССССС (ЛУ ΙΌ N0: 16), while degenerate 5'-terminal primers were:

8as1MK - SSSASSTSSAAUATTSTSMT8ASMSAKASTMS (LY ΙΌ N0: 17) for the light chain and an equal mixture of:

ЕСОКГМН1 - СТТССССААТТС8АКСтаМАССТС8АС8АСТС (ЛЕО ΙΌ N0: 18) and

ЕСОКГМН2 - СТТСС66ЛЛТТС8ЛК6ТХМЛ6СТ68Л68Л6ТСА66 (ЛЮ ΙΌ N0: 19) for the heavy chain.

In the above primer sequences, mixed bases are defined as follows: H = A + T + C, 8 = C + C, Y = C + T, K = C + T, M = A + C, K = A + C, A = A + T, Y = A + C + C.

PCR reactions were performed using the following program: 1) 94 ° C for 3 min, 2) 94 ° C for 15 s, 3) 45 ° C for 1 min, 4) 72 ° C for 2 min, 5) the cycle again to step No. 29 times, 6) the end with the final stage of elongation at 72 ° C for 10 minutes

PCR products were cloned into ρΒ1ικΝπρΙ ΙΙ 8K + (LpIadepe) using restriction enzymes (restrictase enzymes) created by these PCR primers.

Several individual clones of the light and heavy chains were sequenced by conventional methods for identification and in order to avoid possible errors in the polymerase generated sequence (FIGS. 12 and 13). Using the definitions of the canonical classification of Failure! WAS, the RNS of three light chains and heavy chains were identified (Figs. 12-14).

A search using the NСΒI ΙβΒΕικΙ database showed that the variable region of the light chain of the anti-JRL receptor antibody may have originated from the mouse germline gene Ι ^ νΕ Cr1, while the variable region of the heavy chain may have come from the gene of the germline Ι§ν6 1558.s (Fig. 15).

The murine EM164 antibody protein sequencing was performed to confirm the sequences shown in FIG. 12 and 13. The heavy and light chain protein bands of the purified EM164 antibody were transferred onto a ΡνΩΕ membrane from gel (SDS-PAGE, reducing conditions), excised from a ΡνΩΕ membrane, and analyzed by sequencing of the protein sequence. It was determined by Edman sequencing that the L-terminal sequence of the light chain is: PUBMT0TB8 (8E0 ΙΌ N0: 20), which corresponds to the terminal sequence of the cloned light chain gene from the EM164 hybridoma.

It was found that No. the end of the heavy chain is blocked for protein sequencing according to Edman. The peptide fragment of trypsin heavy chain cleavage with a mass of 1129.5 (M + H +, monoisotopic) was fragmented using the rock! Coes besau (Ρ8Ό), and it was determined that its sequence is SCREUSUSLH (8Е0 ΙΌ N0: 21). Another peptide fragment of tryptic cleavage of the heavy chain with a mass of 2664.2 (M + H +, monoisotopic) was also fragmented using the rock! Coes besau (P8O), and it was determined that its sequence is 888TAΥM ^^ 88 ^ T8E ^ 8АVΥΥΕΆΚ (8Е0 ΙΌ N0: 22. Both of these sequences correspond exactly to the sequences С0К3 and framework 3 (PK3) of the cloned heavy chain gene obtained from EM164 hybridoma.

I. Recombinant expression of antibody EM164.

Paired sequences of light and heavy chains were cloned into a single mammalian expression vector (FIG. 16). PCR primers for human variable sequences created restriction sites that allowed the human signal sequence to join in the cloning vector pB1ie8sprSh, and these variable sequences were cloned into a mammalian expression plasmid using the Ε ^ ΒΙ and ΒδίΧνί or NTbSh and Ara1 sites for the light chain or heavy chain, respectively (Fig. 16). The light chain variable sequences were cloned into the human 1dK constant region in the reading frame, and the heavy chain variable sequences were cloned into the human 1d1 constant region sequence. In the final expression plasmids, human SMU promoters triggered the expression of cDNA sequences of both light and heavy chains. The expression and purification of the recombinant mouse antibody EM 164 was performed in accordance with methods that are well known in the art.

Example 2. Humanized version of the antibody EM 164.

The surface reconstruction of the EM164 antibody to provide humanized versions as therapeutic or diagnostic agents was usually performed in accordance with the principles and methods described in US Pat. No. 5,639,641, as follows.

A. Prediction of the surface.

The availability of solvent residues of the variable region for a number of antibodies with decrypted structures was used to predict surface residues for the variable region of a murine antibody against the 1CP-1 receptor (EM164). The availability of amino acids for the solvent for a set of 127 files of unique antibody structures (Table 2) was calculated using the MS software package (Rebegzep e1 a1., 1994, G. 1ο1. Sit., 235, 959-973). The ten most similar amino acid sequences of the light chain and heavy chain from this set of 127 structures were determined using sequence alignment. The average solvent availability for each variable region residue was calculated and considered that positions with greater than 30% average availability were surface residues. Positions with average availability between 25 and 35% were further investigated by calculating the availability of an individual residue only for structures with two identical flanking residues.

Table 2. 127 antibody structures from the Β ^ οοкЬЬаνеη database used to predict the surface of an antibody against the 1CP-1 receptor (EM 164)

Vgokyauep structure files used for sequence predictions

2GSZ ZY1 ZIP 1aI 1aZg 43s9 4 £ ab 6 £ ab 7 £ ab 2d> 2yr 2nd 1a61 1x1 L0 £ 2kg 2] e1 2tsr 2pc 1y11 26 £ ν 2s§g 8 £ a3 1ae6 ΙΒνΙ 26Y 2P9 2 £ b4 2 £ b] 15tZ IE1 1ν & e1b2 1a4] 1s1u 1 “§е 1ue 1ueb 1ue 1P5P 1or§ 1O5r 1a] 7 1au1 1C12 1p1e 1rzk 1gt £ L3b3 1psb 1n £ b 1P £ P 1asu 1a £ y 1съу 1p1b 1pt 1pt ΙηςΒ 1tsr 1t1b 1pp 15s8 1a5 £ 1ahz 1t1b 1tra Ιιών 1ps Chb 1k5 1ke1 1ar2 1Β2 \ ν 1abd Sch Shg 11νε 1tat N§t ΐί§ι 1abO 1a £ Ιοίν Ik £ Ce1 Tsy 1 draw W1 1 boo 1aOc Put 1s1o Nag Nd 11 £ C 1ίβ £ Ιίρί 1 £ gd Ιίνο 1ack 1yp 165b 1da £ Ιββί 1st 1 £ a1 1st 1G61 1ab9 1bb 1 £ 58 1ί§ ^ν 1yg 1 £ og 16Y 1b £ b 1a31 1b £ o car NZG 16νί

B. Molecular modeling.

The molecular model of the mouse EM 164 was generated using the software package ΟχΓογ6 ΜοΕαιΓίΓ κοΓΐ decay ALM. The antibody framework was constructed from structural file for antibodies with the most similar amino acid sequences, which were 2_) e1 for the light chain and 1ps | b for the heavy chain. Non-canonical CMEAs were embedded using a search in the database of C-a structures containing non-redundant decrypted structures. The residues that lie 5A from the CMEA were determined.

C. Selection of AB person.

The surface positions of murine EM164 were compared with the corresponding positions of the sequences of human antibodies in the database KaLa1 (ΙοΠΗδοό, C. apb ^ and, T.T. (2001)

- 21 009807

Vekeagsy, 29: 205-206). Antibody database management program 8B (8eag1e 1998) was used to extract and compare surface antibody residues from natural pairs of human heavy and light chains. The surface of the human antibody with the most identical surface residues, with particular attention to positions that are within 5A of the COB, were chosen to replace the surface residues of the murine anti-ΙΟΕ-Ι receptor antibody.

Ό. PCR mutagenesis.

PCR mutagenesis was performed on the cDNA clone of mouse EM164 (see above) to construct the human EM 164 converted on the surface of the antibody (here, IIEM164). Primer sets were designed to obtain the 8 amino acid substitutions required for all tested versions of IIEM164, and additional primers were designed to alternatively create two residue substitutions within 5A (Table 3). PCR reactions were performed using the following program: 1) 94 ° C for 1 min, 2) 94 ° C for 15 sec, 3) 55 ° C for 1 min, 4) 72 ° C for 1 min, 5) cycle again to step No. 229 times , 6) the end with the final stage of elongation at 72 ° C for 4 minutes PCR products were digested with their corresponding restriction enzymes and cloned into cloning vectors pB1ie8spr1. as described above. Clones were sequenced to confirm the desired amino acid substitutions.

Table 3. PCR primers used to construct 4 humanized antibodies EM164

Primer Sequence 8Er GO ΝΟ: Ειη1641ιονν SASOTSTASASTSSSSAOOTSSAASTOOTOSAOOTSTOOOO STOAOOTOOTOAAOOSSTO 23 Tue 16411044 ^ 011 SAATSAOAAOOTTSSAOOSbOAASSSSASAS 24 Yesh] 641ΐ € ςς§ο12 SbTSSSSTOOAASgTSTOATTOTASTTAOOTAS 25 ET1641stZ SAOSTbTASASTSSOATbTTSTSATOASSSSAAASGSS 26 ESh1641S13 SAOOTSTASASTSSOATbTpTSATOASSSSAAASTSS 27 Yesh1641sr18 SASgASATSTOSAAOAOATOSASSSgSOATSTSSAAWAS 28 Et1641cb§12 TTOSAOATSTAOTSAOAOOSATASTASATAOOTAATO 29th ET164t45 BAATOOTASSgOSASAAASSASSSSSSAOOTSTSSAASSSGS STSATSTAS thirty Et164a67o11 STOSSAOOTOOAOOS6OOASAOATTTSAS 31 ET164a67o12 SAAATST6TSSSTSSTSSAST6SSASTS 32

E. Remains of the surface of the variable region.

Antibody surface remodeling methods described by Rebegkep e! a1. (1. Vu1. Siet., 235, 959-973, 1994) and Vodikka e! a1. (Progo Ep., 9, 895-904, 1996), begin by predicting the remnants of the surface of the variable sequences of mouse antibodies. The remainder of the surface is considered an amino acid that has at least 30% of its total surface area available to the water molecule.

The 10 most homologous antibodies were identified in a set of 127 antibody structure files (FIGS. 17 and 18). Solubility of each position of KaBa! were averaged for these aligned sequences, and the distribution of relative availability for each residue was as shown in FIG. 19. Both light and heavy chains have 26 residues with average relative availability of at least 30% (Fig. 19); thus, these residues are predicted surface residues for EM164. Several residues had average availability between 25 and 35%, and they were further investigated by averaging only antibodies with two identical residues flanking each side of this residue (Tables 4 and 5). After this additional analysis, the initial set of surface residues that was identified above remained unchanged.

- 22 009807

Table 4. Surface residues and average availability (cfr) for the variable sequences of light and heavy chains of antibody EM 164

Remains of surface EM 164 Light chain Heavy chain EM164 No. KaBa1 Wed dost. EM 164 No. Ka! Wed dost. ϋ one 45.89 Ω one 58.19 B 3 41.53 Ω 3 34.08 T 7 31.40 Ω 5 34.36 B nine 50.08 BUT nine 38.01 B fifteen 35.45 B eleven 47.72 0 eighteen 39.79 TO thirteen 46.51 TO 24 34.36 R 14 31.49 8 26 32.63 ABOUT fifteen 31.42 Ω 27 34.35 to nineteen 34.41 N 28 36.38 to 23 31.23 R 40 43.05 t 28 36.24 ABOUT 41 46.56 R 41 44.01 0 42 34.92 ABOUT 42 42.62 to 45 30.58 Ω 43 46.85 8 52 30.40 E 61 46.68 8 56 41.46 TO 62 44.87 ABOUT 57 42.41 TO 64 38.92 ABOUT 60 45.96 TO 65 40.06 8 67 38.20 TO 73 35.92 TO 77 42.61 8 74 48.91 E 81 38.46 8 82V 32.72 V 95E 34.83 8 84 35.21 TO 103 10/31 E 85 39.62 TO 107 36.94 ϋ 98 36.00 TO. 108 60.13 BUT 106 37.65 BUT 109 53.65 8 113 43.42

Table 5

Remains of surface boundary Light chain Heavy chain EM164 No. Ka! Wed dost. EM164 No. Ka! Wed dost. t 5 28.68 Ω 3 31.62 t 7 30.24 Ω 5 36.07 R 12 26.59 R 14 29.88 about sixteen 25.20 ABOUT fifteen 30.87 about 17 25.73 8 17 25.64 8 twenty 25.37 TO nineteen 35.06 TO 24 36.73 TO 23 31.48 8 26 31.00 ABOUT 26 30.53 <2 27 32.29 8 31 12/27 8 27A 29.78 TO 56 ΝΑ V 27C 05/29 t 68 27.71 V 29th ΝΑ t 70 24.65 Ω 42 34.92 8 75 18.80 to 45 32.24 8 82V 32.87 8 52 02/30 R 97 ΝΑ TO. 54 29.50 Υ 99 ΝΑ ϋ 70 03/26 V 103 ΝΑ TO. 74 ΝΑ t 111 25.95 E 79 26.64 BUT 80 29.61 V 95E 42.12 ABOUT one hundred 29.82 TO 103 10/31 E 205 25.78

Residues that have an average availability between 25% and 35% were further analyzed by averaging a subpopulation of antibodies that have two identical residues flanking each side of the residue in question. NA denotes residues that do not have identical flanking

- 23 009807 residues in the 10 most similar antibodies.

E. Molecular modeling to determine which residues fall within 5A of the RNS.

The molecular model described above, generated using the ABM software package, was analyzed to determine what remnants of the EM164 surface were within 5A of the RNS. To remodel the surface of the murine EM164 antibody, all surface residues that are outside the RNC must be replaced with human copies, but residues within 5A of the RNC were treated with extreme caution, as they can also contribute to antigenic specificity. Thus, these last vestiges must be identified and should be treated with caution during the entire process of humanization. The definitions of RNS used to remake the surface combine the ABM definition for RNS 2 of the heavy chain and the definition of KaLa! for the remaining 5 JUICE (Fig. 14). Tab. 6 shows residues that are within 5 A of any RNS residue in the sequence of either the light or heavy chain of the EM164 model.

Table 6. The remains of the surface of the frame of the antibody EM164 within 5A of the RNS

Light chain Heavy chain W T28 thirteen K73 T7 574 P40 6) 42 K45 057 060 E81

C. Identification of the most homologous surfaces of human antibodies.

Candidate surfaces of human antibodies for reshaping the EM164 surface were identified within the KaLa !, antibody database using 8K software, which provided a search for only the indicated positions of residues against this database. To preserve natural pairs, surface residues of both light and heavy chains were compared together. The most homologous human remains from the KaBa database! matched in a ranked order of sequence identity. The top 5 surfaces are given in table. 7. Then these sequences were compared to identify which ones might require the smallest substitutions of 5 A from the SEC. The anti-leukemia B-cell antibody, Cb 1.69, required the smallest number of substitutions of surface residues (10 in total), and only two of these residues were within 5 A of the CEC.

The full-length variable region sequence for EM164 was also compared against the KaLa !, database, and Cb 1.69 was again identified as the most similar human variable region sequence. Together, these sequence comparisons identified an antibody of leukemic B cells Bb 1.69 as the preferred choice as a human surface for EM164.

Table 7. The top 5 human sequences extracted from the KaLa database!

5 most homologous surfaces of human antibodies Antibody Light chain 8Ef GO N0: MiEM164 0ТЬб} Р0 <) К <Э08К. ЕККЫ.А 33 SE69 Т Util ΡΟςΚΟϋΑΚΕΚΚΚ- 34 M8b5 ϋ f8 b I R P69K0O8V.OKKV.A 35 COP571 OM88UKRO9KO888OKKV.- 36 BSARV EUZORKROfyOOZKEKKV- 37 88ЪРВ E U8 0 RIR0 () K0O8KEKKk- 38 Antibody Heavy chain 8Ε (} Ι0Ν0: MiEM164 0 IFAKROKKT ROFEKKKK888EA8 39 SI .69 0 FUAUKROKKTROFOKOFOK888Er8 40 M8b5 P ffr LKROKKT RΟΚΟϋΚΟΤ8ΝΝΕ08 41 COP571 0 0νΑνΚΡΟΚΚΤΡΟ00ΚΚΟΚ888Ε08 42 BSARV - FUAUKROKKTROFFKOOKZ88Ef8 43 88РРВ - C) V A V K P O KT P 0 ζ) f K ¢) O E 8 8Ef8 44

Comparisons were obtained using 8K (Rebegzep 1993). Remains of the EM164 surface, which are within 5 A of the SEC, are underlined.

H. Construction of humanized EM164 genes.

Ten substitutions of surface residues for EM164 (Table 7) were made using PCR mutagenesis methods as described above. Since eight of the surface residues for Cbb 1.69 were not within 5 A from the CEC, these residues were replaced from mouse to human in all versions of humanized EM164 (Tables 8 and 9). Two residues of the surface of the light chain, which were within 5 A from the SEC, (positions KaLa! 3 and 45), were either replaced by human ones or left as murine. Together, these options generate four humanized versions of EM164 that were designed (Figs. 22 and 23).

Of these four humanized versions, version 1.0 has all 10 human surface residues. The most conservative version for substitutions close to SIV is version 1.1, which retained both of the mouse surface residues, which were within 5 A from the SIV.

All four humanized EM 164 antibodies were cloned into an antibody expression plasmid (FIG. 16) for use in transient and stable transfections.

Table 8. Residue substitutions for versions 1.0-1.3 of the humanized antibody EM164 __________________ | Changes in all versions

Light chain: tyts! 8 in R&D 18; ti867 in yiA67

Heavy chain ty (25 in Cu5; Ti1 in NiVI1; TiE611 ° Ci61; TiK641i NiC) 64;

TiB65 in AIs65; TIAJUB in O&O 106

LiM164 changes

Amino Acid 45 Light Chain Amino Acid 3 Light appreciate Total Mi Neither shi yi Mouse leftovers νΐ.0 V TO 0 νΐ.1 B TO 2 νΐ.2 B IN one νΐ.3 V TO one

Ι. Comparison of the affinities of the humanized versions of the EM164 antibody with the affinities of the murine EM164 antibody with respect to binding to the full-sized JU-Ereceptor and the shortened alpha chain of the UE-Ereceptor.

The affinities of the humanized versions 1.0-1.3 of the EM164 antibody were compared with the affinities of the mouse EM164 antibody using competitive binding assays using the biotinylated full-sized human JU-Ereceptor or the labeled tus-epitope tag of the shortened JU-Ereceptor alpha chain described above. Samples of humanized EM164 antibodies were obtained by transient transfection of suitable expression vectors in 293T cells of a human embryonic kidney, and antibody concentrations were determined using an ELISA using purified humanized antibody standards. In order to measure competitive binding with ЕЗЫ, the mixture of samples of humanized antibodies and various concentrations of mouse antibodies ЕМ164 was incubated with a bitinylated full-sized Ι6Ε-receptor or a tus-epitope labeled truncated SE-Ereceptor alpha-chain labeled by indirect method. After equilibration, bound humanized antibody was detected using a conjugate of goat anti eab _'2 human horseradish peroxidase. Graphs ([bound mouse AB] / [bound humanized AB]) against ([mouse AB] / [humanized AB]), which give a theoretically straight line with a slope = (K _from humanized ab / Kv mouse ab), were used to determine the relative affinities of humanized and murine antibodies.

An example of competitive analysis is shown in FIG. 11. Itshi1οη-2HB EYZA plate was coated with 100 μl of 5 μg / ml streptavidin per well in carbonate buffer at ambient temperature for 7 hours. Streptavidin-coated wells blocked 200 μl of blocking buffer (10 mg / ml BSA in TBZ-Buffer) in for 1 h, washed with TBZ-T buffer and incubated with a biotinylated Ι6Ε-цеп receptor (5 ng per well) overnight at 4 ° С. Then, the wells containing the indirectly captured biotinylated UE-receptor were washed and incubated with mixtures of the humanized EM164 antibody (15.5 ng) and mouse antibody (0 ng or 16.35 ng or 32.7 ng or 65.4 ng or 163.5 ng) in 100 μl of blocking buffer for 2 hours at ambient temperature and then incubated overnight at 4 ° C. The wells were then washed TΒ3-T buffer and incubated with the conjugate goat anti eab _'2 Human-horseradish peroxidase for 1 hour (100 l; 1 .mu.g / ml in blocking buffer), followed by washes and detection using ΑΒT3 / Η ₂ substrate O ₂ at 405 nm.

The graph ([linked mouse AB] / [linked humanized AB]) against ([mouse AB] / [humanized AB]) gave a straight line (g ² = 0.996) with a slope (= K _{C of the} humanized ab / K .. .. _AB ) 0.52. Thus, version 1.0 of the humanized antibody bound to the Ι6Ε-Ι receptor more strongly than the murine EM164 antibody. Similar values for a gradient in the range of about 0.5 to 0.8 were obtained for competition between versions 1.0, 1.1, 1.2, and 1.3 of humanized EM164 antibodies with murine EM164 antibody for binding to the full-length Ι6Ε-Ι receptor or to the shortened alpha chain of UE -Receptor, which indicated that all of these humanized versions of the EM164 antibody had similar affinities, which were all better than the affinity of the original mouse EM164 antibody. The chimeric version of the EM164 antibody with a 92E ^ C mutation in the heavy chain showed a slope of approximately 3 in a similar competition for binding to the murine EM164 antibody, indicating

- 25 009807 that the mutant 92E ^ C EM164 had 3 times lower affinity than the murine antibody EM164 with respect to the binding of the UE-1 receptor. The humanized EM164 ν1.0 antibody showed similar inhibition of the UE-1-stimulated growth and survival of MCE-7 cells compared to the murine antibody EM 164 (Fig. 24). Inhibition of serum-stimulated growth and survival of MCE-7 cells with the humanized EM164 ν1.0 antibody was similar to inhibition with the murine EM164 antibody.

Table 9

Segment Light chain Heavy chain RK1 1 -23 (with a random residue at 0 and a deletion at 10 in the chains ν _λ ) 1-30 (with a random remainder at 0) JUICE! 24-34 (with possible insertions numbered as 27A, B, C, ϋ, E, G) 31-35 (with possible insertions, numbered as 35A, B) RK2 35-49 36-49 SOK2 50-56 50-65 (with possible insertions, numbered as 52A, B, C) gkz 57-88 66-94 (with possible insertions numbered 82A, B, C) SPK 89-97 (with possible insertions numbered 95A, B, C, ϋ, E, G) 95-102 (with possible insertions, numbered as 100A, B, C, ϋ, E, G, O, Η, I, ί, K) RK4 98-107 (with a possible insertion, 103-113

numbered as 106A)

We used the KaBa numbering system! for polypeptides of the variable regions of the light and heavy chains of various versions of EM 164 AB. Amino acid residues are grouped into framework (EC) and complementarity determining regions (SICs) in accordance with the position in the polypeptide chain.

Taken from KaLa! e! A1., 8ec. | епepse5 Г Proc. ΕίΓιΗ Εάίίίοη, 1991, и РиЬйсаΐΐοη No. 91-3242.

1. A method of providing improved antibodies against the SE-1 receptor based on the sequences of the mouse and humanized antibodies described herein.

The amino acid and nucleic acid sequences of the anti-JU-1 receptor EM 164 antibody and its humanized versions were used to develop other antibodies that had improved properties and which are also within the scope of this invention. Such improved properties include increased affinity for the SE-1 receptor. Several studies have examined the effects of introducing one or more amino acid substitutions at different positions in an antibody sequence, based on knowledge of the sequence of the primary antibody, on their properties, such as binding and expression level (Wapd, ^ .P. E! A1., 1995, 1. Mo1. Wu1, 254, 392-403; Kayeg, S e! A1., 1998, Proc. No. 1. Asai. 8c1. I8A, 95, 8910-8915; Waidap, T.1. E! A1., 1998 , No. 11 and Vue! Esbpoodu, 16, 535-539).

In these studies, primary antibody variants were obtained by changing the sequences of the heavy and light chain genes in the CICT, CIC2, CEE3 or frame regions using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutator strains E. soi (Waidap, T.1. E! A1., 1998, No. 11 hey! Esbpoodu, 16, 535-539; Ayeu, Ν.Β. e! A1., 1996, Chap! 16, pp. 277 -291, w Räde E18r1au oG Reryez apy Rgo! Et8, Eis. Kau, V.K. e! A1., Asayetu Rgezz). These methods of changing the sequence of the primary antibody have led, through the use of standard screening methods, to improved affinities of such secondary antibodies (Cgat, H. e! A1, 1992, Proc. No.! 1. Asai. 8cE I8A, 89, 35763580; Voyeg, E. That is, ea1., 2000, Proc. No. 11. Asai. 8cE I8A, 97, 10701-10705; ^ aν ^ e8, 1. apy Ktestapp, b., 1996, 1tipododu, 2, 169-179; 1. e! A1., 1996, 1. Mo1. Vu1., 256, 77-88; 8Boy, M.K. e! A1., 2002, 1. Vu1. Sb., 277, 16365-16370; Eigika ^ a, K. e! a1., 2001, 1. Vu1. Sb., 276, 27622-27628).

Using a similar directed strategy for changing one or more amino acid residues of an antibody, the antibody sequences described in this invention can be used to develop antibodies against the UE-1 receptor with improved functions, for example, antibodies having suitable groups such as free amino groups or thiols, at convenient attachment points for covalent modification, for example, for use in the attachment of therapeutic agents.

K. An alternative expression system for murine, chimeric and other antibodies against the UE receptor.

A murine anti-JU-1 receptor antibody was also expressed using mammalian expression plasmids similar to those used to express a humanized antibody (see above). Expressing plasmids are known to have murine constant regions, including the sequences of the Kappa light chain and the gamma-1 heavy chain (McLeap e! A1., 2000,

- 26 009807

Mo1 ^ type 37, 837-845). These plasmids are designed to accept any variable region of an antibody, for example, a murine anti-YP-L receptor antibody, by simple restriction enzyme digestion and cloning. Usually, additional PCR of this anti-UR receptor antibody is required to create a restriction compatible with the restriction in this expression plasmid.

An alternative approach for the expression of a fully murine anti-JuP-receptor antibody is to replace human constant regions in a plasmid expressing a chimeric anti-JuP-receptor antibody. This chimeric expression plasmid (FIG. 16) was constructed using cassettes for the variable regions and for the constant regions of both light and heavy chains. Just as the variable sequences of an antibody were cloned into this expression plasmid by restriction enzyme digestions, individual restriction enzyme digestion products were used to clone in any constant region sequences. For example, kappa light chain cDNA and gamma-1 heavy chain cDNA were cloned from murine hybridoma RNA, such as RNA, described above for cloning variable regions of an anti-JP-receptor antibody. Similarly, suitable primers were constructed from the sequences available in the KaLa1 database (see Table 10). For example, RT-PCR was used to clone the constant region sequences and to create the restriction sites necessary for cloning these fragments into a plasmid expressing a chimeric anti-YRL receptor antibody. This plasmid was then used to express a fully murine anti-JR-L receptor antibody in standard mammalian expression systems, such as CHO cell line.

Table 10. Primers designed for cloning the mouse constant region of gamma-1 and the mouse constant region of kappa, respectively

Mouse constant region primers Primer Name Primer sequence 8ΕΟΙΟΝΟ: ΜιιΙβΟΙ SZspbH TTTTOAOOSTSTTATTTASSASAOAOASTOSTAOAO OOSTSTG 45 MiTsO1 S5epbN TTTTAAOSTTSSSSAAAASSASASSSSSSATSTSTSTAT 46 MiCKar SzepbV TTTTOOOOSTSAAASASTSATTSSTOTTSAAOS 47 Mi! §Kar S5ep <1E TTTT6AATTSOOSST6AT6STSSASSASSAASTO 48

These primers were designed on the basis of the sequences available in the database KaBa1 Tsbkkop, 6. apb Au, T.T. (2001) Exodus Aslbk Kekeagsy, 29: 205-206).

Deposit Statement

The hybridoma producing the EM164 mouse antibody was deposited with the American Type Culture Collection, PO Boch 1549, Mapakkak, UA 20108, on June 14, 2002, according to the Budapest Treaty, and received the ATCC access number (ATCC accessory) PTA-4457.

In this description reference has been made to some patents and printed publications, the contents of which, in each case, are incorporated herein by reference in their respective full form.

Although the invention has been described in detail and with reference to its specific embodiments, a person skilled in the art will understand that various changes and modifications can be made therein without deviating from its idea and scope.

Claims (31)

CLAIM 1. A composition comprising: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody or an epitope binding fragment thereof, and wherein said antibody or said fragment specifically binds to an insulin-like growth factor receptor Ι selected from the group consisting of: (ί) an antibody or an epitope-binding fragment thereof having the same amino acid sequence as a mouse EM164 antibody produced by a mouse hybridoma EM164 (ATCC accession number PTA-4457), (ίί) a surface-remodeled antibody or an epitope-binding fragment thereof having the same the most specific binding as the murine antibody EM164, (ίίί) a human or humanized antibody or epitope-binding fragment thereof having the same binding specificity as the murine antibody EM164, (ίν) functional equiv. the valent of the antibody or its epitope-binding fragment having the same binding specificity as the mouse antibody EM164, (ν) variant of the mouse antibody EM164 or its epitope-binding fragment having at least one mutation, deletion or insertion of a nucleotide in comparison with the murine antibody EM164 and having the same binding specificity as the mouse antibody EM164, and (νί) the mouse antibody EM164 produced by the mouse hybridoma EM164 (access number ATCC PTA-4457), or its epitope-binding fragment, and (b) a second therapeutic sky agent. - 27 009807 2. The composition according to claim 1, where the specified second therapeutic agent is selected from the group consisting of such therapeutic agents as docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Negersipe), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant , exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Аνа8ΐ ^ η), 5-fluorouracil, leucovarin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorostinrethrozenterbene, elastroinzartetin, ebrinetrozeltarbin, emetronetrostanetrobin, emetronetrostanetrobin, mitroceltrostanetrotin, emetronetrostanetrobin, mitroceltrastin, citronetrozarpetin, ebrinetrozeltarbin, ebrinetrozeltarbin, ebronetrozeltarbin, emetropin qing, rituximab (Kyihap), idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine, ibritumumab (Behav1 ^ η), tositumomab (Wehhag), interferon alfa-2b, melphalam, bortezabecamine, (aspetzeminum), asparzamin, amphibia, asparabimabe, tezibecinum, asparabenum, amphibian , erlonitib (Ta ^ ceva), an antibody to the ECE receptor (Cehhkta, ABx-ECE) and epothilone. 3. The composition according to claim 1, where the specified second therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine and camptothecin. 4. The composition according to claim 1, where the specified first therapeutic agent is administered to the patient at a dose of about 1 to about 2000 mg / sq. And where the specified second therapeutic agent is administered at a dose of about 10 to about 2000 mg / sq. m 5. The composition according to claim 1, where the specified first therapeutic agent is administered to the patient at a dose of about 10 to about 1000 mg / sq. m and where the specified second therapeutic agent is administered at a dose of about 50 to about 1000 mg / sq. 6. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier or diluent. 7. A composition comprising: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody or an epitope-binding fragment of an antibody comprising at least one complementarity determining region having an amino acid sequence selected from the group consisting of 8ΥΨΜΗ ΕΙΝΡ8ΝΟΚΊΝΥΝΕΚΡΚΚ. ΟΚΡΌΥΥΟδδΚΧνΥΡϋν Κ88ζ) 8ΓνΗ8ΝνΝΤΥΙ, Ε Κν8ΝΚΡ8 ΡφΟδΗΥΡΡΤ (5ES2 GO N0: 1), (8Er GO N0: 2), (8Ε0 GO N0: 3), (8ΕρΐϋΝΟ N0: 5), (8EO GO N0: 5) and (8Ε9№ΝΟ: 6) η (b) the second therapeutic agent, where, when 8Eff ΙΌ N0: 5 is selected, said antibody or antibody fragment specifically binds to an insulin-like growth factor receptor Ι (UE-U). 8. A composition comprising: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody or an epitope-binding fragment of an antibody comprising at least one variable region of the heavy chain and at least one variable region of the light chain, wherein said variable region of the heavy chain contains three consecutive complementarity determining regions having the amino acid sequence represented by 8Ef ΙΌ N0: 1-3, respectively: 8ΥΑΥΜΗ (8ΕφΙΟΝΟ: 1), ΕΙΝΡ8Ν0ΚΤΝ ΥΝΕΚΡΚΚ 18Е0 GO N0: 2), ΟΚΡΟΥΥΟ88Κ \ νΥΡΏν (8Ер ГО N0: 3), and where the indicated variable region of the light chain contains three consecutive complementarity determining the region having the amino acid sequences represented by 8Еф ΙΌ N0: 46, respectively: ΚδδςδίνΗδΝνΝΤΥί, Ε (8Е0 GO N0: 4), ΚνδΝΚΡδ (8Е0 GO N0: 5), ROSNURRT (8ΕΓ) ΙΟΝΟ: 6) π (b) is the second therapeutic agent. 9. A composition comprising: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody or an epitope binding fragment thereof, said antibody or said fragment comprising a variable region of a heavy chain that has at least 90% sequence identity with respect to the amino acid sequence represented by 8Ef ф N0: 7: (.) U01.908SLEEUKROA8UKG.8 SKAKSUP-! 8UCH ⁷ MNS ^g K () KRSOSYEETOE1 ΝΡ8ΝαΚΤΝΥΗΕΚΡΚΚΚΑΤΕΤν0Κ888ΤΑΥΜ0Ε88ΕΤ8ΕΟ8ΑνΥΥΡΑΚ0ΚΡΟΥ ΥΟ88Κ5νΥΡθνΐνθΑΟΊΤντν88 (8EC GO N0: 7), and (b) a second therapeutic agent. 10. The composition according to claim 9, where the specified variable region of the heavy chain has at least - 28 009807 95% sequence identity with respect to the indicated amino acid sequence represented by WE / ΙΌ N0: 7. 11. The composition according to claim 9, where the specified variable region of the heavy chain has an amino acid sequence that is represented by WE / N0: 7. 12. A composition comprising: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody or an epitope-binding fragment thereof, said antibody or said fragment containing a light chain variable region that has at least 90% sequence identity with respect to the amino acid sequence represented by CE / N0: 8: ОУМТТТР8ЬРУ8ЬООРА818СК88р81УН8ПУМТУЬЕ1 ¥ УррКР098РКШ ¥ Κν8ΝΚΓ80νΡ0ΚΓ8Ο8080Τ0ΡΤΕΚ.Ι8Κ.νΕΑΕ0Ε <3ΙΥΥ0Ρρ08ΗνΡΡΤΡ €> Ο0ΤΚ L1Q. (8E0, 10N0: 8), and (b) a second therapeutic agent. 13. The composition of claim 12, wherein said light chain variable region has at least 95% sequence identity with respect to said amino acid sequence represented by WE / ΙΌ N0: 8. 14. The composition of claim 12, wherein said variable region of the light chain has an amino acid sequence that is represented by 3E / ΙΌ N0: 8. 15. A composition comprising: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody or an epitope binding fragment thereof, said antibody or said fragment containing a variable region of a light chain having a sequence selected from the group consisting of: θννΜΤ4ΤΡΕ8ΕΡν8ΕΟΏΡΑ8Ι80Κ8808ΐνΗ8ΝνΝΤΕΕΐνΥΕ0ΚΡ608ΡΡΕΕΙΥ Κν8ΝΚΓ8σνΡΟΚΡ8Ο8ΟΑΟΊΌΡΤΕΚΙ8ΚνΕΑΕΟΕΟΙΥΥ0Ρ0Ο8ΗνΡΡΊΤΟΟ0ΤΚΕΕΙΚ K. (8Εζ) GO N0: 9); θνΕΜΤ0ΤΡΕ8ΕΡν8ΕΟΏΡΛ8Ι8ΟΚ8808ΐνΗ8ΝνΝΤΥΈΕ \ νΥΕ0ΚΡ608ΡΚΕΕΙΥ Κν8ΝΚΡ8ανΡΟΚΡ8Ο8ΟΑΟΤΟΡΤΕΚΙ8ΚνΕΑΕΠΕΰΙΥΥ0Ρ0Ο8ΗνΡΡΤΓαα6ΤΚΕΕΙΚ К (8Е0 ГО N0: 10); θνΕΜΤ0ΤΡΕ8ΕΡν8ΕΟ “ΡΛ8Ι8ΟΚ8808ΐνΗ8ΝνΝΤΥΓ.ΕΨΥΕ0 ^; 08ΡΚΕΕΙΥ Κν8ΝΚΡ8θνΡΟΚΡ8Ο8ΟΑΟΊΌΡΤΕΚΙ8ΚνΕΑΕΟΕΟΙΥΥ0Ρ0Ο8ΗνΡΡΤΡΟΟΟΤΚΕΕΙΚ К (8Е (Э ГО N0: 11); ΠννΜΤ0ΊΤΕ8ΕΡν8ΤΌΠΡΛ8Ι8ΟΛ8808ΐνΗ8ΝνΝΤΥΕΕν7ΥΕΟΚΡΟ08ΡΚΕΕΙΥ Κν8ΝΚΡ8θνΡΓ ^ ΚΡ8Ο8ΓιΑΟΤΟΓΤΙΚΙ8Τ <νΕΑΕηΐ.0ΤΥΥ0Ε0Ο8ΠνΡΡΤΕΟΟΟΤΚΕΕ1Κ К (8Е0 ГО N0: 12), and (b) a second therapeutic agent. 16. A composition comprising: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody or an epitope-binding fragment thereof, said antibody or said fragment containing a variable region of a heavy chain having the sequence represented by CE / N0: 13: 0νρΕν08ΟΑΕννΚΡΟΑ8νΚΕ80ΚΑ8ΟΥΤΡΤ8ΥΨΜΗ \ ννΚ0ΚΡΟ0ΟΕΕλνΐΟΕΙ ΝΡ8ΝΟΚΤΝΥΝ9ΚΡ9ΟΚΑΤΕΤνθΚ888ΤΑΥΜ0Ε88ΕΤ8ΕΟ8ΑνΥΥΡΑΚ0ΚΡΟΥ Υ088ΚννΥΗ0ν \ ν090ΤΤντν88 (8Εζ) ГО N0: 13), and (b) the second therapeutic agent. 17. The composition according to any one of claims 7-16, wherein said second therapeutic agent is selected from the group consisting of docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (NegersII), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab. (AuakIi), 5-fluorouracil, leucovarin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronate, streptozocin, rituximab, ylaihambin, ibidambhambin, ibidambhambin, ibidambhambin, ibidebamide, ibidebam, arabicamide, ibido (2еу1ш), tositumomaba. (Wehhag), interferon alfa-2b, melphalam, bortezomib (Ve1sabe), altretamine, asparaginase, gefitinib Zhecka), erlonitib (Tagseua), antibodies against the ECE receptor (Ce! Ichgut, ABx-ECE) and epotine. 18. A composition according to any one of claims 7-16, wherein said second therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine and camptothecin. 19. A method of inhibiting the growth of cancer cells, comprising contacting said cell with a composition according to claim 1. 20. A method of treating a patient having cancer, comprising administering to said patient an effective amount of a composition according to claim 1. 21. A method of treating a patient having cancer, comprising administering to said patient an effective amount of a pharmaceutical composition according to claim 6. 22. The treatment method according to any one of claims 19-21, wherein said cancer is a cancer selected from the group consisting of breast cancer, colon cancer, ovarian cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial cancer, pancreatic cancer, melanoma, multiple myeloma, neuroblastoma, and rhabdomyosarcoma. 23. A kit containing: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody having the same amino acid sequence as the mouse EM164 antibody produced by the mouse hybridoma EM164 (ATCC accession number PTA-4457), or an epitope-binding fragment thereof, wherein said antibody or said fragment specifically binds to an insulin-like growth factor I receptor, (b) a second therapeutic agent and (c) instructions for use. 24. A method of inhibiting the growth of cancer cells, comprising contacting said cell with: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody having the same amino acid sequence as a murine EM164 antibody produced by mouse hybridoma EM164 (ATCC accession number PTA-4457), or an epitope-binding fragment thereof, wherein said antibody or said fragment specifically binds to an insulin-like growth factor I receptor I, (b) a second therapeutic agent. 25. A method of treating a patient having cancer, comprising administering to said patient an effective amount: (a) a first therapeutic agent, wherein said first therapeutic agent is an antibody having the same amino acid sequence as a murine EM164 antibody produced by mouse hybridoma EM164 (ATCC accession number PTA-4457), or an epitope binding fragment thereof, wherein said antibody or said fragment specifically binds to the receptor of an insulin-like growth factor I, (b) of a second therapeutic agent. 26. The method according to paragraph 24, where the specified cell is in contact with the specified first therapeutic agent and the specified second therapeutic agent at the same time. 27. The method according to paragraph 24, where the specified cell is in contact with the specified first therapeutic agent and the specified second therapeutic agent sequentially and in any order. 28. The method according A.25, where the specified first therapeutic agent and the specified second therapeutic agent is administered simultaneously. 29. The method of claim 25, wherein said first therapeutic agent and said second therapeutic agent are administered sequentially and in any order. 30. The method according to paragraph 24 or 25, where the specified second therapeutic agent is selected from the group consisting of such therapeutic agents as docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Negsserip), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Aνa5ΐ ^ π), 5-fluorouracil, leykovarin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix, zoledronate , strept ozocin, rituximab (Kyihap), idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine, ibritumum tozitumomab (Vechkhag), interferon alfa-2b, melpholam, bortezomib (Uelcaye), altitrate (tibeta, aminobenzoate), altrete ^ ceva), anti-EOE-receptor antibody (Cehhit, ABx-EOE) and epothilone. 31. The method according to paragraph 24 or 25, where the specified second therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine and camptothecin.