KR20140125351A - Anti-cd98 antibodies and methods of use thereof - Google Patents

Abstract

The invention provides antibodies that bind to CD98, and methods of using the antibodies in the diagnosis and treatment of cancer.

Description

ANTI-CD98 ANTIBODIES AND METHODS OF USE THEREOF < RTI ID = 0.0 >

The present invention generally relates to anti-CD98 antibodies and methods of using such antibodies.

CD98 (CD98 heavy chain; 42F heavy chain; also referred to as SLC3A2) is a type II transmembrane glycoprotein composed of 529 amino acid residues. This protein contains a C-terminal extracellular domain consisting of a cytoplasmic domain in N-terminal cells consisting of 75 amino acids, a single transmembrane domain, and 426 amino acids (Parmacek et al., Nucleic Acids Res. 17: 1915-1931 , 1989). CD98 is covalently linked to one of several light chains (SLC7A5, 6, 7, 8, 10 or 11) which are L amino acid transporters through disulfide bonds. This interaction is required for light chain cell surface expression and amino acid transport function. CD98 also associates with integrin subunits to regulate integrin beta signaling, which regulates cell proliferation, survival, migration, and epithelial adhesion / polarity (Cai et al., J. Cell Sci. 118: 889-899, 2005) .

CD98 was initially identified as a cell surface antigen associated with lymphocyte activation (Haynes et al., J. Immunol. I26: 1409-1414, 1981). CD98 has since been identified in all types of cells except platelets and is expressed at the highest levels in the gastrointestinal (GI) tract and the renal tubule (Verrey et al., Pflugers Arch. 440: 503-512, 2000). Upregulation of CD98 was observed in intestinal inflammation. Recently, it has been shown that intestinal CD98 expression plays an important role in regulating homeostatic and innate immune responses in the intestine. Thus, modulation of CD98 expression in intestinal epithelial cells has been suggested as an expected therapeutic strategy for the treatment and prevention of inflammatory bowel disease, such as inflammatory bowel disease (IBD) and colitis-related cancer (Nguyen et al., J. Clin Invest., 121: 1733-1747, 2011). In addition, CD98 is overexpressed on the cell surface of almost all tumor cells regardless of the origin (Itoh et al., Jpn. J. Cancer Res. 92: 1313-1321, 2001).

Increased expression of L-amino acid transporter 1 (LAT1; also known as SLC7A5), one of the light chains that bind to CD98, is associated with many types of human, including breast, colon, ovarian, ovarian, Cancer cells (Fan et al., Biochem. Pharmacol. 80: 811-818, 2010). Increased amino acid supply may be required to provide an amino acid building block for protein synthesis and to support the high growth rate of cancer cells by stimulating growth through the mammalian target (mTOR) of rapamycin (Fan et al., Supra ; Imai et al., Anticancer Res. 30: 4819-4828, 2010). Expression of LAT1 and CD98 is significantly higher in the metastatic site of human cancer than in the primary site suggesting that overexpression of LAT1 / CD98 may be essential for the progression and metastasis of human cancer. In particular, overexpression of LAT1 / CD98 appears to be required for tumor metastasis in patients with colon cancer (Kaira et al., Cancer Sci. 99: 2380-2386, 2008).

Expression patterns and function of CD98 and LAT1 suggest that these proteins are expected targets for the treatment of various human cancers. Inhibitors of LAT1 activity include, but are not limited to, non-small cell lung cancers (see Imai et al., Oda et al., Cancer Sci. 101: 173-179, 2010), oral cancer cells (Kim et al., Biol. Pharm LAT1 has been shown to have antitumor activity in many types of cancer including breast cancer cells (Shennan and Thomson, Oncol. Rep. 20: 885-889, 2008) (Fan et al.)). ≪ / RTI >

Murine monoclonal antibodies to CD98, identified as HBJ127, inhibit lymphocyte proliferation (Yagita and Hashimoto, J. Immunol. 136: 2062-2068, 1986) and inhibit the growth of bladder tumors and lymphoma cells (Yagita et al. , Cancer Res. 46: 1478-1489, 1986). The epitope for the HJ127 antibody was found to be residue ₄₄₂ AFS ₄₄₄ of human CD98 (Itoh et al., 2007). Different murine monoclonal antibodies to CD98 have been shown to significantly inhibit tumor cell growth in vitro in glioma, prostate, and colon cancer cells (Papetti and Herman, Am. J. Pathol. 159: 165-178, 2001). Additional monoclonal antibodies to human CD98 are disclosed in U.S. Patent Publication No. 20100143367. These monoclonal antibodies bind to an epitope within the amino acid region 372-530 or 104-371 of CD98. Five of these antibodies have been found to inhibit amino acid uptake in bladder cancer cells and three of these antibodies have been shown to inhibit tumor growth in mouse models.

As described herein, a patient's fresh primary acute myeloid leukemia (AML) tumor sample is analyzed using surface tagged antigen profiling (sTAg) of cell surface proteomes to determine whether the dermal protein CD98 is present at high density on the surface of AML tumor cells . Thus, CD98 is a target for the treatment of AML, for example, by the use of binding agents, such as antibodies specifically binding to CD98. In addition, specific binding agents for CD98, such as anti-CD98 antibodies, can be used to treat AML as well as various cancers such as sarcoma, lymphoma, non-small cell lung cancer (NSCLC) and colorectal cancer in a variety of in vivo xenograft models ≪ / RTI >

The present inventors provide antibodies against CD98 useful for the diagnosis and treatment of various types of human cancers.

Using a high-resolution solution-based liquid chromatography-coupled in-line mass spectrometry (LC-MS / MS) after labeling in the solution of intact AML tumor cell surface, the majority Lt; RTI ID = 0.0 > AML < / RTI > cell subtypes. Thus, the present invention provides methods of using such antibodies in the treatment of anti-CD98 antibodies and AML and other cancers including but not limited to lymphoma, sarcoma, non-small cell lung cancer and colorectal cancer.

In one embodiment, the invention provides an isolated antibody or functional fragment thereof that specifically binds human CD98, wherein said antibody or functional fragment comprises residues A377, D397, I398, G400 and A401 of said human CD98 Lt; / RTI > In some embodiments, the epitope further comprises residues D374 and L378 of human CD98. In some embodiments, the epitope further comprises residues P379 and G380 of human CD98. In some embodiments, the epitope further comprises residues F395 and P396 of human CD98. In some embodiments, the epitope further comprises residues Q381, P382 and P399 of human CD98. In some embodiments, the epitope further comprises any one or more additional residues selected from the group consisting of D374, L378, P379, G380, Q381, P382, F395, P396 and P399 of human CD98.

In one embodiment, the invention provides an isolated antibody or functional fragment thereof that specifically binds human CD98, wherein the antibody binds to an epitope comprising residues P379, G380, D397 and I398 of human CD98. In some embodiments, the epitope further comprises residues F395 and P396 of human CD98. In some embodiments, the epitope further comprises residues Q381, P382, P399, G400 and A401 of human CD98. In some embodiments, the epitope further comprises residues D374, A377 and L378 of human CD98. In some embodiments, the epitope further comprises any one or more additional residues selected from the group consisting of D374, A377, L378, Q381, P382, F395, P396, P399, G400 and A401 of human CD98.

In some embodiments, the invention provides an isolated antibody that binds to an epitope comprising residues D374, A377, L378, P379, G380, Q381, P382, F395, P396, D397, I398, P399, G400 and A401 of human CD98 or And provides functional fragments thereof.

In some embodiments, the invention provides an isolated antibody or functional fragment thereof that specifically binds human CD98, wherein the antibody or functional fragment binds to an epitope contained within amino acid residues 369 to 405 of human CD98. In some embodiments, the invention provides an isolated antibody or functional fragment thereof that specifically binds human CD98, wherein said antibody or functional fragment binds to an epitope consisting of amino acid residues 369 to 405 of human CD98.

In some embodiments, monoclonal antibodies of the invention are humanized, human or chimeric antibodies. In some embodiments, the antibody functional fragment of the invention is a Fab, F (ab ') 2, Fv or scFv fragment.

In one embodiment, the invention provides all three heavy chain complementarity determining regions from the heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 31 and SEQ ID NO: CDRs) and / or isolated light chain variable region comprising all three light chain CDRs from the light chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: Antibody or functional fragment thereof.

In one embodiment, the invention provides all three heavy chain CDRs from a heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 31 and SEQ ID NO: The present invention provides isolated antibodies or functional fragments thereof comprising all three light chain CDRs from a light chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 33 and SEQ ID NO: 37 . In some embodiments, the antibody or functional fragment thereof comprises (a) an antibody designated 8-34B; (b) an antibody named 18-2A 2.2; (c) an antibody named 18-2A 7.1; (d) an antibody designated 1-47C; Or (e) all heavy and light chain complementarity determining regions (CDRs) from an antibody named 1-115A. In some embodiments, the antibody or functional fragment thereof comprises all heavy and light chain CDRs from an antibody named 8-34B. In some embodiments, the antibody or functional fragment thereof comprises all heavy and light chain CDRs from an antibody named as 18-2A 2.2. In some embodiments, the antibody or functional fragment thereof comprises all heavy and light chain CDRs from an antibody named as 18-2A 7.1. In some embodiments, the antibody or functional fragment thereof comprises all heavy and light chain CDRs from an antibody named 1-47C. In some embodiments, the antibody or functional fragment thereof comprises all heavy and light chain CDRs from an antibody named 1-115A.

In some embodiments, the antibody comprises a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 31 and SEQ ID NO: In some embodiments, the antibody comprises a light chain variable domain sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 33 and SEQ ID NO: In some embodiments, the antibody comprises a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 31 and SEQ ID NO: 35 and comprises SEQ ID NO: 6, SEQ ID NO: , SEQ ID NO: 33, and SEQ ID NO: 37.

In one embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 4 and the light chain variable domain sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 8 and the light chain variable domain sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 12 and the light chain variable domain sequence of SEQ ID NO: 14. In one embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 31 and the light chain variable domain sequence of SEQ ID NO: 33. In one embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 35 and the light chain variable domain sequence of SEQ ID NO: 37.

In one embodiment, the invention provides a humanized antibody. In some embodiments, the humanized antibody comprises a heavy chain variable domain sequence selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22 and SEQ ID NO: Some embodiments of tantalum, humanized antibodies comprise light chain variable domain sequences selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: In some embodiments, the humanized antibody comprises a heavy chain variable domain sequence selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22 and SEQ ID NO: 23 and comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: Lt; RTI ID = 0.0 > 21 < / RTI > In some embodiments, the humanized antibody comprises a light chain variable domain sequence selected from SEQ ID NO: 15 and SEQ ID NO: 16 and a heavy chain variable domain sequence selected from SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: In one embodiment, the humanized antibody comprises the light chain variable domain sequence of SEQ ID NO: 15 and the heavy chain variable domain sequence of SEQ ID NO: 18.

In one embodiment, the humanized antibody comprises the light chain variable domain sequence of SEQ ID NO: 20 and the heavy chain variable domain sequence of SEQ ID NO: 22. In one embodiment, the humanized antibody comprises the light chain variable domain sequence of SEQ ID NO: 21 and the heavy chain variable domain sequence of SEQ ID NO: 23. In one embodiment, the humanized antibody comprises the light chain variable domain sequence of SEQ ID NO: 20 and the heavy chain variable domain sequence of SEQ ID NO: 23. In one embodiment, the humanized antibody comprises the light chain variable domain sequence of SEQ ID NO: 21 and the heavy chain variable domain sequence of SEQ ID NO: 22. In one embodiment, the invention provides an antibody that binds to the same epitope as the epitope bound by the humanized antibody comprising the light chain variable domain sequence of SEQ ID NO: 21 and the heavy chain variable domain sequence of SEQ ID NO: 22. In an alternative embodiment, the invention includes a binding agent that binds essentially the same epitope as an epitope bound by an antibody from bin 1 or bin 3 to 7, as shown in Fig.

In a further embodiment, the invention includes a binding agent that binds essentially the same epitope as an epitope bound by any of the antibodies disclosed above. In some embodiments, the binding agent inhibits the growth of tumors expressing CD98. In some embodiments, the binding agent is an antibody or functional fragment thereof. In another embodiment, the binder is selected from the group consisting of anticalin, adnectin, affibody, DARPin, fynomer, affitin, affilin, avimer, , Cysteine-rich knottin peptides or engineered Kunitz type inhibitors.

In one embodiment, the present invention provides a binding agent capable of binding to CD98, wherein any one of the antibodies disclosed above replaces the binding agent in a competitive binding assay. In some embodiments, the binding agent is an antibody or functional fragment thereof. In another embodiment, the present invention provides a binding agent capable of binding to CD98, wherein said binding agent replaces any of the antibodies disclosed above in a competitive binding assay. In some embodiments, the binding agent is an antibody or functional fragment thereof.

In some embodiments, the invention provides an antibody that binds to CD98, wherein the antibody is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: At least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% and at least 98% amino acid sequence identity to the amino acid sequence selected from SEQ ID NO: Or greater or 99% or more sequence identity to a heavy chain variable domain. In some embodiments, the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 6, 10, 14, 15, 16, 20, 21, 33, A light chain variable domain having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% do. In some embodiments, the antibody comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, A heavy chain variable domain having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% At least 90% of the amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the invention provides an antibody that retains the biological function of the antibody and is a variant of any one of the above antibodies having one or more amino acid substitutions, deletions, insertions or alterations. In some embodiments, the invention provides an antibody that binds to CD98 expressed on the cell surface and inhibits cell growth. In some embodiments, the anti-CD98 antibody binds to CD98 expressed on the cell surface and inhibits cell proliferation. In some embodiments, the anti-CD98 antibody binds to CD98 expressed on the cell surface and induces apoptosis. In some embodiments, the present invention provides methods of modulating, modulating, and / or modulating one or more of the properties, such as binding affinity, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, An antibody that is any one of the above antibodies.

In some embodiments, the invention provides any of the above antibodies or functional fragments, wherein the antibody or fragment is conjugated to a cytotoxic agent. In various embodiments, cytotoxic agents are selected from chemotherapeutic agents, drugs, growth inhibitors, toxins and radioactive isotopes. In some embodiments, the invention provides any of the above antibodies or functional fragments, wherein the antibody or fragment is conjugated to a detectable marker. In various embodiments, the detectable marker is selected from a radioactive isotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound.

In one embodiment, the invention provides a hybridoma that produces a monoclonal antibody of the invention. In one embodiment, the invention provides a transgenic animal that produces a monoclonal antibody of the invention.

In some embodiments, polynucleotides encoding any of the above antibodies are provided. In one embodiment, a vector comprising the polynucleotide is provided. In one embodiment, host cells comprising the vector are provided. In one embodiment, the host cell is a prokaryotic cell. In one embodiment, the host cell is a human. Coli cells. In another embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell. In one embodiment, a method of producing an anti-CD98 antibody is provided, wherein the method comprises culturing the host cell under conditions suitable for expression of the polynucleotide encoding the antibody and isolating the antibody.

In one embodiment, the invention provides a pharmaceutical composition comprising the antibody or functional fragment thereof, an antibody conjugate, and a binding agent of the invention. In a further embodiment, the invention provides a method of inhibiting the growth of cancer cells expressing CD98, comprising exposing said cells to any one or more of said antibodies or functional fragments thereof, antibody conjugates and binding agents of the invention . In various embodiments, the cancer cells are selected from bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma and leukemia, It is derived from the transition of any one of these arms.

In one embodiment, the invention provides a method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition comprising the antibody or a functional fragment thereof, an antibody conjugate and a binding agent of the invention do. In various embodiments, the cancer is selected from bladder cancer, breast cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma and leukemia, . ≪ / RTI > In some embodiments, the cancer is acute myelogenous leukemia. In some embodiments, the subject has relapsed or refractory acute myelogenous leukemia. In some embodiments, the cancer is associated with increased expression of CD98 on the surface of the cell.

In some embodiments, a subject is administered one or more chemotherapeutic compounds with an antibody or functional fragment, wherein the chemotherapeutic compound is selected from the group consisting of vendamustine hydrochloride, cyclophosphamide, ifosfamide, fludurabine, Is selected from cytotoxic, cytotarin, cytarabine, gemcitabine, prednisone, prednisolone, methylprednisolone, paclitaxel, docked cell, vinorelbine, vincristine, etofoside, irinotecan, anthracycline, adriamycin, cisplatin, carboplatin and rituximab.

In one embodiment, there is provided a method of detecting the presence of CD98 in a biological sample comprising contacting said biological sample with any one of said antibodies under conditions that permit binding of the antibody to CD98, The method comprising the steps < RTI ID = 0.0 > of: < / RTI > In some embodiments, the biological sample is selected from the group consisting of bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma or leukemia, Quot; is a biological sample of a mammal suspected of having or having cancer of a cell or tissue, including, but not limited to, any one of the following.

In one embodiment, a method of diagnosing cancer associated with increased expression of CD98 comprises contacting a test cell with any one of the antibodies; Detecting the binding of the antibody to CD98 and measuring the level of expression of CD98; And comparing the level of expression of CD98 by the test cell with the level of expression of CD98 by the control cell, wherein the higher expression level of CD98 by the test cell as compared to the control cell is a CD98 Indicating the presence of cancer associated with increased expression. In some embodiments, the test cell is a cancer selected from bladder cancer, breast cancer, colon cancer, rectal cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, glioma, lymphoma and leukemia, It is a cell of a patient suspected of having one metastasis. In one embodiment, the method comprises the steps of determining the level of expression of CD98 on the surface of the test cell, and comparing the level of expression of CD98 on the surface of the test cell with the level of expression of CD98 on the surface of the control cell . In some embodiments, the test cells are cancer cells and the control cells are normal cells of the same tissue type.

In one embodiment, the invention provides the use of any of the above antibodies or functional fragments in the manufacture of a medicament, wherein the medicament is for use in a method of inhibiting the growth of cancer cells expressing CD98 . In various embodiments, the cell is a cancer selected from bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma and leukemia or It is derived from the transition of any one of these arms.

In one embodiment, the invention provides any of the above antibodies or functional fragments for use in inhibiting the growth of cancer cells expressing CD98. In various embodiments, the cell is a cancer selected from bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma, Or from the transfer of either of these arms.

In one embodiment, the invention provides the use of a pharmaceutical composition comprising any of the above antibodies or functional fragments in the manufacture of a medicament, wherein the medicament is a medicament for use in a method of treating cancer in a subject to be. In various embodiments, the cancer is selected from bladder cancer, breast cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma and leukemia, . ≪ / RTI > In some embodiments, the cancer is acute myelogenous leukemia. In some embodiments, the subject has relapsed or refractory acute myelogenous leukemia. In some embodiments, the cancer is associated with increased expression of CD98 on the surface of the cell. In some embodiments, the subject is administered one or more chemotherapeutic compounds with the antibody or functional fragment, wherein the chemotherapeutic compound is selected from the group consisting of vendamustin hydrochloride, cyclophosphamide, ifosfamide, fluaravarin, , Gemcitabine, prednisone, prednisolone, methylprednisolone, paclitaxel, docked cells, vinorelbine, vincristine, etofoside, irinotecan, anthracycline, adriamycin, cisplatin, carboplatin and rituximab.

In one embodiment, the invention provides a pharmaceutical composition comprising any of the above antibodies or functional fragments and a pharmaceutically acceptable carrier for use in treating cancer in a subject. In various embodiments, the cancer is selected from bladder cancer, breast cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma and leukemia It is the transition of any one of the arms. In some embodiments, the cancer is acute myelogenous leukemia. In some embodiments, the subject has relapsed or refractory acute myelogenous leukemia. In some embodiments, the cancer is associated with increased expression of CD98 on the surface of the cell. In some embodiments, the subject is administered one or more chemotherapeutic compounds with the antibody or functional fragment, wherein the chemotherapeutic compound is selected from the group consisting of vendamustin hydrochloride, cyclophosphamide, ifosfamide, fluaravarin, , Gemcitabine, prednisone, prednisolone, methylprednisolone, paclitaxel, docked cells, vinorelbine, vincristine, etofoside, irinotecan, anthracycline, adriamycin, cisplatin, carboplatin and rituximab.

In one embodiment, the invention provides the use of any of the above antibodies or functional fragments in the manufacture of a medicament, wherein the medicament is for use in a method of detecting the presence of CD98 in a biological sample. In some embodiments, the method comprises contacting a biological sample with any one of the antibodies under conditions that permit binding of the antibody to CD98, and detecting whether a complex is formed between the antibody and CD98 . In some embodiments, the biological sample is selected from the group consisting of bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma or leukemia, Quot; is a biological sample of a mammal suspected of having or having cancer of a cell or tissue, including, but not limited to, any one of the following.

In one embodiment, the invention provides any of the above antibodies or functional fragments for use in a method of detecting the presence of CD98 in a biological sample. In some embodiments, the method comprises contacting a biological sample with any one of the antibodies under conditions that permit binding of the antibody to CD98, and detecting whether a complex is formed between the antibody and CD98 . In some embodiments, the biological sample is selected from the group consisting of bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma or leukemia, Quot; is a biological sample of a mammal suspected of having or having cancer of a cell or tissue, including, but not limited to, any one of the following.

In one embodiment, the invention provides the use of any of the above antibodies or functional fragments in the manufacture of a medicament for use in a method of diagnosing cancer associated with increased expression of CD98 . In some embodiments, the method comprises contacting a test cell with any one of the antibodies; Detecting the binding of the antibody to CD98 and measuring the level of expression of CD98; And comparing the level of expression of CD98 by the test cell with the level of expression of CD98 by the control cell wherein the higher expression level of CD98 by the test cell relative to the control cell is associated with increased expression of CD98 Indicates the presence of cancer. In some embodiments, the test cell is a cancer selected from bladder cancer, breast cancer, colon cancer, rectal cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, glioma, lymphoma and leukemia, It is a cell of a patient suspected of having or having one metastasis. In one embodiment, the method comprises the steps of determining the level of expression of CD98 on the surface of the test cell, and comparing the level of expression of CD98 on the surface of the test cell with the level of expression of CD98 on the surface of the control cell . In some embodiments, the test cells are cancer cells and the control cells are normal cells of the same tissue type.

In one embodiment, the invention provides any of the above antibodies or functional fragments for use in a method of diagnosing cancer associated with increased expression of CD98. In some embodiments, the method comprises contacting a test cell with any one of the antibodies; Detecting the binding of the antibody to CD98 and measuring the level of expression of CD98; And comparing the level of expression of CD98 by the test cell with the level of expression of CD98 by the control cell wherein the higher expression level of CD98 by the test cell relative to the control cell is associated with increased expression of CD98 Indicates the presence of cancer. In some embodiments, the test cell is a cancer selected from bladder cancer, breast cancer, colon cancer, rectal cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, glioma, lymphoma and leukemia, It is a cell of a patient suspected of having one metastasis. In one embodiment, the method comprises the steps of determining the level of expression of CD98 on the surface of the test cell, and comparing the level of expression of CD98 on the surface of the test cell with the level of expression of CD98 on the surface of the control cell . In some embodiments, the test cells are cancer cells and the control cells are normal cells of the same tissue type.

In another embodiment of the invention, a product or "kit" is provided that contains a substance useful for the treatment of the disorders described above. The product includes a container, and a label or instructions on the container or enclosed in the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container may have an antibody or antibody-drug conjugate (ADC) composition effective for the treatment of the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag having a cap which can be perforated by a hypodermic needle Or vial). One or more active agents in the composition are antibodies or ADCs. A label or instructions for use indicates that the composition is used for the treatment of a selected condition, such as cancer. Alternatively or additionally, the article further comprises a second (or third) container comprising a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution can do. The product may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Figure 1 shows the protein expression levels of CD98 identified and quantified by sTAg analysis in AML, CLL and CRC samples, and related normal controls. The line represents the mean of the% normalized spectral potency factor (NSAF) in the positive control sample.
Figure 2 is a graph showing the results of epitope binning for 39 anti-CD98 antibodies.
Figure 3 shows the binding properties of chimeric anti-CD98 monoclonal antibodies 8-34B, 18-2A 2.1, 18-2A 2.2 and 18-2A 2.7. Figure 3A is a graph showing the results of epitope-binning for chimeric anti-CD98 monoclonal antibodies. The four reference antibodies are shown in Fig. "Homologous" is a control antibody of the same isotype that does not bind to CD98. Figure 3b shows the Kd of the chimeric anti-CD98 monoclonal antibody as measured by FACS analysis using the colon cancer cell line DLDl. Figure 3c shows FACS analysis results of three AML primary tumor samples stained with chimeric anti-CD98 monoclonal antibodies and a cell line expressing cynomolgus monkey CD98 (cynCD98).
Figure 4 shows the construction of a humanized 8-34B antibody. Figure 4A shows the sequence of the human acceptor sequence (AC) and the murine 8-34B light chain variable domain (IGN 34) aligned with sequences of humanized light chains L1 and L2. The CDRs according to Kabat numbering are marked in red, and the substitution at L2 relative to L1 is underlined. Figure 4A discloses SEQ ID NOS: 6, 38, 15, 16 and 38, respectively, in the order in which they appear. Figure 4b shows the sequence of the human acceptor sequence (AC) and the murine 8-34B heavy chain variable domain (IGN 34) aligned with the sequence of the humanized heavy chain H1, H2 and H3. CDRs according to Kabat numbering are marked in red, and substitutions in H2 and H3 are underlined as compared to H3. Figure 4b discloses SEQ ID NOS: 4, 39, 17-19 and 40, respectively, in the order they appear.
Figure 5 shows that anti-CD98 antibody treatment induces strong tumor growth inhibition in established < RTI ID = 0.0 > Ramos < / RTI > tumors. At the beginning of treatment, the tumor volume increased from (a) about 75 mm ³ to (b) about 150 mm ³ to (c) about 250 mm ³ . Antibody dosing was discontinued on day 29 (a) or day 22 (b) and tumor regrowth was measured over the course of the study. Rituximab (anti-CD20 antibody) was used as a positive control control antibody and antibody HB121 (ATCC) was used as an IgG2a homologous negative control.
Figure 6 shows that the anti-CD98 antibody significantly delayed the progression time of the treated Ramos tumors. The tumor doubling time of the previous tumor regrowth data (Figures 4a-4c) was calculated and used for further prediction of progression time (TTP). TTP was then extrapolated to each animal in the treatment group and plotted as a Kaplan-Meier curve until 2000 mm ³ was reached.
Figure 7 shows that in vivo tumor growth in lymphoma xenografts was inhibited by anti-CD98 monoclonal antibody 18-2A compared to Rituxan and negative control IgG2a. The arrows indicate administration of antibody therapy.
Figure 8 shows that in vivo tumor growth in acute myelogenous leukemia xenografts was inhibited by anti-CD98 monoclonal antibodies 18-2A and 8-34B compared to negative control IgG2a. The arrows indicate administration of antibody therapy.
Figure 9 shows that in vivo tumor growth in colorectal carcinomatous grafts is more potent than that of erbuxox and negative control IgG2a (first study) and DC101 + CTX (cyclophosphamide) and negative control IgG2a (second study) RTI ID = 0.0 > monoclonal < / RTI > antibody 18-2A. DC101 is a rat anti-mouse VEGFR2 / KDR IgG1 mAb (ATCC No. HB-11534) and serves as a positive control. The arrows indicate administration of antibody therapy.
Figure 10 shows that in vivo tumor growth in non-small cell lung carcinoma xenografts was inhibited by anti-CD98 monoclonal antibody 18-2A relative to erbuxox (anti-EGFR) and negative control IgG2a. The arrows indicate administration of antibody therapy.
Figure 11 shows the effect of anti-CD98 monoclonal antibodies on in vivo tumor growth of lymphoma xenografts in mouse cultures with different immunodeficient backgrounds: (A) NSG mice; (B) NOD.SCID mouse and (C) SCID mouse.
Figure 12 shows the effect of chimeric anti-CD98 monoclonal antibodies (18-2A-ch7.1 and 8-34B-ch) on in vivo tumor growth of lymphoma xenografts in combination with their myilin monoclonal antibodies (18-2A And 8-34B.
Figure 13 depicts the murine CD98 sequence (SEQ ID NO: 1) which forms the 13 mouse-human CD98 chimeric constructs used to map epitopes on human CD98 bound by the humanized monoclonal antibody IGN523 substituted into the human CD98 sequence SEQ ID NO: 96).
Figure 14 shows the binding of humanized monoclonal antibody IGN523 and control antibody and 13 mouse-human CD98 chimeric constructs, respectively, as determined by FACS analysis.
Figure 15 shows the sequence of the region of human CD98 to which IGN523 binds and the position of this sequence within the three dimensional structure of CD98, confirmed by using a mouse-human CD98 chimeric construct. The amino acids T358 to G368 (underlined) are buried in the crystal structure and are unlikely to be part of the binding interface. The non-conserved residues between human and mouse sequences are indicated in bold typeface.
Figure 16 shows the binding of IGN523 to four constructs produced by introducing non-homologous residues of mouse CD98 into the targeted loop region of the human sequence. Construct 4.1 consists of deletions of mutations I371L, D374Q and A375G, and A376. Construct 4.2 consists of the mutations M383A and E384K. Construct 4.3 consists of the mutations D391N, F395I, P396F and D397H. Construct 4.4 consists of the mutations G400R, A401P and A404L. Binding was detected by FACS analysis of CHO cells transfected with each construct.
Figure 17 shows the binding of IGN523 to a single mutant construct of hydrophobic residues within the targeted loop region. Each labeled hydrophobic residue was replaced with a highly charged amino acid as shown. Binding was detected by FACS analysis of CHO cells transfected with each construct.
Figure 18 shows the binding of IGN523 to a construct containing multiple mutations of residues within the targeted loop region, detected by FACS analysis of CHO cells transfected with each construct. M1 contains the mutations D374Q, D397H, G400R and A401P. M2 contains the mutations D374E and A375E. M3 contains the mutations D397S and I398T.
Figure 19 shows the results of a variable length peptide screen for epitope mapping of the humanized monoclonal antibody IGN523. ELISA results for each peptide are indicated as horizontal lines. The starting and ending points of the line are contained within the peptide. The Y value of the line shows the ELISA results obtained for the peptides. The results suggest a predominant binding to ₃₉₅ FPDIPGA ₄₀₁ and a secondary binding to ₃₇₉ PGQP ₃₈₂ (shaded area).
Figure 20 shows the results of a set of best binding single-site alanine substituted peptides. Each residue was replaced with A (G if the original amino acid was A). The height at which the substitution character is constructed in the graph is the obtained ELISA value for the mutated peptide. The center line and the shaded display interval indicate the reference ELISA value.
21 shows a thermal map showing data obtained from a CLIPS conformational matrix structure combining two partial sequences of human CD98 (SEQ ID NOs: 45 to 59 are shown on the X-axis, SEQ ID NOs: 60 to 74 are Y Axis).
Figure 22 shows the results of a mutagenic screen of a strong binding peptide from the matrix assay shown in Figure 21. SEQ1 shows the sequence of the peptide, and DIF1 indicates where the mutation is located in the peptide. The gray areas indicate peptides with non-mutated sequences. The last column shows the difference in ELISA values between the wild type peptide and the mutated peptide. Higher values indicate that the mutation has a strong negative effect on binding.
Figure 23 shows the positions where amino acid residues found to be important in binding of the humanized monoclonal antibody IGN523 are present in the sequence and surface of human CD98. Figure 23a shows the positions in the sequence of residues identified by chimeric and mutagenic studies (bold letters), pepscan analysis (gray), or both (shaded). Figure 23b shows the location (dark gray) of the residues identified by chimeric and mutagenic studies. Figure 23C shows the location (light gray) of the residue identified by the Pepscan analysis. Figure 23d shows the overlap (black) of two sets of residues.
Figure 24 shows that in vivo tumor growth in the Ramis (RA. 1) buckling lymphoma xenograft was inhibited by the humanized monoclonal antibody IGN523 compared to rituximab and negative control IgG. Antibodies were administered intraperitoneally at a dose of 10 mg / kg on day 11, day 17 and day 25. The arrows indicate administration of antibody therapy.
Figure 25 shows that in vivo tumor growth in DAU buckling lymphoma xenografts was inhibited by the humanized monoclonal antibody IGN523 compared to Rituxan and negative control IgG. Antibodies were administered intraperitoneally at doses of 10 mg / kg on days 20 and 26. The arrows indicate administration of antibody therapy.
Figure 26a shows that in vivo tumor growth in the IGN-LNG-12 pulmonary tumor xenografts was inhibited by the humanized monoclonal antibody IGN523 compared to carboplatin and negative control IgG. IGN523 and carboplatin were intraperitoneally administered at a dose of 10 mg / kg or 75 mg / kg on the 17th day, 24th day and 31st day, respectively. The arrow indicates the administration of the treatment. Figure 26b shows a body weight measurement corresponding to the mouse of Figure 26a treated with the indicated reagent. Carboplatin was administered at its maximum dose to induce weight loss in NOD-SCID mice.
Figure 27 shows that in vivo tumor growth in KG-I acute myelogenous leukemia xenografts was inhibited by humanized monoclonal antibody IGN523 compared to Rituxan and negative control IgG. The antibodies were administered intraperitoneally at a dose of 15 mg / kg on days 21, 28, and 34. The arrows indicate administration of antibody therapy.
Figure 28 shows that in vivo tumor growth in lung tumor xenografts was dose-dependently inhibited by the humanized monoclonal antibody IGN523. The antibody was administered intraperitoneally at the indicated doses on day 12 and day 19. The arrows indicate administration of antibody therapy.
29 shows the staining of human and cynomolgus monkey frozen tissue flakes with the humanized monoclonal antibody IGN523. Human and Cynomolgus monkey kidneys, cerebral and placental frozen flakes were stained with 10 [mu] g / ml of IGN523. Using the variants of Tuson, Fung, and Hierck's methods for immunohistochemistry, the need for label attachment of IGN523 was eliminated, and the presence of secondary labeled anti-human IgG, Specific reactivity between intrinsic IgG (Fung 1992, Hierck 1994, Tuson 1990). The flakes were cut to approximately 5 탆. All slides were first assessed for tissue factor and staining suitability, and then evaluated by the pathologist for the intensity of the staining and subjectively graded. Representative images are presented at a magnification of 40 times except for the human brain (20 times).

General technique

The techniques and procedures described or referred to herein are generally well understood and can be performed by those skilled in the art, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3 ^rd . Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Current Protocols in Molecular Biology (FM Ausubel, et al., Eds., (2003)); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Z. An, ed., Wiley, Hoboken NJ (2009)); Monoclonal Antibodies: Methods and Protocols, M. Albitar, ed., Humana Press, Totawa, N.J. (2010)); ^And through the use of widely used methods described in literature (Antibody Engineering, 2 ^nd Ed., Vols 1 and 2, Kontermann and Dubel, eds., Springer-Verlag, Heidelberg, 2010).

Definitions and Acronyms

Justice

To interpret this specification, the following definitions shall apply and terms used singularly as appropriate will also include the plural, and conversely, the terms used in plural will also include the singular. In the event any discrepancy is inconsistent with any of the documents incorporated herein by reference, the definitions set forth below will take precedence.

As used herein, unless otherwise indicated, the term "CD98" refers to a mammal such as a primate (e.g., human, cynomolgus cyno), dogs and rodents And rats). ≪ / RTI > The amino acid sequence and coding nucleic acid sequence of human CD98 are provided below as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The term "CD98" encompasses unprocessed "whole" CD98 as well as any form of CD98 resulting from processing in cells. The term encompasses naturally occurring variants or mutations of CD98, such as splice variants, allelic variants, SNP variants and isoforms. The CD98 polypeptides described herein can be isolated from a variety of sources, such as human tissue types or other sources, or can be prepared by recombinant or synthetic methods. "Natural sequence CD98 polypeptide" includes polypeptides having the same amino acid sequence as the corresponding CD98 polypeptide from nature. Such native sequence CD98 polypeptides can be isolated from nature or can be prepared by recombinant or synthetic means. The term "native sequence CD98 polypeptide" specifically refers to a naturally occurring truncated or secreted form of a particular CD98 polypeptide (e.g., an extracellular domain sequence), a naturally occurring variant form of the polypeptide (e.g., ≪ / RTI > intact forms) and naturally occurring allelic variants.

The term "antibody" is used in its broadest sense and specifically includes, for example, monoclonal anti-CD98 monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full length or intact monoclonal antibodies) CD98 antibody composition, polyclonal antibody, multivalent antibody, multispecific antibody formed from two or more intact antibodies (e. G., As long as it exhibits the desired biological activity), single chain anti-CD98 Antibody, and fragments of the anti-CD98 antibody. The term "immunoglobulin" (Ig) is used interchangeably herein with antibodies. The antibody may be a human antibody, a humanized antibody, and / or an affinity matured antibody.

An "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, a carbohydrate, a nucleic acid, a lipid, a hapten, or other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide.

An antibody that "binds" to an antigen of interest is an antibody that is useful as a therapeutic agent in targeting cells or tissues expressing the antigen and that binds to the antigen with sufficient affinity so that it does not cross- to be. In such embodiments, the degree of binding of the antibody to a "non-target" protein is about 10% of the binding of the antigen and its specific target protein when measured by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) . The terms "specific binding, "" specifically binding to, " or "specific to" for the epitope on a particular polypeptide or target of a particular polypeptide, Means measurable different bonds. Specific binding can be measured, for example, by measuring the binding of a molecule to a binding of a control molecule, which is a molecule of similar structure that generally has no binding activity. For example, specific binding can be measured by competition with a target, e. G., A control molecule similar to an excess of unlabeled target. In this case, the specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of non-labeled target. As used herein, the particular polypeptide or a term for an epitope on a particular polypeptide target "specific binding", "specifically binding to" or "specific for", for example, about 10 to target ^{- 4} M or more, alternatively at least about 10 ^-5 M, alternatively at least about 10 ^-6 M, alternatively at least about 10 ^-7 M, alternatively at least about 10 ^-8 M, alternatively about 10 ^{- 9} M, alternatively greater than about 10 ^-10 M, alternatively greater than about 10 ^-11 M, or alternatively greater than about 10 ^-12 M. In one embodiment, the term "specific binding" refers to the binding when a molecule binds to an epitope on a particular polypeptide or a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

The term "anti-CD98 antibody" or "antibody binding to CD98" refers to an antibody capable of binding to CD98 with sufficient affinity such that the antibody is useful as a diagnostic agent and / or therapeutic agent in targeting CD98 . Preferably, the degree of binding of the non-CD98 protein independent of the anti-CD98 antibody is determined by, for example, binding of the antibody to CD98 when measured by fluorescence activated cell sorting (FACS) analysis or radioimmunoassay (RIA) ≪ / RTI > The terms "specifically binding to CD98" or "CD98 specific" antibodies are as defined above. In certain embodiments, the antibody that binds to CD98 has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, the anti-CD98 antibody binds to an epitope of CD98 that is conserved among CD98 of different species.

An "isolated antibody" is an antibody identified and separated and / or recovered from a component of its natural environment. Contaminants in his natural environment include, but are not limited to, substances that interfere with therapeutic uses for antibodies and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody comprises (1) greater than 95% by weight of antibody as measured by the Lowry method (Lowry et al., J. Bio. Chem. 193: 265-275, 1951) (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a rotating cup sequencer, or (3) Will be purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using staining. An isolated antibody includes an in situ antibody in a recombinant cell since one or more components of the natural environment of the antibody will not be present. Typically, however, the isolated antibody will be produced by one or more purification steps.

The basic four-chain antibody unit is a heterospray glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). For IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are connected to each other by one or more disulfide bonds according to the H chain isoform. Each of the H and L chains has an intra-chain disulfide bridging at regular intervals. Each H chain has a variable domain (V _H ) at the N-terminus followed by three constant domains (C _H ) for each of the alpha and gamma chains and four C _H domains for the [mu] and [epsilon] isoforms. Each L chain has a variable domain (V _L ) at its N-terminus followed by a constant domain (C _L ) at its other terminus. V _L is aligned with V _H, and C _L is aligned with the first constant domain (C _H 1) of the heavy chain. It is believed that certain amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. Pairing of V _H and V _L forms a single antigen binding site. For the structure and properties of antibodies of a different class, for example, in (Basic and Clinical Immunology, 8 ^th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6).

Refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as "V _H ". The variable domain of the light chain may be referred to as "V _L ". The term "variable" refers to the fact that certain fragments of variable domains differ extensively in sequence from antibody to antibody. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed over the 110-amino acid region of the variable domain. Instead, the V region is a backbone region (FR) composed of 15 to 30 amino acids separated by shorter regions that exhibit extreme variability referred to as "hypervariable regions" with 9 to 12 amino acid residues each, Lt; / RTI > stretch. The variable domains of the native heavy and light chains each comprise four FRs connected by three hypervariable regions forming a loop linkage, each adopting a predominantly β-sheet steric structure and, in some cases, forming part of the β-sheet structure . The hypervariable regions in each chain are present in close proximity by the FRs together and contribute to the formation of the antigen binding site of the antibody together with the hypervariable regions of the other chain (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

An "intact" antibody is an antibody that includes an antigen binding site as well as C _L and one or more heavy chain constant domains C _H 1, C _H 2, and C _H 3. The constant domain may be a native sequence constant domain (e. G., A human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has at least one effector function.

"Antibody fragments" comprise a portion of a whole antibody, preferably an antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments; Diabodies and di-diabodies (see, for example, Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. 90: 6444-8) 9, pp. 129-134 (2003)), International Patent Application Publication (International Patent Application Publication) WO 93111161, and U.S. Patent Nos. 5,837,242 and 6,492,123); Single chain antibody molecules (see, for example, U.S. Patent Nos. 4,946,778, 5,260,203, 5,482,858, and 5,476,786); Double variable domain antibodies (see, for example, U.S. Patent No. 7,612,181); Single variable domain antibodies (SdAbs) (e.g., Woolven et al., Immunogenetics 50: 98-101, 1999) and Streltsov et al., Proc Natl Acad Sci USA. 101: 12444-12449 (2004) Reference); And multispecific antibodies formed from antibody fragments.

A "functional fragment " of a therapeutic antibody will exhibit one or more functions, including at least a specific binding to a therapeutic antigen, if not all or a portion of the biological function is due to an intact antibody.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population consisting of substantially homogeneous antibodies (i.e., the individual antibodies that make up the population may contain a possible naturally occurring mutation ≪ / RTI > The modifier "monoclonal" should not be construed as requiring the manufacture of antibodies by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975), or may be produced by a bacterial, (See, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" are described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 ). ≪ / RTI >

The monoclonal antibodies herein may have identity or homology to the corresponding sequences of the antibody or a portion of the light chain and / or light chain from an antibody derived from a particular species or an antibody belonging to a particular antibody class or subclass, but the remainder of the chain (s) Quot; chimeric "antibodies having homology or homology to corresponding sequences of antibodies belonging to another antibody class or subclass, as well as fragments of such antibodies, which have the desired biological activity (See US Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

A "humanized" form of a non-human (eg, rodent) antibody is a chimeric antibody that contains a minimal sequence derived from a non-human antibody. Generally, a humanized antibody is one in which the moiety from the hypervariable region of the recipient is a non-human species antibody (donor antibody) having the desired antibody specificity, affinity, and performance, such as a supernatant of a mouse, rat, rabbit or non-human primate antibody Is a human immunoglobulin (acceptor antibody) substituted with a residue from a variable region. In some cases, the corresponding non-human residue displaces the framework region (FR) residue of the human immunoglobulin. Furthermore, humanized antibodies may comprise residues that are not found in acceptor antibodies or donor antibodies. These changes are made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of one or more, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of a non-human immunoglobulin and all or substantially all All FRs are FRs of the human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321: 522-525 (1986); (Riechmann et al., Nature 332: 323-329 (1988)); And Presta, Curr., Op. Struct Biol., 2: 593-596 (1992). See also the following references, and the patents and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1: 105-115 (1998); (Harris, Biochem. Soc. Transactions, 23: 1035-1038 (1995)); Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,762; U.S. Patent No. 6,180,370; And U.S. Patent No. 6,054,297.

A "human antibody" is an antibody that has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or that has been prepared by using any of the techniques of making a human antibody disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising a non-human antigen binding residue. Human antibodies can be obtained from phage display libraries (Hoogenboom and Winter, J Mol Biol., 227: 381 (1991); Marks et al., J Mol Biol., 222: , Nature Protocols 1: 755-768 (2006)). (Boerner et al., J Immunol., 147 (1): 86-95 (1991)), which is described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, The methods described can also be used for the production of human monoclonal antibodies. See Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). A human antibody can be produced by administering an antigen to a transgenic animal, e. G., A mouse, which has been altered to produce such an antibody in response to an antigenic challenge, but whose endogenous locus has been disabled (e. (Bruggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8 (4)), in a document on XENOMOUSE ™ technology (Jakobovits, A., Curr. Opin. Biotechnol. : 455-8), and U.S. Patent Nos. 6,075,181 and 6,150,584). See, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006)) on human antibodies raised via human B cell hybridoma technology.

The term "hypervariable region "," HVR "or" HV ", as used herein, refers to a region of an antibody variable domain that forms a loop that is hypervariable and / or structurally defined in sequence. In general, the antibody comprises six hypervariable regions (three hypervariable regions (H1, H2 and H3) at VH and three hypervariable regions (L1, L2 and L3) at VL). Numerous hypervariable region representations are used and encompassed herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Chothia refers instead to the location of the structural loop (Chothia and Lesk J. Mol. Biol., 196: 901-917 (1987)). The ends of the multicellular CDR-H1 loops when numbered using the Kabat numbering convention are H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme positions inserts in H35A and H35B; 35A and 35B Loop is terminated at 32; if only 35A is present, the loop is terminated at 33; if both 35A and 35B are present, the loop is terminated at 34). The AbM hypervariable region represents a trade-off between Kabat CDRs and superanti-structured loops and is used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable region is based on analysis of the available complex crystal structure. The residues from each of these hypervariable regions are indicated below.

The hypervariable region may comprise an "extended hypervariable region" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) in VL, and 89-97 or 89 -96 (L3); And 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3) at VH. Variable domain residues are numbered according to Kabat et al., Supra, for each of these definitions. As used herein, the terms "HVR" and "CDR" are used interchangeably.

The "backbone" or "FR" moiety is a variable domain residue other than the hypervariable region residues as defined herein.

The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and their flexion is described (Kabat et al., Sequences of Proteins oflmmunological Interest, 5 ^th Ed. Public Health Service , The National Institutes of Health, Bethesda, MD. (1991)), the numbering system used for the heavy or light chain variable domains of the assembly of antibodies. When using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of the FR or CDR of the variable domain, or insertion into such FR or CDR. For example, the heavy chain variable domain comprises a single amino acid insert (residue 52a according to Kabat) followed by a residue of H2, and a residue inserted after heavy chain FR residue 82 (e.g. residues 82a, 82b and 82c according to Kabat) ). By aligning the sequence of the antibody in the region of homology with the "standard" Kabat numbered sequence, the Kabat numbering of the residue for a given antibody can be determined.

Kabat numbering systems are commonly used to refer to residues within the variable domains (about residues 1 to 107 of the light chain and about residues 1 to 113 of the heavy chain) (see, for example, Kabat et al., Sequences of Immunological 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). "EU numbering system" or "EU index" is commonly used to refer to residues in the immunoglobulin heavy chain constant region (e. G., The EU index reported in Kabat et al. "EU index as in Kabat" refers to residue numbering of human IgG1 EU antibody. Unless otherwise stated herein, reference to a residue number in the variable domain of an antibody means residue numbering by a Kabat numbering system. Unless otherwise stated herein, reference to a residue number in the constant domain of an antibody refers to residue numbering by the EU numbering system.

An "affinity matured" antibody is an antibody having one or more alterations in its one or more HVRs that improve the affinity of the antibody for the antigen relative to the parent antibody that does not possess the alteration (s). Preferred Affinity The matured antibody will have a nanomolar or even picomole affinity for the target antigen. Affinity matured antibodies are prepared by procedures known in the art. For review, see Hudson and Souriau, Nature Medicine 9: 129-134 (2003); (Hoogenboom, Nature Biotechnol. 23: 1105-1116 (2005)); And Quiroz and Sinclair, Revista Ingeneria Biomedia 4: 39-51 (2010).

An "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

As used herein, an "agonist antibody" is an antibody that mimics one or more of the functional activities of a polypeptide of interest.

"Binding affinity" generally refers to the total total intensity of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to a unique binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g., an antibody and an antigen) do. The affinity of the molecule X to its partner Y can generally be expressed as the dissociation constant (Kd). The affinity may be determined by conventional methods known in the art, including those described herein. Low-affinity antibodies generally exhibit a tendency to bind slowly and easily dissociate to an antigen while highly-affinity antibodies generally tend to bind to the antigen more quickly and remain bound for longer. Various binding affinity determination methods are known in the art, and any of these methods can be used for the purposes of the present invention. Specific exemplary embodiments are described below.

Refers to a stronger association between a molecule (e. G., An antibody) and its binding partner when used herein to refer to a binding affinity, and is denoted by a smaller numerical Kd value. For example, for antibodies having "0.6 nM or better" affinity for the antigen, the affinity of the antibody for the antigen may be less than 0.6 nM, such as 0.59 nM, 0.58 nM, 0.57 nM, or less than 0.6 nM It is an arbitrary value.

In one embodiment, a "Kd" or "Kd value" according to the present invention is calculated by equilibrating a Fab with a minimal concentration of ( ¹²⁵ I) -labeled antigen in the presence of a titration series of non- Fab analysis was performed by using the Fab version of the antibody of interest and its antigen as described by the following assays to determine the solution binding affinity of the Fab for the antigen by capture with a plate coated with anti-Fab antibody (RIA) (Chen, et al., (1999) J Mol Biol 293: 865-881). According to another embodiment, the Kd or Kd value can be determined, for example, using the BIAcore 占 -2000 or Biacore 占 -3000 (BIAcore, Inc., Piscataway, NJ, USA) 0.0 > Plasmon < / RTI > Resonance Assay).

"Response time" or "dissociation rate" according to the invention, or "association rate" or "k _on", for example, BIAcore ™ -2000 or a BIAcore ™ -3000 (BIAcore, Inc., New Jersey, USA Or a surface plasmon resonance technique described above using a microfluidic device (e.g., a primary Piscataway).

As used herein, the phrase "substantially similar" or "substantially the same" means that the skilled artisan will be able to determine the difference between the two values biologically and / or statistically in terms of biological properties as measured by the values (e.g., Kd values) Means a sufficiently high degree of similarity between two numerical values (generally numerical values associated with the antibodies of the present invention and numerical values associated with the reference antibody) so as to be regarded as being of little or no significance. The difference between the two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 20%, as a function of the value for the reference antibody Less than about 10%.

As used herein, the phrase "substantially reduced" or "substantially different" means that a skilled artisan will appreciate that differences between the two values are not statistically significant in terms of biological properties as measured by the values (e.g., Kd values, Quot; means a sufficiently high degree of difference between two numerical values (generally the numerical value associated with the antibody of the present invention and the numerical value associated with the reference antibody) to the extent that it is considered significant. The difference between the two values is preferably greater than about 10%, preferably greater than about 20%, preferably greater than about 30%, preferably greater than about 40%, preferably greater than about 40% Is greater than about 50%.

An antibody or "growth inhibiting" antibody that inhibits the growth of tumor cells expressing a CD98 polypeptide is an antibody that induces measurable growth inhibition of cancer cells that express or overexpress an appropriate CD98 polypeptide. The CD98 polypeptide may be a dendritic polypeptide expressed on the surface of cancer cells or may be a polypeptide produced and secreted by cancer cells. A preferred growth inhibitory anti-CD98 antibody typically inhibits the growth of CD98 expressing tumor cells by 20% or more, preferably from about 20% to about 50%, even more preferably, (For example, from about 50% to about 100%). In one embodiment, the inhibition of growth may be measured at an antibody concentration of from about 0.1 [mu] g / ml to 30 [mu] g / ml in the cell culture or from about 0.5 nM to 200 nM, wherein the growth inhibition is It is measured after 1 to 10 days. Inhibition of growth of tumor cells in vivo can be measured in various ways as described below. The antibody can be administered in an amount of from about 1 [mu] g to about 100 mg of anti-CD98 antibody per kg of body weight, within about 5 to 3 months from the first administration of the antibody, preferably within about 5 to 30 days, It is possible to inhibit growth in vivo.

An "antibody that induces apoptosis" is a protein that has been programmed to cause apoptosis when measured by binding of annexin V, fragmentation of DNA, cell shrinkage, expansion of the endoplasmic reticulum, cell fragmentation, and / or formation of membrane vesicles (referred to as apoptotic bodies) Lt; / RTI > Cells are cells that overexpress CD98 polypeptides. Preferably, the cell is a tumor cell. Various methods of assessing intracellular events associated with apoptosis can be used. For example, the phosphatidylserine (PS) potential can be measured by annexin binding; DNA fragmentation can be assessed through DNA laddering; Nuclear / chromatin condensation with DNA fragmentation can be assessed by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is about 2- to 50-fold, preferably about 5-fold to 50-fold, and most preferably about 2-fold to about 500-fold greater than that of the non-treated cells in the annexin binding assay 10-fold to 50-fold induction.

An "antibody that induces apoptosis " is an antibody that makes viable cells non-viable. A cell is a cell type that specifically expresses or overexpresses a CD98 polypeptide. The cell can be a cancer cell or a normal cell of a certain cell type. The CD98 polypeptide may be a dendritic polypeptide expressed on the surface of cancer cells or may be a polypeptide produced and secreted by cancer cells. In vitro cell death may be measured in the absence of complement and immune effector cells to distinguish from cell-induced cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) induced apoptosis. Thus, an assay for apoptosis can be performed using heat-inactivated serum (i.e., in the absence of complement) in the absence of immune effector cells. (PI), trypan blue (see Moore et al., Cytotechnology 17: 1-11 (1995)) or by the ingestion of 7AAD to determine if the antibody can induce apoptosis The loss of assessed membrane integrity can be assessed relative to non-treated cells. A preferred cell death-inducing antibody is an antibody that induces PI uptake in a PI uptake assay in BT474 cells.

Antibody "effector function" refers to a biological activity that may be attributable to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody and is dependent upon the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity; Fc receptor binding; Antibody-dependent cell mediated cytotoxicity (ADCC); Phagocytosis; Down regulation of cell surface receptors (e. G., B cell receptors); And B cell activation.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain will be different, it is defined that the human IgG heavy chain Fc region typically extends from the amino acid residue present at position Cys226 or Pro230 to its carboxyl-terminal end. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the preparation or purification of the antibody, or can be removed by recombinant manipulation of the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibody may comprise an antibody population in which all K447 residues have been removed, an antibody population in which no K447 residues have been removed, and a population of antibodies with a mixture of antibodies with K447 residues and antibodies without K447 residues .

"Functional Fc region" retains the "effector function" of the native sequence Fc region. An exemplary "effector function" CDC; Fc receptor binding; ADCC; Phagocytosis; Down regulation of cell surface receptors (e. G., B cell receptor; BCR), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e. G., An antibody variable domain) and may be assessed, for example, through the use of the various assays disclosed in the definition herein.

The "native sequence Fc region" includes the same amino acid sequence as the amino acid sequence of the Fc region found in nature. The native sequence human Fc region comprises a native sequence human IgGl Fc region (non-A homologous and A homologous); Native sequence human IgG2 Fc region; Native sequence human IgG3 Fc region; And native sequence human IgG4 Fc regions as well as naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by one or more amino acid alterations, preferably by one or more amino acid substitution (s). Preferably, the variant Fc region comprises at least one amino acid substitution relative to the native sequence Fc region or the Fc region of the parent polypeptide, e. G., From about 1 to about 10 amino acid substitutions in the Fc region of the native sequence Fc region or of the parent polypeptide, Preferably from about 1 to about 5 amino acid substitutions. The variant Fc region herein is preferably at least about 80% homologous, more preferably at least about 90% homologous, most preferably at least about 95% homologous to the native sequence Fc region and / or the Fc region of the parent polypeptide .

"Antibody-dependent cell mediated cytotoxicity" or "ADCC" refers to secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) Refers to the cytotoxicity of these cytotoxic effector cells that specifically bind to antigen-bearing target cells and then kill the target cells with cytotoxins. Antibodies are "absolutely necessary" for "killing" cytotoxic cells and for this killing. NK cells that are primary cells mediating ADCC express only Fc [gamma] RIIII, whereas mononuclear cells express Fc [gamma] RI, Fc [gamma] RII and Fc [gamma] RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of the literature (Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991)). To assess ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, for example, the ADCC assays described in U.S. Pat. Nos. 5,500,362 and 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal models such as those described in Clynes et al. (USA) 95: 652-656 (1998) .

"Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, preferred FcRs are FcRs (gamma receptors) that bind to IgG antibodies and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activation receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains (M. in Daeron, Annu. Rev. Immunol. 234 (1997)). FcR has been described by Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991), Capel et al., Immunomethods 4: 25-34 (1994) J. Lab. Clin. Med. 126: 330-41 (1995)). Other FcRs, including FcRs to be identified in the future, are encompassed by the term "FcR" herein. The term also includes FcRn, a neonatal receptor that is responsible for the transfer of parental IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 1994). Antibody variants with improved or reduced binding to FcR are described, for example, in International Patent Application Publication No. WO 2000/42072, and U.S. Pat. Nos. 7,183,387, 7,332,581 and 7,335,742. See also, for example, Shields et al. J Biol. Chem. 9 (2): 6591-6604 (2001).

"Complement dependent cytotoxicity" or "CDC" refers to the dissolution of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component (C1q) of the complement system to the antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay as described in, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) can be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides having variant Fc regions) and increased or decreased C1q binding capabilities are described, for example, in U.S. Patent No. 6,194,551 B1 and International Patent Application Publication No. WO 1999/51642 . See, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

CD98 polypeptide "extracellular domain" or "ECD" refers to a form of CD98 polypeptide that is essentially free of the epidermal and cytoplasmic domains. Typically, a CD98 polypeptide ECD will have less than 1% of such a transmembrane domain and / or a cytoplasmic domain, preferably less than 0.5% of such domains. The transmembrane domain of CD98 contains amino acid residues 76-103 (Parmacek et al., Nucleic Acids Res. 17: 1915-1931, 1989). The exact boundaries of the transmembrane domain may be different, but it is most likely to be no more than about five amino acids present at either end of the domain as first identified. Thus, optionally, the extracellular domain of the CD98 polypeptide may comprise amino acids from about 98 to 108 to 529 of the sequence of CD98, as disclosed in that document (Parmacek et al.).

"Percent (%) amino acid sequence identity" for a reference polypeptide sequence means that the sequence is aligned and a gap is introduced to achieve maximum percent sequence identity if necessary, and any conservative substitution is not considered part of the sequence identity, Is defined as the percentage of amino acid residues of the same candidate sequence as the amino acid residue of the sequence. Alignment to determine percent amino acid sequence identity may be accomplished in a variety of ways within the skill of the art, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software . Those skilled in the art will be able to determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

As used herein, "alteration" of an amino acid residue / position refers to a change in the primary amino acid sequence relative to the starting amino acid sequence, wherein the change results from altering the sequence comprising the amino acid residue / position. For example, a typical alteration may include substitution (e.g., conservative or non-conservative substitution) of the residue by another amino acid, one or more adjacent to the residue / position (generally less than five or three ) Amino acid insertion, and deletion of the residue / position.

An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and stereostructured epitopes.

When two antibodies recognize identical or sterically overlapping epitopes, the antibody binds to "essentially the same epitope" with the epitope bound by the reference antibody. The most widely used rapid method of confirming whether two antibodies bind to identical or sterically overlapping epitopes is the competition assay (which may consist of any number of different formats) using the labeled antigen or labeled antibody. Typically, the antigen is immobilized on a 96-well plate and the ability of the non-labeled antibody to block binding of the labeled antibody is determined using radioactive or enzymatic labels.

"Epitope mapping" is the process of identifying binding sites or epitopes of an antibody on their target antigen. The antibody epitope may be a linear epitope or a conformational epitope. Linear epitopes are formed by consecutive amino acid sequences in proteins. A stereostructural epitope is formed in the amino acids that are discontinuous in the protein sequence but coexist with the folding of the protein into its three-dimensional structure.

As defined herein, "epitope binding" is the process of classifying antibodies based on epitopes recognized by antibodies. More specifically, epitope vining includes methods and systems for distinguishing epitope recognition properties of different antibodies and identifying antibodies with different binding specificities, in combination with a computerized method of classifying antibodies based on their epitope recognition properties.

"Disability" is any condition or disease that would benefit from treatment with the substance / molecule or method of the present invention. This includes chronic and acute disorders, including pathological conditions that make mammals susceptible to the disorder. Non-limiting examples of disorders to be treated herein include cancerous conditions such as bladder, breast, colon, rectal, stomach, esophageal, lung, laryngeal, renal, oral, ovarian, prostate, sarcoma, melanoma, , Lymphoma or leukemia, as well as metastasis of these cancers.

The terms " cell proliferative disorder "and" proliferative disorder "refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

As used herein, "tumor" refers to all neoplastic cellular growth and proliferation, whether malignant or benign, and to all prodrug and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive, as referred to herein.

The terms "cancer" and "cancerous" refer to or describe the pathological condition of a mammal, which is typically characterized by non-regulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoma. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer); Small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung; Peritoneal cancer; Hepatocellular carcinoma; Gastric or abdominal cancer including gastrointestinal cancer; Pancreatic cancer; Glioblastoma; Cervical cancer; Ovarian cancer; Oral cancer; Liver cancer; Bladder cancer; Urinary tract cancer; Hepatocellular carcinoma; Breast cancer; Colon cancer; Rectal cancer; Colorectal cancer; Endometrial or uterine carcinoma; Salivary gland carcinoma; Renal or neoplastic; Prostate cancer; Mun Mun; Thyroid cancer; Liver cancer; Anal carcinoma; Penis carcinoma; Melanoma; Multiple myeloma and B cell lymphoma; Brain cancer as well as head and neck cancer; And related transitions.

"CD98 expressing cell" is a cell expressing CD98 that is endogenous or transfected on the cell surface. A "CD98 expressing cancer" is a cancer comprising cells having a CD98 protein present on the cell surface. A "CD98 expressing cancer" produces sufficient levels of CD98 on its cell surface so that the anti-CD98 antibody binds to it and has a therapeutic effect on cancer. Cancer that "overexpresses" CD98 is a cancer that has significantly higher levels of CD98 on its cell surface than non-cancerous cells of the same tissue type. Such overexpression may be caused by gene amplification, or by increased transcription or translation. CD98 overexpression can be measured in a diagnostic or prognostic assay by assessing an increased level of CD98 protein present on the surface of the cell (e.g., via immunohistochemistry analysis; FACS analysis). Alternatively, or additionally, the nucleic acid can be detected by, for example, fluorescence in situ hybridization (FISH; see International Patent Application Publication No. WO98 / 45479 published October 1998), Southern blotting, Northern blotting, PCR) techniques, such as real-time quantitative PCR (RT-PCR), to determine the level of CD98-encoding nucleic acid or mRNA in the cells. In addition to the above assays, a variety of in vivo assays can be used by those skilled in the art. For example, the body cells of a patient can be exposed to an optionally labeled label, for example, an antibody labeled with a radioactive isotope, and the binding of the antibody and the cell in the patient can be monitored, for example, Scanning, or analysis of a biopsy taken from a patient previously exposed to the antibody. The CD98 expressing cancer may be any one of bladder cancer, breast cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma or leukemia, But are not limited to, transitions.

Refers to a clinical procedure in an attempt to alter the natural course of a subject or cell being treated, and includes, but is not limited to, a " treatment " Can be performed for prevention or can be performed during the course of clinical pathology. Preferred effects of the treatment include preventing the occurrence or recurrence of the disease, alleviating the symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis in the case of cancer, reducing the rate of disease progression, , And remission or improved prognosis. In some embodiments, the antibodies of the invention are used to slow the development of a disease or disorder or slow the progression of a disease or disorder.

These parameters for evaluating successful treatment and improvement of the disease can be easily measured by an idiomatic procedure familiar to the physician. In the case of cancer treatment, the efficacy can be measured, for example, by assessing the disease progression time (TTP) and / or measuring the rate of response (RR). Other endpoints for measuring efficacy include, for example, total survival (OS), disease free survival (DFS), and remyelinating (or recurrent) survival (RFS). Metastasis can be identified by staging and by bone scan and examination of calcium levels and other enzymes to confirm spread to the bone. CT scans can also be performed to find the pelvis and spread to the lymph nodes within this area. Measurements of liver enzyme levels by chest X-ray and known methods are used to find metastases to the lungs and liver, respectively. Other common methods of monitoring disease include transrectal ultrasound (TRUS) and transrectal needle biopsy (TRNB).

"Entity" is a vertebrate animal. In certain embodiments, the vertebrate animal is a mammal. Mammals include, but are not limited to, farm animals (eg, cows), sports animals, pets (eg, cats, dogs and horses), primates, mice and rats. In certain embodiments, the mammal is a human.

"Effective amount" refers to an amount effective to achieve the desired therapeutic or prophylactic result for the required period of time at the required dose. The "therapeutically effective amount" of the substance / molecule of the present invention may vary depending on factors such as the disease state, age, sex and body weight of the individual, and the ability of the substance / molecule to elicit the desired response in the individual. A therapeutically effective amount encompasses an amount when the therapeutically beneficial effect exceeds any toxic or deleterious effect of the substance / molecule. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result over a required period of time at the required dose. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since the prophylactic dose is used in the subject prior to or at an early stage of the disease. In the case of cancer, the therapeutically effective amount of the drug can, for example, reduce the number of cancer cells and / or; Reduce tumor size and / or; May inhibit (i.e., delay to some extent, preferably stop and / or inhibit) cancer cell invasion into the peripheral organs; May inhibit (i.e., delay to some extent, preferably stop and / or inhibit) tumor metastasis; To some extent inhibit tumor growth and / or; One or more of the symptoms associated with cancer can be alleviated to some extent. See above definition of "treatment ". The drug may exhibit cell proliferation inhibition and / or cytotoxicity to the extent that the drug may interfere with the growth of existing cancer cells and / or may kill existing cancer cells.

"Chronic" administration refers to the administration of the agent (s) in a chronic manner, as opposed to an acute mode, to maintain an initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration is not performed continuously without interruption, but rather is periodic treatment in nature.

Administration together with one or more additional therapeutic agents includes simultaneous (concurrent) administration and sequential administration in any order.

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that do not exhibit toxicity to cells or mammals exposed thereto at the dosages and concentrations employed. Often, the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt formation counter ions, such as sodium; And / or non-ionic surfactants such as TWEEN ™, polyethylene glycol (PEG) and PLURONICS ™.

The term "pharmaceutical formulation" refers to a formulation that exists in a form that makes the biological activity of the active ingredient effective and does not contain additional ingredients that exhibit unacceptable toxicity to the subject to which the formulation is to be administered. Such formulations may be sterile.

A "sterile" preparation is an aseptic condition that does not have all the surviving microorganisms and their spores. An "effective amount" of an antibody disclosed herein is an amount sufficient to achieve the specifically mentioned purpose. An "effective amount" may be determined in an empirical and generic manner in connection with the stated purpose.

The term " therapeutically effective amount "refers to the amount of an antibody or other drug effective in" treating " a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; Reduce tumor size and / or; May inhibit (i.e., delay to some extent, preferably stop and / or inhibit) cancer cell invasion into the peripheral organs; May inhibit (i.e., delay to some extent, preferably stop and / or inhibit) tumor metastasis; To some extent inhibit tumor growth and / or; One or more of the symptoms associated with cancer can be alleviated to some extent. See the definition of "treatment" herein. The drug may exhibit cell proliferation inhibition and / or cytotoxicity to the extent that the drug may interfere with the growth of existing cancer cells and / or may kill existing cancer cells. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result over a required period of time at the required dose. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since the prophylactic dose is used in the subject prior to or at an early stage of the disease.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term includes radioactive isotopes such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu; Chemotherapeutic agents such as, for example, methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, Melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents; Enzymes and fragments thereof, such as nucleolytic enzymes; Antibiotic; Bacterial, fungal, plant or animal-derived toxins, such as small molecule toxins or enzyme active toxins, including fragments and / or variants thereof; And various anti-tumor agents or anti-cancer agents disclosed below. Other cytotoxic agents are described below. Tumor agents cause destruction of tumor cells.

A "toxin" is any substance that may have deleterious effects on cell growth or proliferation.

A "chemotherapeutic agent" is a compound useful in the treatment of cancer regardless of the mechanism of action. Chemotherapeutic agents include those used in "targeted therapy" and conventional chemotherapy. Examples of chemotherapeutic agents are, but are not limited to, following materials: alkylating agents, e. G., Thiotepa (thiotepa), between toksan (CYTOXAN) ^® cyclophosphamide; Alkyl sulfonates such as busulfan, improsulfan and piposulfan; Aziridine, such as benzodopa, carboquone, meturedopa, and uredopa; Ethyleneimine and methylamelamine including altretamine, triethylenemelamine, triethylenepoformamide, triethylenethiophosphoramide, and trimethylolromelamine; Acetogenins (in particular, bullatacin and bullatacinone); Delta-9-tetrahydro-car butterfly play (play draw butterfly (dronabinol), horses play (MARINOL) ^®); Beta-lapachone; Lapachol; Colchicines; Betulinic acid; Camptothecin (camptothecin) (including the synthetic analogue topotecan (topotecan) (hayikamtin ^{(HYCAMTIN) ®)), CPT} -11 ( irinotecan (irinotecan), Kam neoplasm? Sar (CAMPTOSAR) ^®), acetyl camptothecin, Scotland Scopolectin and 9-aminocamptothecin); Bryostatin; Callystatin; CC-1065 (including its analogues adozelesin, carzelesin and bizelesin synthetic); Podophyllotoxin; Podophyllinic acid; Teniposide; Cryptophycins (especially cryptophycin 1 and cryptophycin 8); Dolastatin; Duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); Eleutherobin; Pancratistatin; Sarcodictyin; Spongistatin; Nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine, Norembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, and the like; Nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and lanimnustine; Antibiotics such as enediyne antibiotics (e.g. calicheamicin, especially calicheamicin gamma II and calicheamicin omega II (see, for example, Angew. Chem Intl. Ed. Engl Dynemicin including esperamicin and neocarzinostatin chromophore and related chromogenic protein enediene (see, for example, Dynemicin A, 33: 183-186 (1994) Acacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, and the like), antibiotics (such as antibiotics), aclacinomysins, actinomycin, , Carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L - norleucine, adriamycin ^®, doxorubicin (morpholino-poison Epirubicin, esorubicin, idarubicin, and marcelolone (including but not limited to rubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin) Marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, The compounds of the present invention include quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin); Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azacitidine, 6-aza azauridine, carmofur, cytarabine, dideoxy vaculin, doxifluridine doxifluridine, enocitabine, floxuridine; Androgens, such as, for example, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; Anti-adrenals, such as aminoglutethimide, mitotane, trilostane; Folic acid supplements, such as frolinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfornithine; Elliptinium acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pyrarubicin; Losoxantrone; 2-ethylhydrazide; Procarbazine; PSK ^® polysaccharide conjugate (JHS Natural Products, Eugene, OR); Razoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid; Triaziquone; Trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine), urethanes, urethanes, ; Vindesine (ELDISINE ^® , FILDESIN ^® ); dacarbazine; mannomustine; mitobronitol; mitolactol; million (pipobroman); lyrics Ito Shin (gacytosine); arabinoside ( "Ara-C");thiotepa; such a modified toxin, for example, Taxol ^® paclitaxel (paclitaxel) (Bristol-Myers Squibb on Blossom (Bristol (Myers Squibb Oncology, Princeton, NJ), ABRAXANE ™ Cremophor free, albumin-engineered nanoparticle preparations of paclitaxel (American Pharmaceutical Partners, USA Illinois syaum bugs material) and takso terephthalate (TAXOTERE) ^® dock, laundry cells (Rhone-poul Lenk in Sultanahmet (Rhone-Pou lenc Rorer), Antony, France material); claw is stale (chloranbucil); gemcitabine (Gem cut (GEMZAR) ^®); 6- thioguanine; Murray captopril purine; methotrexate; platinum analogs such as cisplatin and carboplatin (carboplatin ); vinblastine (belban (VELBAN) ^®); platinum; etoposide (VP-16); epoch spa imide; mitoxantrone; vincristine (on Corbin (ONCOVIN) leuco bobbin (; ^®); oxaliplatin (oxaliplatin) leucovovin); vinorelbine (vinorelbine) (or belbin (NAVELBINE) ^®); roadbed anthrone (novantrone); eda track glyphosate (edatrexate); daunomycin; aminopterin (aminopterin); ibandronate (ibandronate); Topo iso Topoisomerase inhibitors RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; Capecitabine (gel Roda (XELODA) ^®); A pharmaceutically acceptable salt, acid or derivative of any of the foregoing substances; And combinations of two or more of the foregoing substances, such as CHOP (acronym for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone), and FOLFOX (oxaliplatin combined with 5-FU and leucovorbin (Abbreviation for therapeutic treatment with ELOXATIN (TM)). Additional chemotherapeutic agents include cytotoxic agents useful as antibody drug conjugates, such as maytansinoids (e.g., DM1 and DM4) and auristatins (e.g., MMAE and MMAF).

The following materials are also included in the definition of "chemotherapeutic agent": (i) wherein that act to regulate hormone action on tumors, or inhibit-hormone, for example, tamoxifen (tamoxifen) (nolba index (NOLVADEX) ^® tamoxifen citrate Raloxifen, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON. ^® anti-estrogens and selective estrogen receptor modulator (SERM), including (toremifene (toremifene) citrate); (ii) aromatase inhibitors that inhibit the adrenal enzyme aromatase (aromatase) that control estrogen production in, for example, 4 (5) -imidazole, amino glue teti imide, MB's (MEGASE) ^® (methoxy guest roll acetate), aromatic Shin (AROMASIN) ^® (exo shemesh tan (exemestane); Pfizer), formate scalpel Tani (formestanie), sol (fadrozole), Libby low (RIVISOR) in Pas ^® (Boro sol (vorozole)), pemara (FEMARA) ^® (letrozole (letrozole); Novartis), and ahrimidekseu (ARIMIDEX) ^® (anastrozole (anastrozole); AstraZeneca); (iii) troxacitabine (1,3-dioxolanucleoside cytosine analog) as well as anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; (iv) protein kinase inhibitors such as MEK inhibitors (International Patent Application Publication No. WO 2007/044515); (v) lipid-phosphorylase inhibitors; (vi) antisense oligonucleotides, such as antisense oligonucleotides, such as PKC-alpha, Raf and H-Ras, such as oblimersen, which inhibit the expression of genes in the signal transduction pathways involved in abnormal cell proliferation, (agent or sense (GENASENSE) ^®,, Inc. (Genta Inc.) Genta); (vii) ribozyme, e.g., VEGF expression inhibitors (e.g., are not geo-atom (ANGIOZYME) ^®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, egg bektin (ALLOVECTIN) ^®, Leu bektin (LEUVECTIN) ^® and a back oxide (VAXID) ^®; PROLEUKIN ^® rIL-2; Topoisomerase 1 inhibitors such as, Ruhr totekan (LURTOTECAN) ^®; Abarelix (ABARELIX) ^® rmRH; (ix) anti-angiogenic agent, for example, bevacizumab (bevacizumab) (Avastin (AVASTIN) ^®, Genentech (Genentech)); And a pharmaceutically acceptable salt, acid or derivative of any of the foregoing.

As used herein, the term "prodrug" refers to a compound that may exhibit lower cytotoxicity to a cell than the parent compound or drug and may be activated in a mode that has higher activity by enzymatic or hydrolysis, Refers to a precursor or derivative form of a compound of the present invention that can be used. See, for example, Wilman, Prodrugs in Cancer Chemotherapy Biochemical Society Transactions, 14, pp. 375-382, 615 ^th Meeting Belfast (1986) and Stella et al., Prodrugs: A Chemical Approach to Targeted Drug Delivery, "Directed Drug Delivery, Borchardt et al., (Ed), pp. 247-267, Humana Press (1985). The prodrug of the present invention may be selected from the group consisting of a phosphate containing prodrug, a thiophosphate containing prodrug, a sulfate containing prodrug, a peptide containing prodrug, a D-amino acid-modified prodrug, a glyco A prodrug, a pyrogenic prodrug, a prodrug containing a? -Lactam, a prodrug containing an optionally substituted phenoxyacetamide, a prodrug containing an optionally substituted phenylacetamide, 5-fluorocytosine and another 5-fluoro uridine bulb But are not limited to, drugs. Examples of cytotoxic drugs that can be derivatized in the form of prodrugs for use in the present invention include, but are not limited to, the compounds of the present invention and chemotherapeutic agents such as the aforementioned chemotherapeutic agents.

"Small molecule" is defined herein as having a molecular weight of less than about 500 daltons.

"Isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or a mixed polymer that is substantially separated from a protein or complex, such as a ribosome or a polymerase, as well as other genomic DNA sequences that are naturally associated with the native sequence . The term encompasses nucleic acid sequences that have been removed from its naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. Substantially pure molecules include molecules in isolated form.

 "Polynucleotide" or "nucleic acid ", as used interchangeably herein, refers to a nucleotide polymer of any length and includes DNA and RNA. The nucleotide can be any substrate that can be introduced into the polymer by deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or by DNA or RNA polymerase or by synthetic reactions. Polynucleotides may include modified nucleotides, such as methylated nucleotides and analogs thereof. As used herein, "oligonucleotide" refers to short (generally) single-stranded (generally) synthetic polynucleotides having generally less than about 200 nucleotides in length, but not necessarily. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides can be equally applied to oligonucleotides as a whole.

The cells that produce the anti-CD98 antibody of the present invention may be selected from the group consisting of a hybridoma cell into which the nucleic acid encoding the antibody has been introduced, for example, a hybridoma cell deposited with the ATCC as well as a bacterial host cell and an eukaryotic host cell .

The term "package insert" is a conventionally included instruction in a commercial package of a therapeutic product, and includes information about symptoms, usage, dosage, administration, reasons for use, and / Used to refer to the instructions contained.

Composition and process

Antibodies that bind to CD98 are provided. An immunoconjugate comprising an anti-CD98 antibody is provided. The antibodies and immunoconjugates of the invention are useful, for example, for the diagnosis or treatment of disorders associated with altered expression of, for example, increased expression of CD98. In certain embodiments, the antibodies or immunoconjugates of the invention are useful for the diagnosis or treatment of cell proliferative disorders, such as cancer.

Anti-CD98 antibody

In one embodiment, the invention provides anti-CD98 antibodies that can be used as therapeutic agents herein. Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, bispecific antibodies and heteroconjugate antibodies as well as variants thereof with improved affinity or other properties.

1. polyclonal antibody

The antibody of the present invention may comprise a polyclonal antibody. Methods for producing polyclonal antibodies are known to those skilled in the art. A polyclonal antibody can be generated in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or adjuvant will be injected into the mammal by subcutaneous or intraperitoneal injection. The immunizing agent may comprise a CD98 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to exhibit immunogenicity in the mammal to be immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, sorbitol globulin, and soybean trypsin inhibitor. Examples of antigen adjuvants that may be used include Freund ' s complete antigen adjuvant and MPL-TDM antigen adjuvant (monophosphoryl lipid A, synthetic trehalose decolinomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. The mammals can then be bled and the serum analyzed for CD98 antibody titer. If desired, the mammal can be boosted until the antibody titer increases or stagnation is reached.

2. Monoclonal antibodies

The antibodies of the present invention may alternatively be monoclonal antibodies. Monoclonal antibodies can be prepared by using the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975) or by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567) . ≪ / RTI >

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit a lymphocyte capable of producing or producing an antibody that will specifically bind to the protein used for immunization. Alternatively, the lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).

The hybridoma cells thus produced are preferably seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of non-fused osteoma cells (also referred to as fusion partners). For example, when the amacrine cell cell lacks the enzyme hypoxanthine guanine phospholibosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridoma typically contains hypoxanthine, a substance that interferes with the growth of HGPRT deficient cells , Aminopterin and tamidine (HAT medium).

Preferred fusion partner myeloma cells are myeloma cells that exhibit sensitivity to a selection medium that is efficiently fused to support stable high-level production of antibodies by the selected antibody-producing cells and selected for non-fused parent cells. Preferred myeloma cell lines are murine myeloma cell lines, such as myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center (San Diego, CA) And SP63 and derivatives, such as the X63-Ag8-653 cell, available from the American Type Culture Collection (Manassas, Va.). Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the preparation of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987)).

The culture medium in which the hybridoma cells grow is analyzed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbant assay (ELISA). The binding affinity of monoclonal antibodies can be measured, for example, by the Scatchard analysis described in Munson et al., Anal. Biochem., 107: 220 (1980).

Once hybridoma cells are identified that produce antibodies with the desired specificity, affinity and / or activity, the clones can be subcloned into a limiting dilution procedure and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, for example, hybridoma cells can be injected into the abdominal cavity of mice to in vivo grow the hybridoma cells as multiple tumors in an animal.

The monoclonal antibody secreted by the subclone can be isolated and purified by common antibody purification procedures such as affinity chromatography (using, for example, Protein A or Protein G Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, Electrophoresis, dialysis or the like, from the culture medium, multiple liquids or serum.

DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to the genes encoding heavy and light chains of murine antibodies) do. Hybridoma cells are used as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and host cells that do not otherwise produce the antibody protein, e. The synthesis of monoclonal antibodies in recombinant host cells is obtained by transfecting the vector into E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells. References to recombinant expression of antibody-encoding DNA in bacteria include those described in Skerra et al., Curr Opinion in Immunol. 5: 256-262 (1993) and Pluckthun, Immunol. Revs. -188 (1992)).

In a further embodiment, an antibody-phage library generated using techniques described in, for example, Antibody Phage Display (Methods and Protocols, PM O'Brien and R. Aitken, eds, Humana Press, Totawa NJ, 2002) Monoclonal antibodies or antibody fragments may be isolated. In principle, a synthetic antibody clone is selected by screening a phage library containing phage displaying various fragments of the antibody variable region (Fv) fused to the phage coat protein. These phage libraries are screened against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus are separated from the non-binding clone in the library. The binding clones can then be eluted from the antigen and further concentrated with additional cycles of antigen adsorption / elution.

The variable domain is a single-chain Fv (scFv) fragment in which VH and VL are covalently linked via short flexible peptides as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994) , Or they may be functionally displayed on the phage as Fab fragments that are each fused to an invariant domain and interact non-covalently.

The repertoire of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, followed by a search for antigen binding clones as described in Winter et al. can do. The library from the immunized source provides high affinity antibodies to the immunogen without requiring the construction of a hybridoma. Alternatively, the inexperienced repertoire can be cloned to generate a single, non-autologous, and a single, human antibody to autoantigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993) Source. Finally, non-rearranged V gene segments from stem cells were cloned and coded for highly variable CDR3 regions as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992) And in vitro libraries can be synthetically prepared by using PCR primers containing random sequences to achieve in vitro rearrangement.

Screening of the library can be accomplished by a variety of techniques known in the art. For example, CD98 can be used to coat the wells of an adsorption plate, can be expressed on host cells that are fixed on an adsorption plate or used in cell sorting, or can be used for capture using streptavidin-coated beads May be conjugated to biotin, or may be used in any other way of panning the display library. The selection of antibodies with a slow dissociation kinetics (and good binding affinity) can be carried out using a long wash as described in Basset al., Proteins, 8: 309-314 (1990) and International Patent Application Publication No. WO 92/09690 1 phage display and the use of low coating densities of the antigens described in the literature (Marks et al., Biotechnol., 10: 779-783 (1992)).

After designing the appropriate antigen screening procedure to select for the phage clone of interest, the Fv sequence of the phage clone of interest and the Fv sequence of interest (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD CD98 antibody can be obtained by constructing a full-length anti-CD98 antibody clone using a suitable constant region (Fc) sequence as described in Klein et al., 1991, vol. 1-3.

3. Antibody fragments

The present invention encompasses antibody fragments. In certain circumstances, it is advantageous to use antibody fragments rather than whole antibodies. Smaller size fragments allow rapid removal and can lead to improved access to solid tumors. For review of specific antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived through proteolytic degradation of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992)) and Brennan et al. , Science, 229: 81 (1985)). However, these fragments can now be produced directly into recombinant host cells. The Fab, Fv, and ScFv antibody fragments are shown in FIG. Can be expressed both in E. coli or yeast cells, and secreted from such cells, thus facilitating the production of large quantities of these fragments. The antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab ' (Carter et al., Biol / Technology 10: 163-167 (1992)). The F (ab ') ₂ fragment can be recovered and chemically coupled directly from E. coli. According to another method, the F (ab ') ₂ fragment can be isolated directly from the recombinant host cell culture. Fab and F (ab ') ₂ fragments with increased in vivo half life including Salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv). International Patent Application Publication Nos. WO 93116185, US 5,571,894 and US 5,587,458. Since Fv and scFv are the only species with intact combining sites that lack constant regions, they may be suitable for reduced non-specific binding during in vivo use. The scFv fusion protein can be constructed to generate fusion of the effector protein at the amino or carboxy terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may be, for example, a "linear antibody" as described in the above cited references. Such linear antibodies may be monospecific or multispecific, e.g., bispecific.

The smallest binding construct derived from the antibody is a separate variable domain (V domain), also referred to as a single variable domain antibody (SdAb). Certain kinds of organisms, camel and cartilage fish, possess high-affinity single V-like domains mounted on Fc equivalent domain structures as part of their immune system (Woolven et al., Immunogenetics 50: 98-101, 1999; Streltsov et al., Proc Natl Acad Sci USA 101: 12444-12449, 2004). V-like domains (referred to as VhH in camels and referred to as V-NARs in sharks) typically display long surface loops that allow penetration of the cavity of the target antigen. In addition, they stabilize the isolated VH domain by shielding the hydrophobic surface patch. These VhH and V-NAR domains are used to manipulate the sdAb. Human V domain variants were designed using selection from phage libraries and other methods of generating stable high-binding VL-derived domains and VH derived domains.

4. Humanized antibodies

The present invention encompasses humanized antibodies. Various methods of humanizing non-human antibodies are known in the art. For example, the humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically derived from "import" By replacing the corresponding sequence of a human antibody with a hypervariable region sequence, it is essentially as described by Winter and co-workers (Jones et al. (1986) Nature 321: 522-525), Riechmann et al. (1988) Nature 332 : 323-327) and human (Verhoeyen et al. (1988) Science 239: 1534-1536).

In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six complementarity determining regions (CDRs) of the parent rodent antibody are grafted onto a human antibody backbone. (Padlan et al., FASEB J. 9: 133-139, 1995) confirmed that only about one-third of the residues in the CDRs were actually in contact with the antigen, and identified them as " . In SDR transplantation techniques, only SDR residues are grafted onto human antibody scaffolds (Kashmiri et al., Methods 36: 25-34, 2005).

The choice of human variable domains (both light and heavy chains) to be used in the production of humanized antibodies may be important in reducing antigenicity. According to the so-called "optimal-fit" method, the sequence of the variable domain of the rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence most similar to the rodent sequence is then accepted as a human framework for humanized antibodies (Sims et al. (1993) J. Immunol. 151: 2296; Chothia et al. 196: 901). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same backbone can be used for several different humanized antibodies (Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. 2623). In some cases, the backbone is derived from a consensus sequence of the most abundant human subclasses, V _L κ subgroup I (V _L κI) and V _H subgroup III (V _H III). In yet another method, the human germline gene is used in a source of skeletal region.

In an alternative paradigm based on a comparison of CDRs (referred to as hyper-humanization), FR homology is irrelevant. This method consists of comparing the non-human sequence with the functional repertoire of human germline genes. Next, a gene encoding a regular construct that is identical or closely related to the murine sequence is selected. Then, among the genes sharing the normal structure with the non-human antibody, the gene having the highest homology in the CDRs is selected as the FR donor. Finally, non-human CDRs are grafted onto these FRs (Tan et al., J. Immunol. 169: 1119-1125, 2002).

It is generally preferred that the antibody be humanized while retaining high affinity for the antigen and other favorable biological properties. To accomplish this goal, humanized antibodies are prepared according to one method, using a three-dimensional model of the parent and humanized sequences to analyze the parent sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are routinely available and will be familiar to those skilled in the art. A computer program can be used to illustrate and display the possible three-dimensional conformation of the selected candidate immunoglobulin sequence. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, Swiss PDB viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Investigations of these displays enable the analysis of possible roles of residues in the function of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from the acceptor sequence and the insertion sequence such that the desired antibody characteristics, such as increased affinity for the target antigen (s), is achieved. In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Another method of antibody humanization is based on the measurement of antibody humanity referred to as Human String Content (HSC). This method compares the mouse sequence to the repertoire of human germline gene and scales the difference as HSC. The target sequence is then humanized by maximizing its HSC and generating a number of different humanized variants (Lazaret al., Mol. Immunol. 44: 1986-1998, 2007), rather than utilizing the overall identity scales.

In contrast to the methods described above, humanized antibodies can be generated and selected using experimental methods. These methods are based on the generation of large libraries of humanized variants and selection of the best clones using enrichment techniques or high throughput screening techniques. Antibody variants can be isolated from phage, ribosome and yeast display libraries as well as by bacterial colony screening (Hoogenboom, Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24 : 523-529, 2006; Feldhaus et al., Nat Biotechnol. 21: 163-70, 2003; Schlapschy et al., Protein Eng. Des Sel 17: 847-60, 2004).

In the FR library method, a library is selected to introduce a collection of residue variants at a particular location in the FR and then select the FR that best supports the transplanted CDR. The moiety to be substituted may be part or all of the " Vernier "moieties identified as residues potentially contributing to the CDR structure (Foote and Winter, J. Mol. Biol. 224: 487-499, 1992) (Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997)).

In FR shuffling, instead of creating a combinatorial library of selected residue variants, the entire FRs are combined with non-human CDRs (Dall'Acqua et al., Methods 36: 43-60, 2005). The library can be screened for binding in a two-step selection process that first humanizes VL and then humanizes VH. Alternatively, a one-step FR shuffling process may be used. This process has been found to be more efficient than two-step screening because the antibodies generated exhibit improved biochemical and physicochemical properties including improved expression, increased affinity and thermal stability (Damschroder et al., Mol. Immunol. 44: 3049-60, 2007)

The "humaneering" method is based on the experimental confirmation of the essential minimum specificity determinants (MSDs) and is based on the evaluation of sequential substitution and binding of non-human fragments into the library of human FRs. The method begins with the region of CDR-3 of the non-human VH and VL chains and begins by progressively transferring other regions of the non-human antibody (including CDR-1 and CDR-2 of both VH and VL) . This method typically enables epitope retention and identification of antibodies from multiple subclasses with different human V intercept CDRs. Humaneering enables the isolation of antibodies with 91% to 96% homology to human germline gene antibodies (Aifenito, Cambridge Health & Institute of Third Annual PEGS, The Protein Engineering Summit, 2007).

5. Human Antibodies

The human anti-CD98 antibodies of the invention can be constructed by combining the Fv clone variable domain sequence (s) selected from human derived phage display libraries with known human constant domain sequence (s) as described above. Alternatively, the human monoclonal anti-CD98 antibodies of the invention can be prepared by the hybridoma method. Human myeloma cell lines and mouse-human xenogeneic myeloma cell lines for the production of human monoclonal antibodies are described, for example, in Kozbor J. Immunol., 133: 3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Marcel Dekker, Inc., New York, 1987) and Boerner et al., J. Immunol., 147: 86 (1991)).

It is also possible to produce transgenic animals (e. G., Mice) capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production upon immunization. Transgenic mice expressing human antibody repertoires have been used to generate high affinity human sequence monoclonal antibodies against a wide variety of potential drug targets. 1995, 6 (5): 561-6), Bruggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8 (4): 455- 8), U.S. Patent Nos. 6,075,181 and 6,150,584, and Lonberg et al., Nature Biotechnol. 23: 1117-1125, 2005.

Alternatively, human antibodies can be produced through the immortalization of human B lymphocytes producing antibodies directed against the target antigen (such B lymphocytes may be recovered from the individual or may have been immunized in vitro). (Boerner et al., J. Immunol., 147 (1): 86-95 (1986)), as described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, (1991)) and U.S. Patent No. 5,750,373.

Gene shuffling may be used to induce a human antibody from a non-human, e. G., Rodent antibody, wherein the human antibody has affinity and specificity similar to the starting non-human antibody. According to this method, also referred to as "epitope imprinting" or "guided selection ", the heavy or light chain variable region of the non-human antibody fragment obtained by the phage display technique described herein is replaced with a repertoire of human V- To produce a population of human chain / human chain scFv or Fab chimeras. Selection with an antigen will isolate the non-human chain / human chimeric scFv or Fab, wherein the human chain restores the antigen binding site that is destroyed upon removal of the corresponding non-human chain in the primary phage display clone (i. The epitopes guide (select) the selection of human chain partners. When the above procedure is repeated to replace the remaining non-human chain, human antibodies are obtained (see International Patent Application Publication No. WO 93/06213 and Osbourn et al., Methods., 36, 61-68, 2005 ) Reference). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides a complete human antibody that does not have non-human derived FR or CDR residues. Examples of guided selection for humanizing mouse antibodies to cell surface antigens include folate binding proteins (Figini et al., Cancer Res., 58, 991-996, 1998) present on ovarian cancer cells and high- CD147 (Bao et al., Cancer Biol. Ther., 4, 1374-1380, 2005).

A potential disadvantage of the guided selection method is that shuffling one antibody can result in epitope variability while keeping other antibodies constant. CDR retention can be applied to retain epitopes recognized by non-human antibodies (Kiimka et al., Br. J. Cancer., 83, 252-260, 2000; Beiboer et al., J. Mol Biol., 296, 833-49, 2000). In this method, non-human CDR-H3 is typically retained because this CDR is located at the center of the antigen binding site and has proven to be the most important region of the antibody in antigen recognition. However, in some cases, CDR-H3, CDR-L3 and CDR-L2 as well as CDR-H3 and CDR-L3 of non-human antibodies can be retained.

6. Bispecific antibodies

Bispecific antibodies are monoclonal antibodies with binding specificities for two or more different antigens. In certain embodiments, the bispecific antibody is a human or humanized antibody. In certain embodiments, one of the binding specificities is the binding specificity for CD98, and the other is the binding specificity for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of CD98. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing CD98. These antibodies possess a CDR98 binding arm and an arm that binds to a cytotoxic agent such as, for example, saponin, anti-interferon-alpha, vinca alkaloid, lysine A chain, methotrexate or radioisotope hapten. Bispecific antibodies can be produced as whole-length antibodies or antibody fragments (e. G., F (ab ') ₂ bispecific antibodies).

Methods for producing bispecific antibodies, such as the simultaneous expression of two immunoglobulin heavy-chain pairs with different specificity for two heavy chains, are known in the art (Milstein and Cuello, Nature, 305: 537 (1983) ). Further details on the generation of bispecific antibodies can be found, for example, in Bispecific Antibodies, Kontermann, ed., Springer-Verlag, Hiedelberg (2011).

7. polyvalent antibody

A polyvalent antibody can be internalized (and / or catabolized) more quickly than a divalent antibody by cells expressing the antigen to which the antibody binds. Antibodies of the present invention include multivalent antibodies (except for the antibodies of the IgM class) (e.g., having four or more antigens) with three or more antigen binding sites that can be readily generated by recombinant expression of a nucleic acid encoding the polypeptide chain of the antibody Antibody). The polyvalent antibody may comprise a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain comprises (or consists of) the Fc region or hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites at the amino-terminus of the Fc region. In certain embodiments, the polyvalent antibody comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, the polyvalent antibody comprises (or consists of) four antigen binding sites. The polyvalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein said polypeptide chain (s) comprises two or more variable domains. For example, the polypeptide chain (s) may comprise VD1- (X1) n-VD2- (X2) n-Fc, wherein VD1 is the first variable domain, VD2 is the second variable domain, and Fc Is one polypeptide chain of the Fc region, X1 and X2 are amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain (s) may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain or a VH-CH1-VH-CH1-Fc region chain. The polyvalent antibody herein may further comprise two or more (e.g., four) light chain variable domain polypeptides. The polyvalent antibody herein may comprise, for example, from about 2 to about 8 light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally further comprise a CL domain.

8. Operating the effect function

For effector function, it may be desirable, for example, to modify the antibody of the invention to enhance antigen-dependent cellular mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. For example, Lazaret al., Proc. Natl. Acad. Sci. USA 2006, 103 (11): 4005-4010); Presta, L. G., Curr. Opin. Immunol. 2008,20 (4): 460-70); And U.S. Patent Nos. 7,183,387, 7,332,581, and 7,335,742.

Alternatively or additionally, the cysteine residue (s) can be introduced into the Fc region to enable interchain disulfide bond formation in this region. The resulting allogeneic antibody may have improved internalization performance and / or increased complement mediated cell death and antibody dependent cellular mediated cytotoxicity (ADCC). See, for example, Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Heterozygous cross-linking agents described in Wolff et al., Cancer Research 53: 2560-2565 (1993) may be used to prepare homodimeric antibodies with improved anti-tumor activity. Alternatively, by having a double Fc region, antibodies capable of improved complement dissolution and ADCC performance can be engineered. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989). To increase the serum half-life of the antibody, a Salvage receptor binding epitope can be introduced into an antibody (particularly an antibody fragment), for example, as described in U.S. Patent No. 5,739,277. As used herein, the term "Salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3 or IgG4) responsible for increasing the in vivo serum half- Quot;

9. Alternative binders

The invention encompasses a non-immunoglobulin binding agent that specifically binds to the same epitope as the epitope bound by the anti-CD98 antibody disclosed herein. In some embodiments, the binding agent is identified as a substance that replaces the anti-CD98 antibody of the invention or is replaced by the anti-CD98 antibody of the invention in a competitive binding assay. These alternative binders can include, for example, any engineered protein scaffold of engineered protein scaffolds known in the art. Such scaffolds include, for example, anticalins based on lipocalin scaffolds, a protein construct characterized by a rigid beta-barrel backing of four hypervariable loops forming a ligand binding site. New binding specificities are engineered with targeted random mutagenesis in the loop region with functional display and guided selection (Skerra (2008) FEBS J. 275: 2677-2683). Other suitable scaffolds include, for example, adnexin or monobodies based on the 10th extracellular domain of human fibronectin III (Koide and Koide (2007) Methods Mol. Biol. 352: 95-109); ApiBody based on the Z domain of Staphylococcus protein A (Nygren et al. (2008) FEBS J. 275: 2668-2676); DARPin based on the ankyrin repeat protein (Stumpp et al. (2008) Drug. Discov. Today 13: 695-701); (Grabulovski et al. (2007) J. Biol. Chem. 282: 3196-3204) based on the SH3 domain of human Fyn protein kinase; Apitin based on Sac7d of Sulfolobus acidolarius (Krehenbrink et al. (2008) J. Mol. Biol. 383: 1058-1068); amylin based on yB-cristalline (Ebersbach et al. (2007) J. Mol. Biol. 372: 172-185); Avimers based on the A domain of membrane receptor proteins (Silverman et al. (2005) Biotechnol. 23: 1556-1561); Cysteine-rich knottin peptides (Kolmar (2008) FEBS J. 275: 2684-2690); And a modified Kunitz-type inhibitor (Nixon and Wood (2006) Curr. Opin. Drug. Discov. Dev. 9: 261-268). See Gebauer and Skerra (2009) Curr. Opin. Chem. Biol. 13: 245-255 for review.

Antibody variant

In some embodiments, amino acid sequence alterations (s) of the antibodies described herein are contemplated. For example, it is desirable to improve the binding affinity and / or other biological properties of antibodies (including, but not limited to, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity or solubility) can do. It is contemplated that, in addition to the anti-CD98 antibodies described herein, anti-CD98 antibody variants can be produced. Anti-CD98 antibody variants can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or synthesizing the desired antibodies or polypeptides. Those skilled in the art will recognize that amino acid changes can alter the post-translational process of the anti-CD98 antibody, for example, alter the number or location of glycosylation sites or alter the membrane anchoring properties.

A mutation can be a substitution, deletion or insertion of one or more codons that encode an antibody or polypeptide that alters the amino acid sequence relative to a native sequence antibody or polypeptide. Amino acid substitution may be the result of substitution of one amino acid with another amino acid having similar structural and / or chemical properties, e. G., The result of substitution of leucine with serine, i. E. Conservative amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. By permitting the insertion, deletion or substitution of amino acids in a sequence, and by testing the resulting mutant for activity exhibited by a full-length or mature natural sequence, an acceptable mutation can be identified.

Amino acid sequence insertions also include insertions within the sequence of single or multiple amino acid residues, as well as amino-terminal and / or carboxyl-terminal fusions ranging from the length of one residue to the length of the polypeptide containing more than 100 residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include antibodies and enzymes at the N-terminus or C-terminus (e.g., in the case of antibody-induced enzyme prodrug therapy) or fusions of polypeptides that increase the serum half-life of the antibody.

(b) the charge or hydrophobicity of the molecule at the target site, or (c) the effect of their effect on maintaining the volume of the side chain; and (c) the structure of the polypeptide backbone, Substantial changes in the biological properties of the antibody are achieved by selecting significantly different substitutions. Amino acids can be classified according to similarities in the properties of their side chains (A. L. Lehninger, in Biochemistry, 2nd ed., Pp. 73-75, Worth Publishers, New York (1975)):

(1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M);

(2) Non-charged polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q);

(3) Acid: Asp (D), Glu (E);

(4) Basicity: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues can be divided into groups based on common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) Acid: Asp, Glu;

(4) Basicity: His, Lys, Arg;

(5) Residues affecting chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

A non-conservative substitution would involve replacing a member of one of these classes with a member of another class. Such substituted moieties may be introduced into the conservative substitution moiety or into the remaining (non-conserved) moiety.

Mutations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Acid Res., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 1987), for DNA cloned to generate anti-CD98 antibody variant DNA. 10: 6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34: 315 (1985)), restriction selection mutagenesis (Wells et al., Philos, Trans. R. Soc. London SerA, 415 (1986)) or other known techniques.

Any cysteine residues that do not participate in maintaining the proper steric structure of the anti-CD98 antibody can also be substituted with serine in general to improve the oxidative stability of the molecule and prevent abnormal cross-linking. In contrast, the cysteine bond (s) can be added to an anti-CD98 antibody to improve its stability (particularly when the antibody is an antibody fragment, such as an Fv fragment). Cysteine engineered antibodies that can be used to generate antibody-drug conjugates are described, for example, in International Patent Application Publication No. WO 2006/034488.

In one embodiment, the anti-CD98 antibody molecule of the invention is a "de-immunized" antibody. A "de-immunized" anti-CD98 antibody is an antibody that is derived from a humanized or chimeric anti-CD98 antibody having within its amino acid sequence one or more alterations that reduce the immunogenicity of the antibody relative to each original non- de-immunized antibody to be. One of the procedures for generating such antibody mutants involves the identification and removal of T cell epitopes of antibody molecules. In the first step, the immunogenicity of the antibody molecule can be confirmed by a variety of methods known in the art, for example, in vitro confirmation of T cell epitopes or in-silico prediction of such epitopes. Once an important residue has been identified in T cell epitope function, mutations can be made to eliminate immunogenicity and retain antibody activity. For review, see, for example, Jones et al., Methods in Molecular Biology 525: 405-423, 2009.

In vitro affinity mature

In one embodiment, antibody variants having improved properties, such as affinity, stability or expression levels, as compared to the parent antibody can be matured in vitro. Similar to natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. The library of antibodies may be displayed as a Fab, scFv or V domain fragment on a surface of an organism (e.g., a phage, a bacterium, or a yeast) or in a state of being linked (either covalently or non- . The affinity selection of the displayed antibodies enables isolation of organisms or complexes bearing the genetic information encoding the antibody. Display methods, such as two or three rounds of mutagenesis and selection using phage display, typically generate antibody fragments with affinity within the low nanomolar range. Preferred Affinity The matured antibody will have a nanomolar or even picomole affinity for the target antigen.

Phage display is the most widely used antibody display and selection method. Antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions with bacteriophage coat proteins. The selection includes exposure to the antigen, which allows the phage-displayed antibodies to bind to their target (the process referred to as "panning"). The phages bound to the antigen are recovered and infected with the bacteria to generate phage for selection of additional rounds. See, for example, Hoogenboom, Methods. Mol. Biol. 178: 1-37, 2002) and the literature (Bradbury and Marks, J. Immuno. Methods 290: 29-49, 2004).

In the yeast display system (Boder et al., Nat. Biotech. 15: 553-57, 1997; Chao et al., Nat. Protocols 1: 755-768, 2006) And displayed as linked single chain variable fusions (scFv). The scFv is fused to the attachment subunit of the yeast aggregation protein Aga2p attached to the yeast cell wall via disulfide bonds with Aga1p. The display of proteins through Aga2p minimizes potential interactions with other molecules on the yeast cell wall by allowing proteins to protrude from the cell surface. Antibodies with improved affinity or stability are selected by screening the library using magnetic separation and flow cytometry. Yeast is labeled with biotinylated antigen and a secondary reagent, such as streptavidin conjugated to a fluorophore, to confirm binding of the soluble antigen of interest. Mutations in the surface expression of the antibody can be measured by immunofluorescence labeling of the hemagglutinin or c-Myc epitope tag flanking the scFv. Since expression has been shown to correlate with the stability of the displayed protein, antibodies can be selected for improved stability and affinity (Shusta et al., J. Mol. Biol. 292: 949-956, 1999). A further advantage of the yeast display is that the displayed protein folds within the endoplasmic reticulum of eukaryotic yeast cells using an endoplasmic reticulum chaperon and a quality control device. Once maturation is complete, antibody affinity can be conveniently 'titrated' while being displayed on the surface of the yeast, eliminating the need for expression and purification of each clone. The theoretical limit of the yeast surface display is potentially a smaller functional library size than the functional library size of the other display methods, but recent methods use a mating system of enzyme cells to generate a putative diversity estimated at 10 ¹⁴ in size Patent Publication No. 2003 / 0186,374 and Blaise et al., Gene 342: 211-218, 2004).

In the ribosomal display, an antibody-ribosome-mRNA (ARM) complex is generated for selection in a cell-free system. A DNA library encoding a particular library of antibodies is genetically fused to a spacer sequence that lacks a stop codon. This spacer sequence is still attached to the peptidyl tRNA when it is translated and occupies the ribosomal tunnel so that the protein of interest can be folded out of the ribosome and folded. The resulting complex consisting of mRNA, ribosomes and protein binds to the surface-bound ligand, enabling simultaneous isolation of the antibody and its coding mRNA through affinity capture by the ligand. The ribosome-associated mRNA can then be reverse transcribed back to the cDNA, which can then be used to mutate the next round of selection (Fukuda et al., Nucleic Acids Res. 34, e127, 2006). In mRNA display, puromycin is used as the adapter molecule to establish covalent linkage between antibody and mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98, 3750-3755, 2001).

Because these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but is limited only by the number of ribosomes and different mRNA molecules present in the test tube. Second, since the library should not be transformed after an arbitrary diversification step, a random mutation can be easily introduced into each of the selection rounds, for example, with a proofreading polymerase.

The diversity can be introduced into CDRs or whole V genes of the antibody library in a targeted manner or through random introduction. The electron method (targeted approach) is a sequential targeting of all CDRs of antibodies through high or low level mutagenesis, or isolated hot spots of somatic hypermutation (Ho, et al., J. Biol. Chem. 280: 607- 617, 2005) or targeting of residues that are suspected of affecting affinity on an experimental basis or for structural reasons. DNA polymerase (Hawkins et al., J. Mol. Biol. 226: 889-896, 1992) Random mutations can be introduced into the entire V gene using error inducible replication of the E. coli mutant strain. (Lu et al., J. Biol. Chem 278: 43496-43507, 2003; and U.S. Patent Nos. 5,565,332 and 6,989,250 number). Alternative techniques for targeting hypervariable loops that extend into the framework region residues (Bond et al., J. Mol. Biol. 348: 699-709, 2005) use loop deletion and insertion in CDRs, (US Patent Publication No. 2004/0005709). An additional method of generating diversity in CDRs is disclosed in U.S. Patent No. 7,985,840.

Screening of libraries can be accomplished by a variety of techniques known in the art. For example, CD98 can be immobilized on a solid support, column, pin or cellulose / poly (vinylidene fluoride) membrane / other filter, or can be expressed on a host cell affixed to an adsorption plate or used in cell sorting Or it may be conjugated to biotin for capture by streptavidin-coated beads, or it may be used in any other way of panning the display library.

For review of in vitro affinity maturation methods, see Hoogenboom, Nature Biotechnology 23: 1105-1116, 2005 and Quiroz and Sinclair, Revista Ingeneria Biomedia 4: 39-51, 2010, See literature.

Modification of anti-CD98 antibody

Sharing modifications of anti-CD98 antibodies are included within the scope of the present invention. Covalent modification comprises reacting a targeted amino acid residue of an anti-CD98 antibody with an organic derivatizing agent capable of reacting with a selected side chain or N-terminal or C-terminal residue of an anti-CD98 antibody. Other variations include the deamidation of the glutaminyl and asparaginyl moieties to the corresponding glutamyl and aspartyl residues, respectively; Hydroxylation of proline and lysine; Phosphorylation of hydroxyl groups of seryl or threonyl residues; (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)) of lysine, arginine and histidine side chains; Acetylation of the N-terminal amine; And amidation of any C-terminal carboxyl group.

Other types of covalent modifications of the anti-CD98 antibodies included within the scope of the present invention include altering the native glycosylation pattern of the antibody or polypeptide (Beck et al., Curr. Pharm. Biotechnol. 9: 482-501, 2008; Walsh, Drug Discov. Today 15: 773-780, 2010), and in a manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337, One of the polymers, for example polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.

The anti-CD98 antibodies of the invention may also be used in combination with another heterologous polypeptide or amino acid sequence, such as the epitope tag (Terpe, Appl. Microbial Biotechnol. 60: 523-533, 2003) or the Fc region of an IgG molecule (Aruffo, "Immunoglobulin to form a chimeric molecule comprising an anti-CD98 antibody fused to a fusion protein "in Antibody Fusion Proteins, SM Chamow and A. Ashkenazi, eds., Wiley-Liss, New York, 1999, pp. 221-242) It is possible.

Preparation of anti-CD98 antibody

Anti-CD98 antibodies can be prepared by culturing transformed or transfected cells with a vector containing the anti-CD98 antibody encoding nucleic acid. Standard recombinant techniques can be used to obtain a polynucleotide sequence encoding the polypeptide component of the antibody of the invention. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR technique. Once the sequence encoding the polypeptide is obtained, the sequence is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in the host cell. Many vectors available in the art and known in the art can be used for the purposes of the present invention. The selection of the appropriate vector will mainly depend on the size and vector of the nucleic acid to be inserted into the vector and the specific host cell to be transformed. Suitable host cells for expressing an antibody of the invention include prokaryotic organisms including gram negative or gram positive organisms such as archaea and calm bacteria, eukaryotic microorganisms such as filamentous fungi or yeasts, invertebrate cells such as insects or plant cells , And vertebrate cells such as mammalian host cell lines. The host cells are transformed with the expression vectors described above and, if appropriate, cultured in a modified universal nutrient medium to induce a promoter, select transformants or amplify the gene encoding the desired sequence. The antibody produced by the host cell is purified using standard protein purification methods known in the art.

Methods of constructing antibodies, including vector construction, expression and purification, are further described in the following references: Pluckthun et al. (1996) in Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation (McCafferty, J , Hoogenboom, HR, and Chiswell, DJ, eds), 1 Ed., Pp. 203-252, IRL Press, Oxford); (Kwong, K. & Rader, C. E. coli expression and purification of Fab antibody fragments. Current protocols in protein science editorial board John E Coligan et al., Chapter 6, Unit 6.10 (2009)); (Tachibana and Takekoshi, "Production of Antibody Fab Fragments in Escherichia coli, " in Antibody Expression and Production, M. Al-Rubeai, Ed .; Springer, New York, 2011); And Therapeutic Monoclonal Antibodies: From Bench to Clinic (Ed Z. An, John Wiley & Sons, Inc., Hoboken, NJ, USA).

Of course, it is contemplated that anti-CD98 antibodies may be prepared using alternative methods well known in the art. For example, direct peptide synthesis using solid phase techniques can generate the appropriate amino acid sequence or a portion thereof (see, for example, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969) and Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963)). In vitro protein synthesis can be performed using manual techniques or automation. Various portions of anti-CD98 antibodies can be chemically synthesized and combined using chemical or enzymatic methods to produce the desired anti-CD98 antibody. Alternatively, the antibody can be purified from cells or body fluids, such as lactose, of a transgenic animal engineered to express the antibody, for example, as disclosed in U.S. Patent Nos. 5,545,807 and 5,827,690.

Immunoconjugate

The present invention provides immunoconjugates (referred to interchangeably as "antibody drug conjugates" or "ADCs") comprising any of the anti-CD98 antibodies of the invention covalently linked to one or more cytotoxic agents by a synthetic linker ). The ADC combines the high specificity of monoclonal antibodies with the pharmacological efficacy of cytotoxic molecules, allowing cytotoxic agents to be specifically targeted to tumor cells and avoiding the non-specific toxicity of most anti-cancer drugs. For review, see, for example, Carter and Senter, Cancer J. 14: 154-169 (2008); (Ducry and Stump, Bioconjugate Chem. 21: 5-13 (2010)); And Beck et al., Discov. Med. 10: 329-339 (2010)).

The cytotoxic agent used in the immunoconjugate of the present invention may be any of the aforementioned chemotherapeutic agents, drugs or growth inhibitors, toxins (for example, enzyme-activated toxins derived from bacteria, fungi, plants or animals, or fragments thereof) or radioactive isotopes . ≪ / RTI > In some embodiments, the immunoconjugate comprises a DNA binding agent (e. G., Calicheamicin) or a tubulin depolymerizing agent (e. G., Maytansinoid or auristatin). The present invention also contemplates an immunoconjugate formed between an antibody and a compound having nuclear lysis activity (for example, a ribonucleic acid degrading enzyme or a DNA nucleic acid internal degrading enzyme such as a deoxyribonuclease degrading enzyme; DNase).

Enzyme active toxins and fragments thereof that can be used in the immunoconjugates of the present invention include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (of Pseudomonas aeruginosa ), lysine A chain, Aleurites fordii protein, dianthine protein, Pitolaca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor , Kursin, croutin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restricin tosin, phenomycin, enomycin and tricothecene. See, for example, International Patent Application Publication No. WO 93/21232.

A variety of radioactive isotopes can be used for the production of radiolabeled antibodies. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes of Lu. When a conjugate is used for detection, the conjugate may be labeled with radioactive atoms for stereographic studies, e.g., tc ^99m or I ¹²³ , or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging or MRI) For example, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Radioactive isotopes are known, for example, as described in Reilly, " The radiochemistry of monoclonal antibodies and peptides, " in Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer, RM Reilly, ed., Wiley, Hoboken NJ, Can be introduced into the conjugate.

The linking agent may be a " cleavable linker "that facilitates release of the cytotoxic drug in the cell, but is not considered to be cleavable linkage. The linking agent used in the immunoconjugates of the invention may be selected from the group consisting of acid labile linkers (e.g., hydrazone linkers), disulfide containing linkers, peptidase sensitive linkers (e. G., Peptide linkers such as citrulline (Chari et al., Cancer Research 52: 127-131 (1992) and U.S. Pat. No. 5,208,020), thioether linkers, or multidrug transporters (Kovtun et al., Cancer Res. 70: 2528-2537, 2010). ≪ / RTI >

Various bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMS, Sulfo-GMBS, A conjugate composed of an antibody and a cytotoxic agent can be prepared using -MBS, sulfo-SIB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). The present invention provides a conjugate composed of an antibody and a cytotoxic agent using any suitable method disclosed in the art, for example, in Bioconjugate Techniques, 2 ^nd Ed., GT Hermanson, ed., Elsevier, San Francisco, It is also considered to be able to manufacture.

A universal antibody-drug conjugation strategy was based on a random conjugation chemistry using the ε-amino group of the Lys residue or the thiol group of the Cys residue to generate a heterogeneous conjugate. The recently developed technique enables site-specific conjugation with antibodies, which results in homogeneous drug loading and prevents ADC subpopulations with altered antigen binding or pharmacokinetic properties. These include the manipulation of "thiomaps" which provide reactive thiol groups and contain cysteine substitutions at certain positions on the heavy and light chains that do not destroy immunoglobulin junctions and assembly or alter antigen binding (Junutula et al. Immunol. Meth. 332: 41-52 (2008); Junutula et al., Nat. Biotechnol. 26: 925-932, 2008). In another method, the coding of the stop codon UGA from the termination to the insertion of Selenocysteine allows for the site specific covalent attachment of the selenocysteine in the presence of other natural amino acids to allow site specific covalent attachment of the selenocysteine to the antibody (Hofer et al., Proc Natl Acad Sci USA 105: 12451-12456 (2008); Hofer et al., Biochemistry 48 (50): 12047-12057, 2009).

Pharmaceutical preparation

The antibody or antibody-drug conjugate (ADC) of the present invention may be administered by any route appropriate to the condition being treated. The antibody or ADC will typically be administered parenterally, i. E., Vaginally, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally, and epidurally.

In one embodiment, for the treatment of cancer, the antibody or antibody-drug conjugate is administered via an intravenous route. The dose administered through the route is in the range of about 1 [mu] g / m < 2 > to about 10,000 [mu] g / m < 2 > per dose, generally once per week for a total of 1, 2, 3 or 4 doses. Alternatively, the dosage range may be from about 1 μg / m2 to about 1,000 μg / m2, from about 1 μg / m2 to about 800 μg / m2, from about 1 μg / m2 to about 600 μg / From about 10 μg / m2 to about 200 μg / m2, and from about 1 μg / m2 to about 200 μg / m2, from about 10 μg / m2 to about 500 μg / Mu g / m < 2 >. (But less than once a day), monthly (but less than once a day), monthly (but once a week, once a week) to relieve or alleviate symptoms of the disease, Less than once per day), once a month or intermittently. Administration may continue at any of the intervals disclosed until the tumor being treated or the cancer symptoms are remedied. Administration may continue after remission or alleviation of symptoms is achieved, wherein such remission or relief is extended by such continued administration.

In one embodiment, the invention further provides a pharmaceutical formulation comprising one or more anti-CD98 antibodies of the invention and / or one or more immunoconjugates thereof and / or one or more anti-CD98 antibody-drug conjugates of the invention. In some embodiments, the pharmaceutical agent comprises 1) an anti-CD98 antibody and / or an anti-CD98 antibody-drug conjugate and / or an immunoconjugate thereof, and 2) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical agent comprises 1) an anti-CD98 antibody and / or an immunoconjugate thereof, and optionally 2) one or more additional therapeutic agents.

The pharmaceutical preparation comprising the antibody or immunoconjugate of the present invention or the antibody-drug conjugate of the present invention may be prepared by mixing the antibody or antibody-drug conjugate having the desired degree of purity with an optional physiologically acceptable carrier, excipient or stabilizer (Remington ' s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of aqueous solutions, or lyophilized or other dried formulations. The formulations herein may contain more than one active compound required for the particular condition being treated, preferably compounds having complementary activity that do not adversely affect each other. For example, in addition to an anti-CD98 antibody, additional antibodies, for example, a second anti-CD98 antibody that binds to a different epitope on the CD98 polypeptide, or a growth factor that affects the growth of some other target, It may be desirable to include the antibody in a preparation. Alternatively or additionally, the composition may further comprise a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an anti-hormone agent and / or a cardioprotectant. Such molecules are suitably present together in effective amounts for the intended purpose.

The antibodies or immunoconjugates of the invention may be in any form suitable for delivery to the target cell / tissue, for example, microcapsules or macroemulsions (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. al., Molecules 10: 146-161 (2005); Malik et al., Curr. Drug. Deliv 4: 141-151 (2007)); Sustained-release preparations (Putney and Burke, Nature Biotechnol. 16: 153-157, (1998)); Or liposomes (Maclean et al., Int. J. Oncol. 11: 235-332 (1997), Kontermann, Curr. Opin. Mol. Ther. 8: 39-45 (2006)).

Treatment method

The antibodies or immunoconjugates of the present invention can be used, for example, in vitro, in vitro and in vivo therapeutics. In one aspect, the invention provides a method of inhibiting cell growth or proliferation in vivo or in vitro comprising exposing the cells to an anti-CD98 antibody or an immunoconjugate thereof, under conditions permitting binding of the immunoconjugate to CD98 . ≪ / RTI > By "inhibiting cell growth or proliferation" is meant a decrease in cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% And includes inducing apoptosis. In certain embodiments, the cell is a tumor cell. In certain embodiments, the cell is a bladder, breast, colon, rectum, stomach, esophagus, lung, larynx, kidney, oral, ovarian or prostate tumor cell, or sarcoma, melanoma, glioma, lymphoma or leukemia cell.

In one embodiment, the antibody or immunoconjugate of the invention is used for the treatment or prophylaxis of a cell proliferative disorder, such as cancer. In certain embodiments, the cell proliferative disorder is associated with increased expression and / or activity of CD98. For example, in certain embodiments, the cell proliferative disorder is associated with increased expression of CD98 on the surface of cancer cells. Examples of cell proliferative disorders to be treated by the antibody or immunoconjugate of the present invention include bladder cancer, breast cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, Glioma, lymphoma or leukemia, or metastasis of any of these cancers.

In one aspect, the invention provides a method of treating a cell proliferative disorder, comprising administering to the subject an effective amount of an anti-CD98 antibody or an immunoconjugate thereof. In certain embodiments, a method of treating a cell proliferative disorder comprises administering to a subject an effective amount of a pharmaceutical preparation comprising an anti-CD98 antibody or an anti-CD98 immunoconjugate, and optionally one or more additional therapeutic agents, such as the additional therapeutic agents provided below . In one embodiment, the anti-CD98 antibody or immunoconjugate can be conjugated to CD98 by forming an antibody or immunoconjugate-antigen complex by contacting the antibody or immunoconjugate with CD98 so that the conjugated cytotoxic agent of the immunoconjugate approaches the interior of the cell, Can be used to target. In one embodiment, the conjugated antibody or immunoconjugate is internalized into cancer cells expressing CD98.

Anti-CD98 antibodies or immunoconjugates can be administered to humans for therapeutic purposes. Moreover, an anti-CD98 antibody or immunoconjugate may be administered to a non-human mammal (e.g., a primate, a pig, a rat or a mouse) expressing CD98 in which the antibody cross-reacts for veterinary purposes or as an animal model of human disease . In the latter case (an animal model of human disease), such an animal model may be useful in assessing the therapeutic efficacy of the antibody or immunoconjugate of the invention (e. G., Dose and time course studies of administration).

The antibodies or immunoconjugates of the invention may be used alone or in combination with other compositions in therapy. For example, an antibody or immunoconjugate of the invention may be administered with one or more additional therapeutic agents and / or adjuvants. In certain embodiments, the additional therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, or a growth inhibitor. In some embodiments, the chemotherapeutic agent is administered in combination with a medicament or combination of agents, such as an alkylating agent (e.g., vendamustine hydrochloride, cyclophosphamide or isopa-midi), a nucleoside analog (e. (Eg, paclitaxel, docked cell or vinorelbine), vinca alkaloids (eg, cyclophosphamide), corticosteroids (eg, prednisone, prednisolone or methylprednisolone) For example, vincristine or etoposide, topoisomerase inhibitors such as irinotecan, antibiotics such as anthracycline or adriamycin, platinum analogs such as cisplatin or carboplatin, A therapeutic antibody (e. G., Rituximab) or a combination of agents (e. G., CHOP or CVP), wherein the combination therapy is useful for the treatment of cancer. In some embodiments, the additional compound is a therapeutic antibody (e. G., Rituximab) other than an anti-CD98 antibody.

Such recognized combination therapies encompass a combined administration wherein the two or more therapeutic agents are included in the same or separate agent, and separate administration, wherein administration of the antibody or immunoconjugate of the invention results in the administration of an additional therapeutic agent and / / RTI > and / or the administration of the adjuvant, concurrently with administration and / or after administration. The antibodies or immunoconjugates of the invention may also be used in conjunction with additional therapies including, but not limited to, radiation therapy and / or bone marrow and peripheral blood transplantation.

The antibody or immunoconjugate of the invention (and any additional therapeutic or adjuvant) may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired, for topical treatment, ≪ / RTI > Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody or immunoconjugate is suitably administered by a pulse tube, particularly reducing the dose of the antibody or immunoconjugate. The dosage may be achieved in part by any suitable route, for example injection, e.g. intravenous or subcutaneous, depending on whether the administration is a short-term or long-term administration.

The antibodies or immunoconjugates of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the method of administration, the schedule of administration, and other factors known to the physician. The antibody or immunoconjugate is optionally formulated with one or more agents currently used for the prophylaxis or treatment of the disorder, but need not be so. The effective amount of such other agents depends on the amount of antibody or immunoconjugate present in the formulation, the nature of the disorder or treatment, and other factors discussed above. They are generally used in the same dosage and route of administration as those described herein, or used in about 1% to 99% of the doses described herein, or as an appropriate dose / route of any of the experimentally / clinically identified Capacity and any path.

The appropriate dose of an antibody or immunoconjugate of the invention (either alone or when used in combination with one or more other therapeutic agents, such as a chemotherapeutic agent) for the prevention or treatment of a disease will depend on the type of disease being treated, the antibody or immunoconjugate The severity and course of the disease, the severity and course of the disease, whether the antibody or immunoconjugate is administered for prophylactic or therapeutic purposes, the pre-treatment, the clinical history of the patient and the response to the antibody or immunoconjugate, and the discretion of the primary care physician. The antibody or immunoconjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an antibody or immunoconjugate of about 1 ug / kg to 100 mg / kg (e.g., 0.1 mg / kg to 20 mg / kg) may be administered, for example, Or an initial candidate dose to be administered to a patient by a continuous conduit. A typical daily dose will be from about 1 [mu] g / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired inhibition of the disease symptoms occurs. An exemplary dose of an antibody or immunoconjugate will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to a patient. Such a dose may be administered intermittently, e. G., Every week or every three weeks, (e. G., To provide the patient with about 2 to about 20 or, for example, about 6 doses of antibody or immunoconjugate) . One or more lower doses may be administered following the loading dose higher than the initial dose. Exemplary dosing regimens include administration of the antibody at an initial loading dose of about 4 mg / kg followed by administration of the antibody at a weekly maintenance dose of about 2 mg / kg. However, other dosing regimens may be useful. The progress of this treatment is easily monitored by universal techniques and analysis.

Diagnostic and detection methods

In one embodiment, the anti-CD98 antibodies and immunoconjugates of the invention are useful for detecting the presence of CD98 in a biological sample. As used herein, the term "detection " encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In certain embodiments, such tissue is a normal and / or cancerous tissue that expresses CD98 at a relatively higher level than other tissues such as bladder cancer, breast cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, lung cancer, Cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma or leukemia, or metastasis of any of these cancers.

In one aspect, the invention provides a method of detecting the presence of CD98 in a biological sample. In certain embodiments, the method comprises contacting a biological sample with an anti-CD98 antibody under conditions that permit binding of the anti-CD98 antibody and CD98, and detecting whether a complex is formed between the anti-CD98 antibody and CD98 .

In one aspect, the invention provides a method of diagnosing a disorder associated with increased expression of CD98. In certain embodiments, the method comprises contacting a test cell with an anti-CD98 antibody; Measuring (quantitatively or qualitatively) the level of expression of CD98 by the test cell by detecting the binding of the anti-CD98 antibody to CD98; And comparing the level of expression of CD98 by the test cell with the level of expression of CD98 by control cells (normal cells derived from the same tissue as the test cells, or cells expressing CD98 at levels comparable to these normal cells) Wherein higher expression levels of CD98 by test cells relative to control cells indicate the presence of a disorder associated with increased expression of CD98. In certain embodiments, increased expression corresponds to a higher density of CD98 expression on the surface of tumor cells relative to normal cells. In certain embodiments, the test cell is obtained from an individual suspected of having a disorder associated with increased expression of CD98. In certain embodiments, the disorder is a cell proliferative disorder, such as cancer or a tumor. Exemplary cell proliferative disorders that can be diagnosed using the antibodies of the present invention include but are not limited to bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, Glioma, lymphoma or leukemia, or metastasis of any of these cancers.

In some embodiments, the diagnostic or detection method, e. G., The method described above, comprises detecting binding of CD98 and anti-CD98 antibodies in a membrane preparation obtained from cells expressing CD98 on CD98 or surface thereof expressed on the cell surface . In certain embodiments, the method comprises contacting the cell with an anti-CD98 antibody under conditions that permit binding of the anti-CD98 antibody and CD98, and detecting whether a complex is formed between the anti-CD98 antibody and CD98 on the cell surface . An exemplary assay for detecting binding of anti-CD98 antibody and CD98 expressed on the surface of a cell is "FACS" assay.

Certain other methods can be used to detect binding of anti-CD98 antibody to CD98. Such methods include, but are not limited to, antigen binding assays well known in the art, such as Western blot, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, But are not limited to, immunohistochemistry (IHC).

In certain embodiments, the anti-CD98 antibody is labeled. A label may be a moiety directly detectable, such as a label or moiety (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radiolabel), as well as a moiety indirectly detected through, for example, enzyme reaction or molecular interaction, , Enzymes, or ligands. Exemplary labels include radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorescent moieties such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbeliferone, (Lysine), such as, for example, the fruit fly luciferase and the bacterium luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazine diion, horseradish peroxidase (HRP), alkaline phosphatase, An enzyme that oxidizes a dye precursor such as HRP using glucose oxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, hydrogen peroxide, Such as, for example, free radicals and xanthine oxidase, lactoperoxidase or microperoxidase, biotin / avidin, spin label, bacteriophage Branch, but are not limited to, such as the stable free radical to contain one of these.

In some embodiments, the anti-CD98 antibody is immobilized on an insoluble matrix. Immobilization involves separating the anti-CD98 antibody from any CD98 that remains free in solution. This is commonly done by insolubilization of the anti-CD98 antibody prior to the analytical procedure, adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Patent No. 3,720,760), (for example using glutaraldehyde cross-linking) CD98 antibody after formation of a complex between the anti-CD98 antibody and the CD98 antibody, or by formation of a complex between the anti-CD98 antibody and CD98, for example by immunoprecipitation.

Any of the above embodiments of the diagnosis or detection can be performed using an immunoconjugate of the invention in place of or in addition to the anti-CD98 antibody.

analysis

Various assays known in the art can characterize the anti-CD98 antibodies and immunoconjugates of the invention in terms of their physical / chemical properties and / or biological activity.

Activity analysis

In one embodiment, an assay is provided to identify an anti-CD98 antibody having biological activity or an immunoconjugate thereof. Biological activity may include, for example, the ability to induce apoptosis, including the ability to inhibit cell growth or proliferation (e.g., "cell death" activity), or programmed cell death (apoptosis). Antibodies or immunoconjugates having such biological activity are also provided in vivo and / or in vitro.

In certain embodiments, the anti-CD98 antibody or immunoconjugate thereof is tested for its ability to inhibit cell growth or proliferation in vitro. Analysis of inhibition of cell growth or proliferation is well known in the art. A specific assay for cell proliferation, exemplified by the "apoptosis" assay described herein, measures cell viability. One such assay is the CellTiter-Glo ™ light-emitting cell viability assay commercially available from Promega (Madison, Wis.). The assay measures the number of viable cells in the culture based on the quantification of the ATP present, which is an indication of metabolic active cells. See Crouch et al (1993) J. Immunol. Meth. 160: 81-88) and U.S. Patent No. 6602677. The assay may be performed in a 96 well or 384 well format suitable for treating it with automated high throughput screening (HTS). See Cree et al., (1995) AntiCancer Drugs 6: 398-404.

Another cell proliferation assay is the colorimetric assay, MTT, which measures the oxidation of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductase "Analysis. Like the Celliter-Wright ™ assay, this assay indicates the number of metabolically active cells present in the cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65: 55-63) and Zhang et al. (2005) Cancer Res. 65: 3877-3882.

In one embodiment, the anti-CD98 antibody is tested for its ability to induce apoptosis in vitro. Analysis of the induction of apoptosis is well known in the art. In some embodiments, such assays are performed by, for example, administration of propidium iodide (PI), trypan blue (see Moore et al. (1995) Cytotechnology, 17: 1-11) Measure the loss of displayed film integrity. In an exemplary PI uptake assay, cells were cultured in Dulbecco's modified Eagle's medium (D-MEM) (50:50 horses F-12 supplemented with 10% heat-inactivated FBS (hypoclone) and 2 mM L- Lt; / RTI > Thus, the assay is performed in the absence of complement and immune effector cells. The cells are seeded in a 100 x 20 mm dish at a density of 3 x 10 ⁶ per dish and allowed to attach overnight. The medium is removed and replaced with fresh medium alone, or medium containing various concentrations of antibody or immunoconjugate. Cells are incubated for 3 days. After treatment, the monolayer is washed with PBS and desorbed by trypsinization. The cells were then centrifuged at 1200 rpm for 5 minutes at 4 ° C and the pellet was resuspended in 3 ml of cold Ca ²⁺ binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl ₂ ) Capsules are aliquoted into 35 mm strainer-capped 12 x 75 mm tubes (1 ml per tube, 3 tubes per treatment group) to remove turbid and cell clumps. PI (10 [mu] g / ml) is then added to the tube. Analyze the sample using a FACSCAN ™ flow cytometer and FACSCONVERT ™ Cell Quest software (Becton Dickinson). This confirms an antibody or immunoconjugate that leads to a statistically significant level of apoptosis as measured by PI uptake.

In one embodiment, the anti-CD98 antibody or immunoconjugate is tested for its ability to induce apoptosis (programmed cell death) in vitro. An exemplary assay for antibodies or immunoconjugates that induce apoptosis is the Annexin binding assay described in, for example, Zhang et al., BioTechniques 23: 525-531, 1997. Another exemplary assay for an antibody or immunoconjugate that induces apoptosis is a histone DNA ELISA colorimetric assay that detects degradation of the nucleomes of genomic DNA. For example, such assays can be performed using a cell death detection ELISA kit (Roche, Palo Alto, Calif., USA).

Cells used in any of the in vitro assays in these in vitro assays include cells or cell lines that have been engineered to express CD98 naturally or to express CD98. These cells include tumor cells that overexpress CD98 relative to normal cells derived from the same tissue. Such cells include cell lines expressing CD98 (including tumor cell lines), and cell lines that do not normally express CD98 but are transfected with nucleic acid encoding CD98.

In one embodiment, the anti-CD98 antibody or immunoconjugate thereof is tested for its ability to inhibit cell growth or proliferation in vivo. In certain embodiments, the anti-CD98 antibody or immunoconjugate thereof is tested for its ability to inhibit tumor growth in vivo. Biometric model systems, such as xenograft models, may be used for such tests. In an exemplary xenograft system, human tumor cells are suitably introduced into immunologically impaired non-human animals, such as SCID mice. The antibody or immunoconjugate of the present invention is administered to an animal. The ability of the antibody or immunoconjugate to inhibit or reduce tumor growth is measured. In certain embodiments of the xenograft system, the human tumor cells are human tumor cells. Such cells useful for preparing xenograft models include, but are not limited to, cells that express endogenous CD98 and cells that naturally express CD98. In certain embodiments, the human tumor cells are introduced into a suitable site of a non-human animal, such as a wired fat pad, suitably immunocompromised by subcutaneous injection or implantation.

Combination and other analyzes

In one embodiment, the anti-CD98 antibody is tested for its antigen binding activity. For example, in certain embodiments, the anti-CD98 antibody is tested for its ability to bind to exogenous or endogenous CD98 expressed on the surface of cells. FACS analysis can be used for these tests.

Panels of monoclonal antibodies raised against CD98 can be classified based on the epitopes they recognize (a process known as epitope vining). Epitope-binning is typically performed by using competitive assays to assess the ability of an antibody to bind to an antigen in the presence of another antibody. In an exemplary competition assay, immobilized CD98 is conjugated to a solution comprising a first labeled antibody that binds to CD98 and a second non-labeled antibody that is tested for binding to CD98 for its ability to compete with the first antibody Lt; / RTI > The second antibody may be present in the hybridoma supernatant. As a control, immobilized CD98 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permitting binding of the first antibody to CD98, the excess unbound antibody is removed and the amount of label associated with immobilized CD98 is determined. If the amount of label associated with immobilized CD98 is substantially reduced in the test sample relative to the control sample, this suggests that the second antibody competes with the first antibody for binding to CD98. In certain embodiments, the immobilized CD98 is present on the surface of the cell or in a membrane preparation obtained from a cell expressing CD98 on its surface.

High-throughput processing of epitope vining is also known in the art. See, for example, Jia et al., J. Immunol. Methods 2004, 288 (1-2): 91-98), which describes a multiplexed competitive antibody bining method for characterization of monoclonal antibodies; And Miller et al., J. Immunol. Methods 2011, 365 (1-2): 118-25) describing epitope recognition of murine monoclonal antibodies by multiplexed pairing analysis.

Epitope mapping

Epitope mapping is the process of identifying the binding site or epitope of an antigen on its target protein antigen. The antibody epitope may be a linear epitope or a conformational epitope. Linear epitopes are formed by consecutive amino acid sequences in proteins. A stereostructural epitope is discrete in the protein sequence but is formed from amino acids that coexist when folding the protein into its tertiary structure.

Various methods of mapping antibody epitopes on a target protein antigen are known in the art. These include mutagenesis methods, peptide scanning methods, display methods, methods involving mass spectroscopy, and structural determinations.

The site-directed mutagenesis method involves targeted site-directed mutagenesis that identifies important amino acids by systematically introducing substitutions along the protein sequence and then confirming the effect of each substitution on antibody binding. This can be carried out by the "alanine scanning mutagenesis" described in the literature (Cunningham and Wells (1989) Science 244: 1081-1085), or some other form of point mutagenesis of amino acid residues in human CD98. However, mutagenesis studies may also show amino acid residues that are important for the overall three-dimensional structure of CD98 but not directly involved in antibody-antigen contact, and other methods may be required to identify functional epitopes determined by this method .

Shotgun mutagenesis mapping uses a comprehensive plasmid-mutation library for the target gene, wherein each clone in the library has a unique amino acid mutation, and the entire library covers all amino acids in the target protein. Clones that constitute the mutant library are individually arranged in microplates, expressed in living mammalian cells, and tested for immunoreactivity with antibodies of interest. Amino acids critical to the antibody epitope are identified by loss of reactivity and then mapped onto the protein structure to visualize the epitope. By automating the analysis, new epitope maps can be derived from days to weeks. Because this technique uses the native structure of proteins in mammalian cells, both the linear epitope structure and the stereostructured epitope structure can be mapped onto the complex protein (Paes et al., J. Am. Chem. Soc. 131 20: 6952-6954 (2009); Banik and Doranz, Genetic Engineering and Biotechnology News 3 (2): 25-28 (2010)).

An epitope bound by an anti-CD98 antibody may be identified by using a peptide scanning method. Libraries of short peptide sequences from overlapping fragments of the target protein CD98 in peptide scanning are tested for their ability to bind to the antibody of interest. The peptides can be synthesized and used in a number of ways, such as, for example, in ELISA or via core, or on a chip on a "pesccan" method (WO 84/03564 and WO 93/09872) The peptide is screened for binding by any of the solid phase screening methods (Reineke et al., Curr. Opin. Biotechnol. 12: 59-64, 2001). Such peptide screening methods are incapable of detecting some discrete functional epitopes, i. E., Functional epitopes comprising amino acid residues that are not contiguous along the primary sequence of the CD98 polypeptide chain.

Lt; RTI ID = 0.0 > CLIPS < / RTI > (the chemical linkage of peptides onto the scaffold) can be used to map the conformational epitopes. The loose ends of the peptides are attached to the synthetic scaffolds so that the scaffold peptides can adopt the same spatial structure as the corresponding sequence of the intact protein. The CLIPS technique is used to immobilize linear peptides in a cyclic structure ('single-loop' format) and to coexist with different portions of the protein binding site ('double-loop' format) to generate a conformational epitope that can be analyzed for antigen binding Loop, " triple-loop " format, etc.) (U.S. Patent No. 7,972,993).

For example, epitopes bound by antibodies of the present invention may be mapped using display techniques including phage display, microbial display and ribosome / mRNA display described above. In these methods, a library of peptide fragments is displayed on the surface of the phage or cell. The epitopes are then mapped by screening the mAbs for these fragments using selective binding assays. A number of computer tools have been developed that allow the prediction of a conformational epitope based on linear affinity-selected peptides obtained using phage display (Mayrose et al., Bioinformatics 23: 3244-3246, 2007). A method for detecting a conformational epitope by phage display can also be used. Microbial display systems can also be used to identify appropriately folded antigenic fragments on cell surfaces to identify conformational epitopes (Cochran et al., J. Immunol. Meth. 287: 147-158, 2004; Rockberg et al., Nature Methods 5: 1039-1045, 2008).

Methods involving protein solubilization and mass spectrometry can also be used to identify antibody epitopes (Baerga-Ortiz et al., Protein Sci. 2002 June; 11 (6): 1300-1308). In limited protein lysis, the antigen is cleaved by different proteolytic enzymes in the presence and absence of antibody, and the fragments are identified by mass spectrometry. The epitope is the region of the antigen that is protected from protein lysis upon binding of the antibody (Suckau et al., Proc. Natl. Acad. Sci. USA 87: 9848-9852, 1990). Additional protein lysis-based methods can be used, for example, by selective chemical modifications (Fiedler et al., Bioconjugate Chemistry 1998, 9 (2): 236-234, 1998), epitope cleavage (Van de Water et al., Clin. Immunol. Immunopathol. 1997, 85 (3): 229-235, 1997) and a recently developed hydrogen-double hydrogen (H / D) exchange method (Flanagan, N., Genetic Engineering and Biotechnology News 3 28, 2010).

Epitopes bound by the antibodies of the present invention can be identified by structural methods such as X-ray crystallographic structure identification (e.g., International Patent Application Publication No. WO 2005/044853), molecular modeling and nuclear magnetic resonance (NMR) spectroscopy (Zinn-Justin et al. (1992) Biochemistry 31: < RTI ID = 0.0 > Zinn-Justin et al. (1993) Biochemistry 32: 6884-6891).

Additional antibodies that bind to the same epitope as the epitope bound by the antibody of the present invention include, for example, those that comprise fragments of human CD98 comprising an epitope sequence, such as by screening antibodies raised against CD98 for binding to an epitope For example, by immunizing an animal with a peptide, or by selecting an antibody for binding to an epitope sequence using phage display. Antibodies that bind to the same functional epitope are expected to exhibit similar biological activity, e. G., Blockade of the biological activity of CD98, and such activity can be ascertained by functional analysis of the antibody.

Additional activity analysis

In one embodiment, the anti-CD98 antibody of the invention is an antagonist antibody that inhibits the biological activity of CD98. The anti-CD98 antibodies of the present invention can be analyzed to ascertain whether they inhibit the biological activity of CD98, for example, binding to light chains. To confirm whether the CD98 antibody of the present invention inhibits binding to a light chain, the ability of CD98 antibody to inhibit amino acid uptake in cancer cell lines was examined (Kim et al., Biochim. Biophys. Acta 1565: 112-122, 2002) . ≪ / RTI >

In one embodiment, the purified anti-CD98 antibodies include, but are not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion Can be further characterized by a series of analyzes that do not.

In one embodiment, the present invention contemplates that although the half-life of an antibody in vivo is important because it possesses some effector function rather than all effector functions, a desirable candidate for many applications where certain effector functions (e.g., complement and ADCC) Lt; / RTI > In certain embodiments, the Fc activity of the antibody is determined to ensure that only the desired properties are retained. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks Fc [gamma] R binding (thereby potentially lacking ADCC activity) but retains FcRn binding capacity. One example of an in vitro assay that assesses ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be determined in vivo, for example, in animal models such as those described in Clynes et al. PNAS (USA) 95: 652-656 (1998) Can be evaluated. Clq binding assays may be performed to confirm that the antibody lacks CDC activity because it is unable to bind to C1q. To assess complement activation, a CDC assay as described in, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) can be performed. FcRn binding and in vivo elimination / half-life measurements can also be performed using methods known in the art.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the foregoing description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific references cited herein are hereby incorporated by reference in their entirety for clarity.

Example

The following examples are illustrative of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced in light of the general description provided above.

Example 1: Identification of CD98 on the surface of acute myelogenous leukemia (AML) tumor cells

A total of 16 primary AML samples were obtained from the Fred Hutchinson Cancer Research Center (FHCRC). Eleven samples of healthy donors were analyzed. Hematoxylin and eosin staining of AML cells was performed to monitor the quality of individual AML samples. Only samples containing 75% or more tumor cells were analyzed. In addition, there were 23 primary chronic lymphocytic leukemia (CLL) samples obtained from the Billings Clinic or the University of Florida, and 22 normal samples obtained from the Collaborative Human Tissue Network (CHTN) or the National Disease Research Interchange (NDRI) Analysis of adjacent colon control samples was performed. Sample handling was optimized to maximize cell viability during sample isolation. Optimal label attachment times for AML, CLL and CRC samples were measured to enable efficient label attachment without compromising cellular integrity.

Sixteen core AML samples, five bone marrow mononuclear cells (BMMC) controls and six peripheral blood mononuclear cell (PBMC) control samples, 20 core CLL samples, 27 with a surface tagged antigen profiling (sTAg) The repertoires of surface proteins on the cells in the CRC samples, and in the 22 normal adjacent colon samples were identified and quantitatively profiled. After chemically tagging the extracellular domain of the protein associated with the AML tumor cell membrane of intact primary tumor cells, the tagged protein was concentrated by chromatography using a solid phase affinity resin. The eluted proteins were stored at -80 캜 before the mass spectrometry analysis described below.

The protein concentrated by the sTAg method was identified and quantified using high resolution shorts liquid chromatography inline mass spectrometry (MS). The sensitivity of linear ion capture and the high resolution and mass accuracy conferred by the innovative Ovitrap mass spectrometer (Olsen et al., Mol. Cell Proteomics 8: 2759-2769, 2009) coupled to a nanofluidic liquid chromatographic apparatus The hybrid thermofixer LTQ-Orbit trap BELLOS mass spectrometer was used for shotgun-based bottoms-up proteomics to determine the nature and quantitative presence of protein in the AML cell surface enriched fraction (Yates et al., Annu. Rev. Biomed. Eng. 11: 49-79, 2009). Since the peptide mass and fragmentation pattern were dynamically recorded by mass spectrometry, trypsin digests of concentrated surface proteins were separated by hydrophobic on-line nano-flow liquid chromatography. To verify the identity of peptides and proteins, the raw MS data was processed using the SEQUEST algorithm implemented on the Rapid Processing Sorcerer 2 platform (Lundgren et al., Curr. Protoc. Bioinformatics, Chapter 13: Unit 13.3, 2009) To confirm an optimal-fit match between the experimental fragmentation pattern and the in-silico confirmed fragmentation pattern from the human proteome. (Keller et al., Anal. Chem. 74: 5383-5392, 2002) and ProteinProphet (Nesvizhskii et al., Anal. Chem. 75: 4646-4658, 2003) By statistically verifying the matches generated using this method, we ensure the lowest possible false detection rate (FDR), ensuring that only the confirmed protein is included in the candidate pool.

Relative quantitative levels of identified proteins in the sTAg sample were determined using a spectrophotometric method (Neilson et al., Proteomics 11: 535-553, 2011). Spectral counting is based on experimental evidence that the number of assigned (positively identified) spectra associated with peptides from each protein is strongly correlated with the relative presence of that protein in the original mixture (Liu et al. , Anal. Chem. 76: 4193-4201, 2004). Spectral counts of identified peptides were obtained from protein bacteriological analysis software platforms, including scaffold (Proteom Software) and Proteo IQ (NuSep), which displays, classifies and filters the results of mass spectrometry data retrieved by SEQUEST. The untreated spectral counts were converted to% normalized spectral presence factor (% NASF) values (Zybailov et al., J. Proteome Res. 5: 2339-2347, 2006) to resolve variability in protein length differences and sample size . Selected monoclonal antibodies were used to validate protein body measurements as an independent external confirmatory measure of sTAg mass spectrometry-based proteomic profiling of primary tumor cell surface expression using quantitative FACS.

Using sTAg, it was confirmed that the heterodimeric type II transmembrane II glycoprotein CD98 having the amino acid sequence of SEQ ID NO: 1 was present at high density on the surface of AML, CLL and CRC tumor cells. As shown in Figure 1, using sTAg, 20 of the 20 primary CLL samples with 7 samples of 16 primary AML samples with an average% NSAF of 0.11 and an average% NSAF of 0.15 and CD98 Respectively. CD98 was identified in five of five bone marrow mononuclear cell (BMMC) samples and five in six peripheral blood mononuclear cell (PBMC) samples with an average% NSAF of 0.05 and 0.06, respectively. Further, CD98 was identified in only one normal adjacent colon sample with 11 samples out of 27 primary CRC samples with an average% NSAF of 0.10 and an average% NSAF below 0.01. Based on this analysis, CD98 is substantially enriched on a significant portion of the patient-derived AML, CLL and CRC primary tumor specimens relative to the relevant normal control.

Example 2: Identification of CD98 in tumor

Antibody titration experiments were performed using anti-CD98 monoclonal antibody 8-34B (see Example 3) and homologous control antibody HB-121 to establish dilution ratios that allow for minimal background and maximal detection of the signal. (FFPE) or freshly frozen tissue using steam-based antigen retrieval (pH 6.0 citrate buffer) at 1:50, 1: 100, 1: 200 and 1: Serial dilutions were performed. Frozen control cell lines (F244 and F244-P) and formalin-fixed control cell lines (F244, RM and F244-P) were now provided by Igenica and manufactured by LifeSpan. For studies on formalin-fixed and paraffin-embedded tissues, a dilution ratio of 8-34B of 1:20 and 1:50 was chosen, while 1: 400 of 8-34B Dilution ratio was selected.

The primary detection system was a vector anti-mouse secondary antibody (BA-2000) and a vector ABC-AP kit (AK-5000) together with the vector red substrate kit (SK-5100) used to generate the fucsia- ). Tissues were also stained with positive control antibodies (for CD31 and Vimentin) to confirm that the tissue antigens were preserved and accessible for immunohistochemical analysis. Only tissues showing positive for CD31 and vimentin staining were selected for the remainder of the study.

Antibodies 8-34B at dilution ratios of 1: 20 and 1: 50 were found in 10 of the 15 malignant melanomas on formalin-fixed and paraffin-embedded tissues and in 4 of 18 lung carcinomas Positive stain. In addition, antibody 8-34B at a dilution ratio of 1: 400 showed positive staining of 6 samples of 6 frozen lung cancer species samples and 2 of 2 frozen melanoma samples.

Example 3: Preparation of monoclonal antibodies against CD98

Monoclonal antibodies were prepared according to the general methods described in the " Antibodies A Laboratory Manual "(Harlow and Lane 1988 CSH Press). Male 129S6 / SvEv mice purchased from Taconic Farms were used for immunization. 10 mice were immunized via lateral subcutaneous injection using ⁶ human CD98 (huCD98) expressing tumor cells. On day 39 after immunization, mice were boosted intraperitoneally using 5 million huCD98 expressing tumor cells. On day 42, the spleen was recovered. Individual splenocytes were prepared and fused with CRL-2016 myeloma cells (ATCC) using the PEG-based method described generally in the "Antibodies A Laboratory Manual" (Harlow and Lane 1988 CSH Press) Respectively.

Hybridomas were grown on 384-well tissue culture plates and the supernatant of individual wells was screened by ELISA for the generation of antibodies recognizing huCD98. The positive wells were then transferred to a 48 well plate and amplified and the supernatant was collected to confirm huCD98 binding by ELISA. Individual hybridomas were established that produced anti-HuCD98 antibodies as identified unique clones that produced monoclonal anti-huCD98 antibodies by plating single hybridoma cells into wells of 96 well plates. These cells were grown with a colony, and the supernatants of these individual colonies were screened by ELISA to identify monoclonal antibodies that bind to huCD98. Clonal hybridomas were injected into Pristane treated Balb / C mice to generate duplicates. Gammabind Sepharose (Gly Healthcare Product Code 17-0885-01), Protein A IgG binding buffer (Thermo Scientific Part Number < RTI ID = 0.0 > 21001) and IgG elution buffer (Thermo Scientific Part No. 21004).

The nucleic acid sequences and amino acid sequences for the heavy chain variable region and the light chain variable region of antibody 8-34B are shown below:

8-34B heavy chain variable region

8-34B light chain variable region

The nucleic acid sequences and amino acid sequences for the heavy chain variable region and the light chain variable region of antibody 18-2A 2.2 are shown below:

18-2A 2.2 heavy chain variable region

18-2A 2.2 Light chain variable region

The nucleic acid sequences and amino acid sequences for the heavy chain variable region and the light chain variable region of antibody 18-2A 7.1 are shown below:

18-2A 7.1 heavy chain variable region

18-2A 7.1 Light chain variable region

The nucleic acid sequence and amino acid sequence for the heavy chain variable region and the light chain variable region of antibody 1-47C are shown below:

1-47C heavy chain variable region

1-47C light chain variable region

The nucleic acid sequences and amino acid sequences for the heavy chain variable region and the light chain variable region of antibody 1-115A are shown below:

1-115A heavy chain variable region

1-115A light chain variable region

Example 4 Homologous Classification and Binding of Monoclonal Antibodies

Specific IgG2a HRP - product # 115-035-206, chlorine x IgG2b HRP - product # 115-035-206, goat x IgG2b HRP - product # 115-035-206, purchased from Jackson Immunologicals, Individual hybridoma supernatants containing antibodies recognizing huCD98 were evaluated for homologs by detection on ELISA using the product # 115-035-207, Chloro x IgG3 HRP-product # 115-035-209.

Competitive ELISA was performed to establish competitive binding bins. Individual hybrid anti-huCD98 homologous sorted antibody-containing hybridoma supernatants were allowed to bind to huCD98 in individual wells of ELISA plates. After 1 hour, the wells were washed and fixed using 4% paraformaldehyde. The hybridoma supernatants containing individual anti-huCD98 isoformed antibodies (antibodies of different isoforms) were then allowed to bind to huCD98 in individual wells of ELISA plates for 1 hour. After washing, the wells were incubated with a specific secondary antibody (Jackson Immunological Calc < RTI ID = 0.0 > 37069). Individual IgG2a allogeneic antibodies that could bind in the presence of IgG1 are considered to be present in a unique epitope bin from that particular IgG1. Individual IgG2a allogeneic antibodies that were unable to bind in the presence of IgG1 are considered to be present in the same epitope bin as that particular IgG1. In this way, multiple epitope bins for the huCD98 binding antibody were defined as illustrated in Fig.

Example 5: Binding Affinity

Purified anti-CD98 monoclonal antibodies are tested for affinity by the general methods disclosed by the literature (Carderelli et al. (2002) Cancer Immunol Immunother 51; 15-24). Briefly, CD98 expressing cells were incubated overnight with different amounts of anti-CD98 monoclonal antibodies followed by incubation with a goat anti-human Fc specific or anti-mouse Fc specific PE-conjugated secondary antibody (Jackson ImmunoLogic ). ≪ / RTI > The FACS data is analyzed using the graph pad prism software and the Kd is measured using the graph pad prism Kd calculation means.

Example 6: Preparation and characterization of chimeric antibodies

Total RNA was extracted from a hybridoma producing the anti-CD98 monoclonal antibody using a Qiagen RNeasy mini kit (Cat. No. 74104) followed by a Qiagen One Step RT-PCR kit (Cat. No. 210210). RT-PCR was performed using a primer set specific for murine heavy and light chain sequences. For each RNA sample, twelve separate heavy and 11 light chain RT-PCR reactions were set up using a degenerate forward primer mixture covering the leader sequence of the murine variable region. The reverse primer was located within the constant region of the murine heavy and light chains. The RT-PCR product from the first round reaction was further amplified in the second round PCR. Twelve individual heavy and 11 light chain RT-PCR reactions were set up using a semi-nested set of primers specific for antibody variable regions. The PCR reaction was run on an agarose gel and the heavy and light chain PCR products were cut from the gel and cloned into a sequencing vector. 10 to 20 clones per hybridoma were sequenced to identify anti-CD98 monoclonal antibody variable regions. The heavy chain variable region was then cloned in-frame into a vector containing the human IgGl heavy chain constant region sequence and the light chain variable region was cloned into a vector containing the human kappa light chain constant region sequence, . Chimera antibodies were generated from transient transfection of HEK293 freestyles cells and purified using the method described in Example 3. [

Purified chimeric antibodies were analyzed for binding as described in Example 4 for mouse monoclonal anti-CD98 antibodies. Figure 3a shows the results of competitive binding assays for 8-34B, 18-2A 2.1, 18-2A 2.2 and 18-2A 7.1 chimeric antibodies. Reference antibodies 1 to 4 are as shown in Figure 2, but "homologous" is a control IgG2a antibody that does not bind to CD98. The results shown in Figure 3A demonstrate that chimeric antibodies retain epitope binding specificity for their origin of murine antibodies. The binding affinity of murine anti-CD98 monoclonal antibody and chimeric anti-CD98 monoclonal antibody was measured by FACS analysis as described in Example 5 using the colon cancer cell line DLD1. The Kd values (0.9 nM to 4.5 nM) are shown in Figure 3b, which suggests that all of these recombinant antibodies retain high binding affinity for CD98, comparable to their mimoline antibodies. The chimeric monoclonal antibodies purified using three AML primary tumor samples and a cell line expressing cynomolgus monkey CD98 (cynCD98) were also analyzed by FACS as described in Example 5. The results presented in Figure 3c demonstrate that all chimeric antibodies retained binding to human CD98 and cynCD98 on AML cells.

CD98 monoclonal antibody and chimeric anti-CD98 monoclonal antibody by measuring the binding / dissociation rate using a Biacore system (reviewed in Lipschultz et al., Methods 20: 310-318, 2000) Binding affinity was measured. As shown in Table 2, the affinities of chimeric antibodies were similar to those of the mimic antibodies. Data for the humanized antibody 8-34B H2 L1 (see Example 7) are also presented.

Example 7: Preparation of humanized antibodies

As shown in Figure 4, the murine CDRs of the murine heavy and light chain variable domains were grafted into human acceptor framework regions to produce murine monoclonal antibody 8-34B in humanized form. The murine light chain CDRs were grafted into the human acceptor sequence (Jinbank Accession No. ACJ71709.1) to construct the humanized 8-34B light chain variable domain L1 (SEQ ID NO: 15). (Amino acid substitutions S63T and D70E according to Kabat numbering; see Fig. 4A), 8-34B humanized light chain variable domain L2 (SEQ ID NO: 16 ). The CDRs of the murine heavy chain were grafted into the human acceptor sequence (Jinbank Accession No. 137782) to construct the 8-34B humanized heavy chain H1 (SEQ ID NO: 17). Substitution of one residue in FR2 of the human acceptor heavy chain and two residues in FR3 with corresponding residues of the murine monoclonal antibody to generate amino acid substitutions I48L, V71K and F78V following carbation numbering to produce the 8-34B humanized heavy chain variable domain H2 (SEQ ID NO: 18) (see Fig. 4B). The 8-34B humanized heavy chain variable domain H3 (SEQ ID NO: 19) added two additional back-substitutions V67L and T73N to the murine residue in FR3 as shown in Figure 4b.

The nucleic acid sequences and amino acid sequences for the H2 heavy chain and L1 light chain variable regions of humanized antibody 8-34B are shown below:

The humanized 8-34B heavy chain variable region H2

The humanized 8-34B light chain variable region L1

The humanized forms of murine monoclonal antibody 18-2A were prepared by grafting the CDRs of the murine heavy and light chain variable domains of chimeric antibody 18-2A 7.1 into the human receptor framework region. The murine light chain CDRs were grafted into the human acceptor sequence (Jinbank Accession No. ACJ71709.1) to construct the humanized 18-2A 7.1 light chain variable domain L1 (SEQ ID NO: 20). A specific human framework residue was replaced with the corresponding residue of a murine monoclonal antibody to construct 18-2A 7.1 light chain variable domain L2 (SEQ ID NO: 21). The CDRs of the murine heavy chain were grafted into human acceptor sequences to construct 18-2A 7.1 humanized heavy chain variable domain H1 (SEQ ID NO: 22). A specific human backbone residue was replaced with the corresponding residue of a murine monoclonal antibody to construct the 18-2A 7.1 humanized heavy chain variable domain H2 (SEQ ID NO: 23).

Nucleic acid and amino acid sequences for the heavy and light chain variable regions of humanized antibody 18-2A 7.1 are provided below:

Humanized 18-2A 7.1 heavy chain variable region H1

Humanized 18-2A 7.1 light chain variable region L1

Humanized 18-2A 7.1 heavy chain variable region H2

Humanized 18-2A 7.1 Light chain variable region H2

Example 8: Anti-CD98 monoclonal antibody mediated inhibition of in vivo tumors

Antibody potency for tumor growth and metastasis formation is studied, for example, in subcutaneous or submucosal xenograft models. The antibody may be non-conjugated or conjugated to a therapeutic agent as is recognized in the art.

Monoclonal antibodies against CD98 are generated as described in Example 1 and purified and characterized as described above. The chimeric or humanized antibodies described above may also be used. Therapeutic monoclonal antibodies, or cocktails comprising a mixture of individual monoclonal antibodies, are used to treat mice treated with subcutaneous or isotopic injections of tumor xenografts.

1 x 10 ⁷ cancer cells in a mixture of 1: 1 (without magnesium or calcium) PBS and BD Matrigel (BD Biosciences) were injected into female right SCID or right flank of nu ^{- / -} mice To produce subcutaneous tumors. The total volume injected per mouse is 200 ml, where 50% is Matrigel (Vidi Biocysis). Once the tumors reach a size of 65 to 200 마우스, the mice are randomized. Antibody is administered weekly, body weight and tumor are measured once and twice each week. The tumor volume is calculated as described above (van der Horst et al., (2009) Neoplasia 11: 355-364). Purified mouse IgG or PBS as a negative control; Alternatively, mice are injected with a purified monoclonal antibody that recognizes an antigen other than CD98.

Example 9: Effect of CD98 monoclonal antibody on growth of B cell lymphoma xenografts in mice

Ramos (B cell lymphoma) cell line was obtained from ATCC and cultured according to the supplier's protocol. Animals were obtained from Charles River Laboratories.

(1: 1) mixture of PBS (without magnesium or calcium) and BD Matrigel (Vidi Biocysis) in the right flank of an immunodeficient SCID female mouse from 4 to 6 weeks of age with CB.17 background x 10 ⁷ viable cells were subcutaneously injected. The total volume injected per mouse is 200 [mu] l, where 50% is Matrigel (Vidi Biocysis). Once the tumors reached a size of 65 to 200 마우스, the mice were randomly assigned. Antibodies were administered weekly, body weight and tumors were measured once and twice weekly, respectively. Tumor volume was calculated as described above (van der Horst et al. (2009) Neoplasia 11: 355-364). All experiments were performed on a group of 7 or more animals per experiment. Animal experiments were now carried out according to a protocol approved by the Committee on Animal Care and Use of the Clinical Trials Board of Nikka Corporation.

Statistical significance was calculated between the treatment group and the control group using the Graph Pad Prism software package and Student's t-test. A p value of less than 0.05 was considered significant. Doubling time and time analysis were calculated as described in Daniel et al. (2007) Blood 110: 4037-4046.

Rituximab (anti-CD20 antibody) was used as a positive control control antibody. Antibody HB121 was an IgG2a negative control. Rituximab and anti-CD98 antibodies 8-93A and 18-3A are IgG1 antibodies, while all other anti-CD98 antibodies are IgG2a antibodies.

Treatment with anti-CD98 antibody has been shown to induce strong tumor growth inhibition in established B cell lymphoma (Ramos) tumors. In particular, anti-CD98 antibody therapy was superior to rituximab in inducing tumor growth inhibition (see Figures 5a and 5b).

In addition, anti-CD98 antibodies have been found to significantly prolong the progression time of the treated Ramos tumors. Tumor doubling times of the tumor regrowth data of Figures 5A-5C were calculated as described above and used for further prediction of progression time (TTP). TTP was then extrapolated to each animal in the treatment group until 2000 ㎣ was reached and plotted as Kaplan-Meier curves as shown in Figures 6a-6c. As shown in FIGS. 6A-6C, various anti-CD98 antibodies are superior to rituximab in prolonging the progression time in Ramos tumors.

The starting tumor volume of established < RTI ID = 0.0 > Ramos < / RTI > tumors was increased to assess the potential maximum therapeutic efficacy of anti-CD98 antibodies. Table 3 shows the therapeutic efficacy of anti-CD98 antibodies in a Ramos xenograft tumor model with an increasing tumor volume starting size. As can be seen, the anti-CD98 antibody retains tumor growth inhibition (TGI) even in increasing tumor volume.

Example 10: Effect of anti-CD98 monoclonal antibody on inhibition of in vivo tumor growth

The effects of anti-CD98 monoclonal antibodies 8-34B and 18-2A using the protocol described in Example 7 were tested in several xenograft models.

Cell lines of DLD-1 (colorectal carcinoma), A549 (non-small cell lung carcinoma), Ramos (B cell lymphoma) and OCI-AML-3 (acute myeloid leukemia) cell lines were obtained from ATCC and cultured according to the supplier's protocol. The animals were obtained from Charles River Ray Borey Torres. Immuno-deficient NOD.SCID female mice with CB.17 background were used for sarcoma tumor models, and immunocompetent SCID female mice between 4 weeks and 6 weeks old with CB.17 background were treated with Ramos and DLD- 1 tumor model and immunoreactive nu ^{- / -} female mice between 4 weeks and 6 weeks of age were used for A549 and OCI-AML-3 tumor models. Rituximab (IgG1 anti-CD20 antibody) or erbitux (IgG1 anti-EGFR antibody) was used as a positive control antibody and IgG2a antibody to an irrelevant antigen was used as a negative control. DC101 is a rat anti-mouse VEGFR2 / KDR IgG1 mAb (ATCC No. HB-11534) and is used as a positive control. Injection, antibody treatment and statistical calculations were performed as described in Example 9.

As shown in Figures 6-9, anti-CD98 monoclonal antibody 18-2A has been shown to be useful in the treatment of colorectal cancer (DLD-1), non-small cell lung cancer (A549), bucket lymphoma (Ramos) and AML (OCI- It is a potent inhibitor of tumor growth in the graft. The effect of 18-2A was advantageously compared with the effects of Rituxan (FIG. 7) and Erbitux (FIGS. 9 and 10).

Anti-CD98 monoclonal antibody 8-34B inhibited tumor growth in Ramos and AML (OCI-AML-3) xenografts as shown in Figures 7 and 8.

Example 11: Anti-CD98 monoclonal antibody in mouse strains with different immunodeficiency backgrounds

Three different anti-CD98 monoclonal antibodies 8-300B, 8-34B and 18-2A were tested in the Ramos xenograft model as described in Example 9 using different mouse strains. IgG2a antibody was used as a negative control. The three immunodeficient mouse varieties are less immunologically impaired mice or highly immunocompromised mice. SCID mice lack functional B cells and T cells but retain natural killer (NK) cell function and some complement function. NOD.SCID mice lack complement function and only partial NK function, while NSG (NOD / SCID / gamma) mice lack NK function.

As shown in Figure 11, the tumor growth inhibitory effect of the anti-CD98 antibody in the Ramos xenograft model increases when it is evaluated in a mouse strain with better immunity ability, which indicates that the in vivo antitumor activity of the anti-CD98 antibody is lower than that of the immune effector ≪ / RTI > function and inhibition of CD98 activity. The results in NSG mice lacking all ADCC and CDC function suggest that the anti-CD98 antibody can also inhibit the biological function of CD98 in Ramos xenografts and that CD98 may be important for the growth and / or maintenance of Ramus tumor in vivo It implies.

Example 12: In vivo efficacy of murine and chimeric anti-CD98 monoclonal antibodies

The in vivo efficacy of the murine anti-CD98 antibodies 8-34B and 18-2A, as described in Example 9, was tested in vivo by the chimeric antibodies 8-34B-ch and 18-2A-ch7.1 in a Ramos xenograft model Respectively. Antibodies were tested at a dose of 0.5 mg / kg (~ 30 nm). The results presented in Figure 12 confirm that chimeric anti-CD98 antibodies inhibit tumor growth in vivo with similar potency to their mimoline counterparts.

Example 13: Epitope mapping of humanized monoclonal antibody IGN523

Materials and methods

reagent. FREESTYLE ™ CHO-S cells (Invitrogen) were maintained in Freestyle ™ CHO expression medium (Invitrogen) supplemented with GlutaMAX ™. Control antibodies 4F2 and MEM108 were obtained from Santa Cruz Biotechnology and Thermosynthetic, respectively. Antibodies were labeled with Alexa Fluor 647 using a suitable Zenon antibody label attachment kit (Invitrogen).

Plasmid construction. (CD98 ECD, CD98 ECD point mutants and CD98 ECD chimera were constructed by GeneWiz and cloned into the pCDNA3.1 vector (Invitrogen). The full-length CD98, CD98 point mutants, Were constructed by gene synthesis and cloned into the pDisplay vector (Invitrogen). All constructs were confirmed by DNA sequencing.

Fluorescence-activated cell sorter (FACS) analysis of IGN523 binding to CD98

CD98 chimeric, CD98 point mutant constructs and human wild type control constructs were transfected electroporated using a Nucleofector 4D unit (loner). The constructs were mixed with the transfection solution and transiently transfected into the Freestyle ™ CHO-S cells. Transfected CHO-S cells were harvested 24 hours after transfection. Cells were quantified and stained with IGN523, MEM108 or 4F2 antibody. Prior to staining, the antibody was labeled with Alexa Fluor 647 using a suitable xenon antibody labeling kit (Invitrogen). Flow data was acquired on a Miltenyi MACSQuant analyzer (Miltenyi Biotec) and analyzed using FlowJo software version 9.5.3 (Tree Star, Inc.) Data analysis was performed.

Identification of the IGN523 binding region on CD98

To identify the region of CD98 involved in IGN523 binding, the region of approximately 40 contiguous amino acids of human CD98 (SEQ ID NO: 1) was replaced with the corresponding region of mouse CD98 (SEQ ID NO: 96) The effects of these substitutions were monitored using FACS analysis. Figure 13 shows regions of the mouse sequence that are substituted into the human sequence to form thirteen mouse-human CD98 chimeric constructs. Figure 14 shows the binding of IGN523 and control antibody and murine CD98 (Mu), human CD98 (Hu) and thirteen mouse-human chimeras. The results demonstrate that the substituted region in chimeric construct 10 is required for the binding of IGN523 and CD98. In contrast, it can be seen that regions responsible for the binding of control antibodies are found in the substituted regions in chimeras 11 and 12.

Figure 15 shows the sequence of the region of human CD98 to which IGN523 binds and the position of this sequence within the three dimensional structure of CD98, confirmed by using a mouse-human CD98 chimeric construct. The region defined by chimeric 10 consists of the amino acid residues T358 to N405 of human CD98. The amino acids T358 through G368 (underlined) are buried in the crystal structure and are unlikely to be part of the binding interface. The non-conserved residues between human and mouse sequences are indicated in bold typeface. Substitution of N at site D391 generates extra glycosylation sites in the mouse sequence relative to humans.

Sophisticated mapping of IGN523 with single mutations and multiple mutations in CD98

To further define the IGN523 epitope, single amino acid changes or multiple amino acid changes were introduced into the region defined by mouse-human chimeric 10. Four constructs were prepared by introducing non-homologous residues from a portion of the mouse CD98 sequence into the human CD98 sequence, respectively. Construct 4.1 contained deletions of mutations I371L, D374Q and A375G, and A376. Construct 4.2 contained the mutations M383A and E384K. Construct 4.3 contained the mutations D391N, F395I, P396F and D397H. Construct 4.4 contained the mutations G400R, A401P and A404L. FACS analysis of CHO cells transfected with each construct showed that the mutants contained in construct 4.1 interfered with the binding of IGN523 and CD98 (Figure 16). The mutations contained in construct 4.4 also affected the binding of IGN523 and CD98 to a lesser degree (Figure 16).

To confirm that the hydrophobic moiety is involved in the binding interface of IGN523 and human CD98, hydrophobic residues in the targeted loop region are replaced with highly charged amino acids such as aspartic acid or asparagine to produce single mutant constructs of these hydrophobic moieties . The single mutant constructs were individually transfected into CHO cells and analyzed by FACS to confirm binding with IGN523. As shown in Figure 17, the mutation of the hydrophobic residues A377 and L378 to the charged amino acid negatively affected IGN523 binding. To a lesser extent mutations of residues I398 and A401 showed a similar negative effect on binding of IGN523.

Additional constructs containing multiple mutations were made to identify additional residues important for the binding of IGN523 and CD98. As shown in Figure 18, a number of mutations (D374Q, D397H, G400R and A401P) in construct M1 completely disrupted the binding of IGN523, suggesting the importance of these residues. Constructs M2 (D374E and A375E) and M3 (D397S and I398T) also reduced binding. Additional experiments (not shown) using constructs involving the deletion of A376 suggested that the presence of this residue appears to be required for correct folding of the epitope loop region.

The following amino acids have been identified as being part of the IGN523 epitope: mouse-human chimeras, mutations of hydrophobic residues and multiple mutations.

Peptide scanning analysis

As a complementary study to these mutation studies, binding epitopes of IGN523 were further analyzed using peptide scanning analysis (Pepscan).

Synthesis of peptides: To reconstruct the discrete epitopes of the target molecule, a library of structured peptides was synthesized using chemically linked peptide (CLIPS) technology (Pepscan) on the scaffold, a proprietary technology of Pepscan. The chemical linkage between the peptide and the scaffold was performed essentially as follows: the side chains of multiple cysteines in the peptide were coupled to one or two CLIPS templates. T2 CLIPS template A 0.5 mM solution of 1,3-bis (bromomethyl) benzene was dissolved in ammonium bicarbonate (20 mM, pH 7.9) / acetonitrile (1: 1 (volume / volume)). This solution was added onto the peptide array to allow the CLIPS template to bind to the side chains of the two cystines present in the solid phase bound peptide of the peptide array (455 well plate with 3 쨉 l wells). The peptide array was shaken vigorously in the solution for 30 to 60 minutes while fully covering with the solution. Finally, the peptide array was extensively washed with excess H ₂ O and sonicated in destruction buffer containing 1% SDS / 0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70 ° C for 30 minutes , And sonicated in H ₂ O for 45 minutes. See also the method described by Timmerman et al. (2007), J. Mol. Recognit. 20: 283-99) and Slootstra et al. (1996), Molecular Diversity 1: 87-96.

ELISA Screening: The binding of each of the synthesized peptides to the antibody was tested in a Pepscan-based ELISA. The peptide array was incubated with the primary antibody solution (4 < 0 > C overnight). After washing, the peptide arrays were incubated with a 1/1000 dilution of the antibody peroxidase conjugate (SBA, Catalog No. 2010-05) at 25 ° C for 1 hour. After washing, peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μl / ml 3% H ₂ O ₂ were added. After one hour, the color development was measured. The color development was quantified by charge coupled device (CCD) -camera and image processing system.

Data Processing: The values obtained from the CCD camera are from 0 mAU to 3000 mAU, similar to a standard 96 well plate ELISA reader. The results were quantified and stored in a Peplab database. Often, wells contain bubbles that result in false positive values. The card is manually inspected and any value caused by bubbles is scored as zero.

Synthetic quality control: To verify the quality of the synthesized peptides, a separate set of positive control peptides and negative control peptides were synthesized simultaneously. They were screened with antibody 57.9 (Posthumus et al., J. Virology, 1990, 64: 3304-3309).

The results of the variable length peptide screening are shown in Fig. The ELISA results for each peptide are indicated by the horizontal line. The starting and ending points of the line indicate which residues are included in the peptide, and the Y value of the line shows the ELISA results obtained for the peptides. The ELISA results for the peptides show the predominant binding to ³⁹⁵ FPDIPGA ⁴⁰¹ (SEQ ID NO: 42) and the secondary binding to ³⁷⁹ PGQP ³⁸² (SEQ ID NO: 43). The overall analysis of 29 single-site alanine substitution sets showed the strongest binding to ³⁹⁴ SFDIPGAVASANMTV ⁴⁰⁷ (SEQ ID NO: 44). Figure 20 shows the result of the best binding single position alanine substituted peptide set wherein each residue of peptide SEQ ID NO: 44 was replaced with alanine or glycine (if the original residue was alanine). The analysis shows the strongest dependence on the residues F395, P396, D397 and I398 which appear to form the core of this epitope.

Next, to analyze the binding of the antibody to the stereostructural epitope, a pair of peptides from the two regions identified in the peptide screen were coexisted using a CLIPS conformational matrix structure. Figure 21 shows a thermal map showing data obtained from a CLIPS conformational matrix structure combining two partial sequences of human CD98 (shown on the X and Y axes). The results suggested a high dependence on the secondary structure. ³⁹⁵ FPDIPGAVSAN ⁴⁰⁵ (SEQ ID NO: 70) and ³⁷² GLDAAALPGQP ³⁸² (SEQ ID NO: 50). These two peptides were used as a reference for the mutagenic screen shown in Fig. SEQ1 shows the sequence of the peptide, and DIF1 indicates where the mutation is located in the peptide. The gray areas indicate peptides with non-mutated sequences. The last column shows the difference in ELISA values between the wild type peptide and the mutated peptide. Higher values suggest that mutations have a strong negative effect on binding. The mutagenic screen identified P379, G380, D397 and I398 as important binding residues.

Figure 23 shows the location of the amino acid residues identified as important amino acid residues in the binding of the humanized monoclonal antibody IGN523 on the human CD98 surface. Figure 23a shows the location of residues identified by chimeric and mutagenic studies, while Figure 23b shows the location of residues identified by pepscan analysis. Figure 23C shows that the two sets of residues are substantially overlapping, confirming that the highlighted loop region is the binding epitope for IGN523.

Example 14: In vivo antitumor activity of the humanized anti-CD98 monoclonal antibody IGN523

The effect of anti-CD98 humanized monoclonal antibody IGN523 using the protocol described in Example 8 was tested in several xenograft models. Injection, antibody treatment and statistical calculations were performed as described in Example 9. IGN523 was compared to the standard control drug rituximab in the Ramos (RA.1) and DAU buckling lymphoma models (Figures 24 and 25). IGN523 was then compared to carboplatin at its maximum drug tolerance dose in patient-derived NSCLC xenograft model IGN-LNG-12 (Figure 26). IGN523 was also tested in the AML xenograft model KG-1 (Figure 27). To preserve the inherent heterogeneity of tumors, the tumors used were subcultured to a minimum in NOD / SCID mice without any intervening cell culture.

In all cases, IGN523 showed significant tumor growth inhibition. Interestingly, tumors derived from IGN-LNG-12 patients caused weight loss in NOD-SCID mice, which correlated with tumor burden (FIGS. 26A and 26B). Carboplatin induced significant tumor growth inhibition at its maximum dose, but also increased weight loss. On the other hand, IGN523 treatment exerted an antitumor effect similar to carboplatin, but reduced weight loss induced by IGN-LNG-12. These data demonstrate that IGN523 treatment is as effective as carboplatin in this NOD.SCID model without inducing weight loss.

The data demonstrate significant tumor growth inhibition in the Burkitt lymphoma model Ramos (RA.1) and DAU, patient derived lung tumor xenograft IGN-LNG-12, and AML model KG-1. Further, inhibition of tumor growth of IGN523 is comparable to the anticancer agent carboplatin or rituximab, respectively (Table 4).

The dose-response relationship of IGN523 in IGN-LNG-12 tumors was also investigated. IGN-LNG-12 was selected to measure the dose response to the antibody using "therapeutic dosing regimen ". On the 12th day and 19th day, 1 mg / kg to 30 mg / kg of IGN523 was administered (Fig. 15). A dose of 10 mg / kg and 30 mg / kg resulted in a maximum tumor growth reduction of 50% to 66% relative to the control group in IGN-LNG-12 pulmonary tumors.

Taken together, in vivo efficacy data demonstrate that IGN523 induces significant tumor growth inhibition that is at least comparable to tumor growth inhibition of standard clinical drugs in various xenograft models.

Example 15: Receptor binding specificity for humanized anti-CD98 monoclonal antibody IGN523

Table 5 shows the percentage of sequence homology of the extracellular domain (ECD) of CD98 and the epitope of CD98 to which IGN523 binds between the indicated species. As shown in Table 5, the homology between the human epitope of CD98 to which IGN523 binds and the cynomolgus monkey epitope is 96%. Receptor binding specificity studies using a variety of methods, such as surface plasmon resonance (SPR, Biacore), bio-layer interferometry (Octet) or ELISA, suggests that IGN523 binds to human and cynomolgus monkey CD98 with high affinity, (Including murines, rats, rabbits, dogs and pig homologues) of CD98 due to the homology of the CD98 (Table 5). Biacore and Octet data demonstrate that the K _D of IGN523 is 2 nM to 6 nM for human CD98 and 8 nM to 14 nM for cynomolgus monkey CD98. ELISA binding data shows that the EC ₅₀ for IGN523 is 9 ng for human CD98 and 39 ng for cynomolgus monkey CD98.

Human, and cynomolgus frozen tissue flakes of the kidney, placenta, and cerebrum were stained with IGN523 (Fig. 29) to confirm that the cynomolgus monkey was the appropriate species for toxicity studies. In the frozen flakes of the human kidney and placenta (positive control), moderate to strong membrane and cytoplasmic staining of the renal tubular epithelium (kidney) and the nutrient epithelial cells (placenta) were observed and the intensity of the staining was measured by the corresponding cynomolgus monkey tissue It was similar but not identical to the staining intensity in the flakes. In the case of human and cynomologus monkey cerebrum, similar weak to intermediate cytoplasmic staining of neural networks and cytoplasmic granular epithelium was observed. Putting it all together, the staining in the cynomolgus monkey tissue seems comparable to the staining in human tissue. Based on affinity and staining data, the cynomolgus monkey is considered to be a species suitable for assessing the safety of IGN523.

Example 16: Single dose pharmacokinetic study in cynomolgus monkeys

Experimental non-GLP monoclonal intravenous pharmacokinetic studies of IGN523 were performed in male cynomolgus monkeys and female cynomolgus monkeys (sex / age) at doses of 1 mg / kg, 3 mg / kg, 10 mg / kg and 100 mg / N = 2 per group). A significant dose-dependent response rate of IGN523 was observed after intravenous administration of 1 mg, 3 mg, 10 mg or 100 mg of IGN523 per kg of body weight to the monkey for a single hour. Thus, the basic assumptions about the linearity implied in the application of the utilized non-compartmental analysis method are not applied to the IGN 523 over the entire capacity range studied, and the parameters are displayed for illustrative comparison between the capacity groups. Only 2 animals per sex were evaluated at each dose level, but there was no apparent difference in PK profiles between male and female animals. The mean concentration-time profiles of male and female animals were similar within each dose group at the dose of 1 mg to 100 mg of IGN 523 per kg body weight (Table 6).

Taken together, there was a 10-fold reduction in the "apparent" plasma CL of IGN 523 over a dose range of 1 mg / kg to 100 mg / kg. The mean "apparent" MRT and the mean "apparent" T _1/2 were increased 12- to 13-fold over the dose range of 1 mg / kg to 100 mg / kg. 1 mg / kg, 3 mg / kg or 10 mg / in capacity kg I did not have a difference in average "apparent" V _ss, 100 mg / in the capacitor group kg average "apparent" V _ss is 1 mg / kg, 3 mg / kg or 10 mg / kg group. All of these findings suggest that the non-linear propensity of IGN523 in monkeys may be due to the target medication propensity (TMDD). The TMDD model has been used conventionally to describe the non-linearity of other monoclonal antibodies. TMDD occurs when an antibody has specificity for densely packed cell surface targets that are abundantly expressed to exhibit quantitatively significant removal pathways at low doses of target-antibody interaction (Mager 2001, Mager 2003, Luu 2012 ).

Example 17: Repeated dose GLP studies in cynomolgus monkeys

The toxicology, PK and immunogenicity of IGN523 are studied in GLP multi-dose IV dose toxicology studies in cynomolgus monkeys. This study provides comprehensive data on clinical endpoints, toxic kinetics, immunogenicity (development of anti-ANG523 antibodies), and histopathology, including extensive tissue listings (including injection sites) This is done with the formulated material that represents the material to be treated. In addition, selected safety pharmacological endpoints (nerve activity, ECG, respiration) are also evaluated.

The dosing regimen is a 60 minute intravenous cycle once a week for 8 weeks. The doses used were IGN 523 at 10 mg / kg, 30 mg / kg and 100 mg / kg once weekly, followed by a recovery period of 4 weeks (Table 5). A four week recovery period is considered to be sufficient time to allow complete elimination of IGN523 and to assess the reversibility of any potential toxicity. The highest dose is expected to approach the MTD and provides significant exposure that far exceeds the expected exposure in the patient. Immunogenicity (anti-ANG523 antibody) and toxic reaction rate are monitored.

Table 8 contains a detailed summary of the study design for multi-dose, cytoplasmic GLP toxicology studies. The selected safety pharmacological endpoints (neurobehavioral, electrocardiographic, respiratory) will be evaluated in relation to the GLP repeat dose study in cynomolgus monkeys.

Example 18: Evaluation of hemolytic activity of IGN523

Cynomolgus whole blood will be used to assess the hemolytic potential of IGN523 in vitro. The results of this test will be used to identify any potential effects on hemoglobin. On the day of whole blood collection, non-human primate whole blood will be used for the study. Table 9 contains a summary of the GLP study design for the hemolytic potential assessment of IGN523.

Example 19: Acute intravenous and perivascular stimulation

Design stimulus and focal tissue resistance studies to assess short-term toxicity of compounds at high concentrations at the direct injection site. As part of this study, compounds are administered intravenously and intravascularly to identify effects on tissues that are expected to encounter initial exposure. Rabbits are standard animal models for this evaluation.

Example 20: Tissue cross-reactivity of IGN523

A full range of human tissues is used to characterize the tissue cross-reactive profile of IGN523 as detailed in the FDA Handbook (Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (February, 1997)). A comprehensive list of organizations studied is included in Table 10.

Example 21: Phase I clinical study to evaluate the safety and pharmacokinetics of IGN523 in patients with relapsed or refractory acute myelogenous leukemia

Primary purpose

The primary objectives of this study are as follows:

재발된 또는 불응성 급성 골수성 백혈병(AML)을 갖는 환자에게 매주 투약으로 시작되는 일정으로 투여된 IGN523의 안전성 및 내약성을 평가하는 것;

Assessing the safety and tolerability of IGN523 administered at a schedule beginning with weekly dosing to patients with relapsed or refractory acute myelogenous leukemia (AML);

매주 4회 투여된 경우 IGN523의 최대 내약 용량(MTD) 및 용량 제한 독성(DLT)을 측정하는 것; 및

Measuring the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of IGN523 administered four times per week; And

안전성, 약동학 및 약력학 데이터에 기초하여 IGN523의 권장된 2 기 용량(RP2D)을 확인하는 것.

Identify the recommended second dose (RP2D) of IGN 523 based on safety, pharmacokinetic and pharmacodynamic data.

Secondary purpose

IGN523에 대한 항체 형성률을 평가하는 것;

Assessing antibody formation rates against IGN523;

재발된 또는 불응성 AML을 갖는 환자에서 IGN523의 약동학을 특징규명하는 것;

Characterizing the pharmacokinetics of IGN523 in patients with relapsed or refractory AML;

재발된 또는 불응성 AML을 갖는 환자에서 IGN523의 항백혈병 활성을 예비적으로 평가하는 것; 및

Preliminary evaluation of the anti-leukemic activity of IGN523 in patients with relapsed or refractory AML; And

IGN523 항백혈병 활성을 예측할 생물학적 마커를 예비적으로 평가하는 것.

IGN523 Preliminary assessment of biological markers to predict anti-leukemia activity.

Way

Approximately 6 dose groups using standard 3 + 3 design, and open label increase of extension groups in MTD or RP2D

Number of subjects

Capacity increase: approximately 21 to 30

Capacity Expansion: 20

Research drug

The IGN523 drug will be supplied as a 20 mg 10 mg / ml solution in a 25 ml disposable glass vial. An appropriate volume of IGN523 drug will be diluted to 250 ml and will be inoculated intravenously (IV) over 1 hour. In the I-dose escalation study (see protocol synopsis in paragraph 10.2.1), groups of patients can be treated with elevated doses up to 30 mg / kg weekly for 8 doses. Continued treatment beyond 8 weeks will be provided to patients with continued clinical benefit (i. E., Absence of disease progression and unacceptable toxicity). To maximize the accumulation of data at relevant doses and to minimize the patient's treatment at potentially less than therapeutic doses, patient dose escalation may be allowed under certain conditions (as described in paragraph 10.2.1).

Diagnostic and Critical inclusion criteria

Relapsed or refractory AML without effective standard therapy

측정가능한 질환을 가져야 함;

Have measurable disease;

18세 이상의 연령;

Age 18 or older;

이스턴 코퍼레이티브 온콜로지 그룹(Eastern Cooperative Oncology Group)(ECOG) 수행 상태 0 내지 2;

Eastern Cooperative Oncology Group (ECOG) performance status 0 to 2;

12주 이상의 기대 수명;

Life expectancy over 12 weeks;

25,000/㎣ 이상의 혈소판 수(수혈에 의해 유지될 수 있음);

Platelet count above 25,000 / ((which may be maintained by transfusion);

2.5배 규격 정상 상한(ULN) 이하의 AST(SGOT)/ALT(SGPT);

2.5 times AST (SGOT) / ALT (SGPT) below the normal upper limit (ULN);

1.5배 규격 ULN 이하의 총 빌리루빈;

Total bilirubin less than 1.5 times ULN;

2배 규격 ULN 이하의 크레아티닌, 또는 50 ㎖/분 이상의 계산된 또는 측정된 크레아티닌 제거;

Creatinine less than or equal to double ULN, or calculated or measured creatinine greater than or equal to 50 ml / min;

임신가능 여성 및 남성의 경우, 연구 참여 전 및 연구 참여의 지속기간 동안 적절한 피임(또는 출산 조절의 호르몬 또는 차단제 방법; 금욕)에 대한 동의; 및

For pregnant women and men, consent to appropriate contraception (or hormone or blocker method of abstinence, abstinence) during pre-study participation and duration of study participation; And

작성된 사전 동의서를 이해하고 서명할 의지를 갖는 능력.

Ability to understand and sign the written consent form.

Exclusion criteria

주기 1의 1일째 날 전, 4주 이내에 단일클론 항체 치료의 사용, 또는 2주 이내에 화학치료 또는 방사선치료의 사용(주기 1의 1일째 날 전 최대 72시간까지 말초 모세포 수를 조절하기 위해 제공되는 하이드록시우레아가 허용됨);

The use of monoclonal antibody therapy within the first day of cycle 1, within 4 weeks, or the use of chemotherapy or radiotherapy within 2 weeks (provided for up to 72 hours prior to the first day of cycle 1, Hydroxyurea is allowed);

선행 항암치료로부터 1 초과의 NCI CTCAE v4.0 등급의 미해결된 급성 독성;

Unresolved acute toxicity of more than 1 NCI CTCAE v4.0 grade from anticancer chemotherapy;

면역억제 치료를 현재 요구하는 선행 동종이계 줄기 세포 이식;

Previous allogeneic stem cell transplantation currently requiring immunosuppressive therapy;

단일클론 항체 치료에 대한 중증 알레르기 또는 아나필락시스 반응의 이력;

History of severe allergic or anaphylactic reactions to monoclonal antibody therapy;

백혈병의 공지된 연수막 또는 CNS 침습

Known leukemia or CNS invasion of leukemia

계속되는 또는 활성 감염, 증상을 나타내는 울혈성 심부전, 불안정한 협심증, 심장 부정맥, 또는 연구 요건에의 충족을 제한할 정신과적 질병/사회적 상황

Continuous or active infection, congestive heart failure with symptoms, unstable angina, cardiac arrhythmia, or psychiatric disease / social conditions that will limit the fulfillment of research requirements

주기 1의 1일 전 4주 이내의 최근 주요 수술

Recent major surgery within 4 weeks 1 day before cycle 1

임신 또는 수유 여성

Pregnant or lactating women

Diagnostic plan

Based on the following reasons, patient AML samples of peripheral blood (or bone marrow) following study enrollment will be retrospectively analyzed for expression of CD98 (not analyzed as part of patient screening and not used as a condition for study eligibility ):

CD98은 정상적인 골수 샘플들의 유사하게 "게이팅된" 세포에 비해 대다수(약 94%)의 AML 환자의 CD34⁺/CD33⁺ 및 CD34⁺/CD33^- 하위집단에서 상이하게 과다발현된다.

CD98 is overexpressed differentially in the CD34 ⁺ / CD33 ⁺ and CD34 ⁺ / CD33 ^- subpopulations of the majority (about 94%) of AML patients compared to similarly "gated" cells of normal bone marrow samples.

제안된 I 기 연구에 참여할 환자를 선택하기에(또는 참여로부터 환자를 배제하기에) 적절한 환자 샘플에 대한 CD98 발현을 검출하고 정량하는 임상적으로 검증된 방법은 없다.

There is no clinically proven method for detecting and quantifying CD98 expression on appropriate patient samples to select patients to participate in the proposed Phase I study (or to exclude patients from participation).

I 기 결과의 어세이 및 분석의 적격화는 환자 선택을 요구할 수 있는 향후 임상 연구의 디자인을 알려주는 데에 이용될 것이다.

Assessment of the I-outcomes and eligibility of analysis will be used to inform future designs of clinical studies that may require patient selection.

Test product, method of administration and starting dose

IGN523

Intravenous line

Starting dose: less than 1/6 of the human equivalent capacity (HED) of NOAEL (No Observed Adverse Effect Level) observed in the GLP multi-dose cynomolgus toxicity study

Military initiation and continuation of treatment

To enable observation of possible severe and / or severe acute (e.g., related) toxicity that will affect subsequent patient registration or medication decisions, the first patient in each new dose group will be treated as any other It will be given at least 1 day before the patient.

The patient will receive an intravenous dose of IGN 523 every 8 weeks (twice the 28 day cycle). Dosages above 8 weeks will be acceptable for patients who meet the criteria for continued clinical benefit (ie, no progression of disease) and acceptable safety for up to one year.

Increased patient capacity

To maximize the accumulation of information at relevant doses and potentially minimize patient exposure to doses below optimal doses, an increase in dose in a patient may be allowed under the following conditions:

환자는 임의의 용량 상승 전에 그들의 원래 배정된 용량 수준에서 적어도 2회 28일 주기에서 완료해야 한다.

Patients should complete at least two 28-day cycles at their original assigned dose levels before any dose increases.

환자는 IGN523 투여의 적어도 1회 28일 주기를 통해 완료된 3개 내지 6개 환자 용량 군에 의해 제거되는 최고 용량 수준까지 그들의 용량을 단지 상승시킬 수 있다.

The patient can only raise their dose to the highest dose level that is eliminated by the 3 to 6 patient dose group completed over at least once a 28 day cycle of IGN523 administration.

모든 환자내 용량 상승 결정은 환자에게 최선의 이익이라고 생각되는지에 대한 의료인과 협의된 연구자의 판단에 기초할 것이다.

The decision to increase the dose in all patients will be based on the judgment of the investigator in consultation with the healthcare provider as to whether or not they are considered to be the best benefit for the patient.

Definition of DLT

DLT will be any of the following side effects considered by the investigator to be associated with IGN 523 (and not due to another clearly identifiable cause) that occur during day 1 to day 28 of cycle 1 :

하기 독성을 제외한 등급 3 또는 4 비-혈액학적 독성:

Grade 3 or 4 non-toxic: Non-hematological toxicity:

- fever (occurring in the absence of signs / symptoms of dyspnea) (occurring within 24 hours or 24 hours after completing the crossing) and resolved within 24 hours by administration of reduced pedicle velocity, supportive treatment and / or corticosteroids; Irritable, and / or hypoxic), reversible grade 3 non-allergic rhododendrity, and / or chronic allergic rhinitis, including, but not limited to,

- Grade 3 or 4 hyperuricemia, hyperphosphatemia, or hyperuricemia in the absence of symptomatic manifestations (ie, 1.5 times ULN or higher creatinine, cardiac arrhythmia, sudden death or seizure), transient (ie, lasting less than 48 hours) Hypocalcemia, or grade 3 hyperkalemia;

출혈을 초래하거나, 혈소판 수혈 없이 2주 이내에 기준 값의 80% 이상까지 개선되지 않는 (수혈 뒷받침을 요구하는 기존 혈소판감소증을 갖지 않는 환자에서) 등급 3 또는 4 혈소판감소증; 및

Grade 3 or 4 thrombocytopenia (in patients who do not have existing thrombocytopenia requiring blood transfusion) that do not lead to bleeding or improve to more than 80% of baseline values within 2 weeks without platelet transfusion; And

발열(100.4℉/38℃의 구강 또는 고실 온도)과 관련되어 있거나 성장 인자 뒷받침 없이 2주 이내에 기준 값의 80% 이상까지 개선되지 않는 (성장 인자 뒷받침을 요구하는 기존 호중구감소증을 갖지 않는 환자에서) 등급 3 또는 4 호중구감소증.

(In patients who do not have conventional neutropenia requiring growth factor support) that are associated with fever (oral or solid temperature at 100.4 ℉ / 38 캜) or that do not improve to more than 80% of the reference value within two weeks without growth factor support Grade 3 or 4 neutropenia.

DLT window

Day 1 to day 28 of cycle 1

Capacity increase method

Capacity Imagery can only occur after each individual in a given group reaches the day of the 28th day. Patients who experienced disease progression and were withdrawn from the study before day 28 without DLT will not be evaluated for DLT and will be replaced. The capacity increase will proceed with a maximum 100% increase rate between the groups (or less if a significant AE is observed) according to the following scheme:

3명의 환자들 중 0명의 환자가 주어진 용량 수준에서 DLT를 갖는 경우, 3명의 환자들은 다음 용량 수준에서 등록될 수 있다.

If 0 out of 3 patients have DLT at a given dose level, 3 patients can be enrolled at the next dose level.

3명의 환자들 중 2명 이상의 환자들이 주어진 용량 수준에서 DLT를 경험하는 경우, 용량 증가는 중단될 것이고, 이 용량은 MTD를 초과하는 용량으로 공표될 것이다.

If two or more of the three patients experience DLT at a given dose level, the dose increase will cease and this dose will be announced at a dose exceeding the MTD.

3명의 환자들 중 1명의 환자가 주어진 용량 수준에서 DLT를 경험하는 경우, 적어도 3명 더 많은 환자들이 동일한 용량 수준에서 등록될 것이다. 이들 3명의 환자들 중 0명의 환자가 DLT를 경험하는 경우, (100% 미만의 용량 증가율일 수 있는) 다음 용량 수준으로 진행된다. 이 군의 1명 이상의 환자가 DLT를 경험한 경우, 용량 상승은 중단되고, 이 용량은 MTD를 초과하는 용량으로서 공표될 것이다.

If one of the three patients experiences DLT at a given dose level, at least three more patients will be enrolled at the same dose level. If 0 of these 3 patients experience DLT, they proceed to the next dose level (which may be a dose increase rate of less than 100%). If more than one patient in this group has experienced DLT, the dose escalation will cease and this dose will be published as a dose in excess of the MTD.

일단 MTD가 초과되면, 이전 용량 상승 증가율이 30% 이하인 경우, 이전 용량 수준을 MTD로서 평가하기 위해 최소 6명의 평가가능한 환자들이 이전 용량 수준에서 등록될 수 있다. 이전 용량 상승 증가율이 30%를 초과한 경우, 2개의 최고 용량 수준 사이의 중간에 있는 하나 이상의 용량 수준이 평가될 수 있다.

Once the MTD is exceeded, a minimum of six assessable patients can be enrolled at the previous dose level to assess the previous dose level as MTD, if the previous dose increase rate is less than 30%. If the previous capacity increase rate exceeds 30%, one or more capacity levels in the middle between the two highest capacity levels can be evaluated.

최소 6명의 환자들 중 3분의 1 미만의 환자에서 DLT를 발생시키는 최고 용량 수준은 MTD로서 공표될 것이다.

The highest dose level that causes DLT in less than one-third of patients in at least six patients will be published as MTD.

Capacity increase committee

At the conclusion of each study group, the Internal Capacity Rise Committee, consisting of healthcare workers, drug safety representatives, and additional immediate research team members, agreed with the researchers to agree to proceed with capacity increases, change capacity- something to do. This committee will review all available research data for the current army and all available safety data for the previous army before determining the capacity increase for subsequent military units. All study subjects will receive research treatments until disease progression, the occurrence of unacceptable toxicity, non-compliance, or withdrawal by subject or researcher decision.

Criteria for evaluation efficacy

Peripheral blood count

Bone marrow aspiration and biopsy

safety

The safety outcome measures are as follows: Incidence and nature of DLT, incidence and severity of side effects.

PK sampling

PK evaluation will be performed according to the following schedule:

Day 1 Dose: 30 minutes, 4 hours, 24 hours and 48 hours (or 72 hours) before, and after dosing,

Day 8, Day 15 and Day 21 Dosage: 30 minutes, 4 hours and 48 hours (or 72 hours) before,

Subsequent doses: before and 30 minutes after dosing

Extension group

To obtain additional safety, tolerability and pharmacokinetic data, and preliminary evidence of clinical activity, a maximum of 20 patients with relapsed or refractory AML will be enrolled in the expansion group at MTD or RP2D. Although the MTD will be largely determined by the safety (DLT) data observed during the DLT observation period I, the RP2D will also consider additional safety data beyond the DLT window and will also collect information (PK and target occupancy data collected during I- May also be included.

                               SEQUENCE LISTING <110> IGENICA, INC.   <120> ANTI-CD98 ANTIBODIES AND METHODS OF USE THEREOF <130> IGN-0001 PCT <140> <141> <150> 61 / 563,443 <151> 2011-11-23 <160> 124 <170> PatentIn version 3.5 <210> 1 <211> 529 <212> PRT <213> Homo sapiens <400> 1 Met Ser Gln Asp Thr Glu Val Asp Met Lys Glu Val Glu Leu Asn Glu 1 5 10 15 Leu Glu Pro Glu Lys Gln Pro Met Asn Ala Ala Ser Gly Ala Ala Met             20 25 30 Ser Leu Ala Gly Ala Glu Lys Asn Gly Leu Val Lys Ile Lys Val Ala         35 40 45 Glu Asp Glu Ala Gla Ala Ala Ala Ala Lys Phe Thr Gly Leu Ser     50 55 60 Lys Glu Glu Leu Leu Lys Val Ala Gly Ser Pro Gly Trp Val Arg Thr 65 70 75 80 Arg Trp Ala Leu Leu Leu Leu Phe Trp Leu Gly Trp Leu Gly Met Leu                 85 90 95 Ala Gly Ala Val Val Ile Ile Val Arg Ala Pro Arg Cys Arg Glu Leu             100 105 110 Pro Ala Gln Lys Trp Trp His Thr Gly Ala Leu Tyr Arg Ile Gly Asp         115 120 125 Leu Gln Ala Phe Gln Gly His Gly Ala Gly Asn Leu Ala Gly Leu Lys     130 135 140 Gly Arg Leu Asp Tyr Leu Ser Ser Leu Lys Val Lys Gly Leu Val Leu 145 150 155 160 Gly Pro Ile His Lys Asn Gln Lys Asp Asp Val Ala Gln Thr Asp Leu                 165 170 175 Leu Gln Ile Asp Pro Asn Phe Gly Ser Lys Glu Asp Phe Asp Ser Leu             180 185 190 Leu Gln Ser Ala Lys Lys Lys Ser Ile Arg Val Ile Leu Asp Leu Thr         195 200 205 Pro Asn Tyr Arg Gly Glu Asn Ser Trp Phe Ser Thr Gln Val Asp Thr     210 215 220 Val Ala Thr Lys Val Lys Asp Ala Leu Glu Phe Trp Leu Gln Ala Gly 225 230 235 240 Val Asp Gly Phe Gln Val Arg Asp Ile Glu Asn Leu Lys Asp Ala Ser                 245 250 255 Ser Phe Leu Ala Glu Trp Gln Asn Ile Thr Lys Gly Phe Ser Glu Asp             260 265 270 Arg Leu Leu Ile Ala Gly Thr Asn Ser Ser Asp Leu Gln Gln Ile Leu         275 280 285 Ser Leu Leu Glu Ser Asn Lys Asp Leu Leu Leu Thr Ser Ser Tyr Leu     290 295 300 Ser Asp Ser Gly Ser Thr Gly Glu His Thr Lys Ser Leu Val Thr Gln 305 310 315 320 Tyr Leu Asn Ala Thr Gly Asn Arg Trp Cys Ser Trp Ser Leu Ser Gln                 325 330 335 Ala Arg Leu Leu Thr Ser Phe Leu Pro Ala Gln Leu Leu Arg Leu Tyr             340 345 350 Gln Leu Met Leu Phe Thr Leu Pro Gly Thr Pro Val Phe Ser Tyr Gly         355 360 365 Asp Glu Ile Gly Leu Asp Ala Ala Leu Pro Gly Gln Pro Met Glu     370 375 380 Ala Pro Val Met Leu Trp Asp Glu Ser Ser Phe Pro Asp Ile Pro Gly 385 390 395 400 Ala Val Ser Ala Asn Met Thr Val Lys Gly Gln Ser Glu Asp Pro Gly                 405 410 415 Ser Leu Leu Ser Leu Phe Arg Arg Leu Ser Asp Gln Arg Ser Ser Lys Glu             420 425 430 Arg Ser Leu Leu His Gly Asp Phe His Ala Phe Ser Ala Gly Pro Gly         435 440 445 Leu Phe Ser Tyr Ile Arg His Trp Asp Gln Asn Glu Arg Phe Leu Val     450 455 460 Val Leu Asn Phe Gly Asp Val Gly Leu Ser Ala Gly Leu Gln Ala Ser 465 470 475 480 Asp Leu Pro Ala Ser Ala Ser Leu Pro Ala Lys Ala Asp Leu Leu Leu                 485 490 495 Ser Thr Gln Pro Gly Arg Glu Glu Gly Ser Pro Leu Glu Leu Glu Arg             500 505 510 Leu Lys Leu Glu Pro His Glu Gly Leu Leu Leu Arg Phe Pro Tyr Ala         515 520 525 Ala      <210> 2 <211> 1590 <212> DNA <213> Homo sapiens <400> 2 atgagccagg acaccgaggt ggatatgaag gaggtggagc tgaatgagtt agagcccgag 60 aagcagccga tgaacgcggc gtctggggcg gccatgtccc tggcgggagc cgagaagaat 120 ggtctggtga agatcaaggt ggcggaagac gaggcggagg cggcagccgc ggctaagttc 180 ggtcgcacc 240 cgctgggcac tgctgctgct cttctggctc ggctggctcg gcatgcttgc tggtgccgtg 300 gtcataatcg tgcgagcgcc gcgttgtcgc gagctaccgg cgcagaagtg gtggcacacg 360 ggcgccctct accgcatcgg cgaccttcag gccttccagg gccacggcgc gggcaacctg 420 gcgggtctga aggggcgtct cgattacctg agctctctga aggtgaaggg ccttgtgctg 480 ggtccaattc acaagaacca gaaggatgat gtcgctcaga ctgacttgct gcagatcgac 540 cccaattttg gctccaagga agattttgac agtctcttgc aatcggctaa aaaaaagagc 600 atccgtgtca ttctggacct tactcccaac taccggggtg agaactcgtg gttctccact 660 caggttgaca ctgtggccac caaggtgaag gatgctctgg agttttggct gcaagctggc 720 gtggatgggt tccaggttcg ggacatagag aatctgaagg atgcatcctc attcttggct 780 gagtggcaaa atatcaccaa gggcttcagt gaagacaggc tcttgattgc ggggactaac 840 tcctccgacc ttcagcagat cctgagccta ctcgaatcca acaaagactt gctgttgact 900 agctcatacc tgtctgattc tggttctact ggggagcata caaaatccct agtcacacag 960 tatttgaatg ccactggcaa tcgctggtgc agctggagtt tgtctcaggc aaggctcctg 1020 acttccttct tgccggctca acttctccga ctctaccagc tgatgctctt caccctgcca 1080 gggacccctg ttttcagcta cggggatgag attggcctgg atgcagctgc ccttcctgga 1140 cagcctatgg aggctccagt catgctgtgg gatgagtcca gcttccctga catcccaggg 1200 gctgtaagtg ccaacatgac tgtgaagggc cagagtgaag accctggctc cctcctttcc 1260 ttgttccggc ggctgagtga ccagcggagt aaggagcgct ccctactgca tggggacttc 1320 cacgcgttct ccgctgggcc tggactcttc tcctatatcc gccactggga ccagaatgag 1380 cgttttctgg tagtgcttaa ctttggggat gtgggcctct cggctggact gcaggcctcc 1440 gacctgcctg ccagcgccag cctgccagcc aaggctgacc tcctgctcag cacccagcca 1500 ggccgtgagg agggctcccc tcttgagctg gaacgcctga aactggagcc tcacgaaggg 1560 ctgctgctcc gcttccccta cgcggcctga 1590 <210> 3 <211> 357 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 3 caggtgcagc tgaaggagtc cggccccggc ctggtggccc cctcccagtc cctgtccatc 60 acctgcaccg tgtccggctt ctccctgacc tcctacggcg tgcactggat ccgccagccc 120 cccggcaagg gcctggagtg gctgggcctg atctgggccg gcggctccat caactacaac 180 tccgccctga tgtcccgcct gtccatctcc aaggacaact ccaagtccca ggtgttcctg 240 aagatgaact ccctggagac cgaggacacc gccatgtact actgcgcccg caagggccac 300 atgtactcct acgccatgga ctactggggc cagggcacct ccgtgaccgt gtcctcc 357 <210> 4 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 4 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr             20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu         35 40 45 Gly Leu Ile Trp Ala Gly Gly Ser Ile Asn Tyr Asn Ser Ala Leu Met     50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Glu Thr Glu Asp Thr Ala Met Tyr Tyr Cys Ala                 85 90 95 Arg Lys Gly His Met Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Ser Val Thr Val Ser Ser         115 <210> 5 <211> 342 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 5 gacatcgtga tgacccagtc cccctcctcc ctgaccgtga ccgccggcga gaaggtgacc 60 atgtcctgca agtcctccca gtccctgctg aactccggca accagaagac ctacctgacc 120 tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc ctccacccgc 180 gagtccggcg tgcccgaccg cttcaccggc tccggctccg gcaccgagtt caccctgacc 240 atctcctccg tgcaggccga ggacctggcc gtgtactact gccagaacga ctactcctac 300 cccccctgga ccttcggcgg cggcaccaag ctggagatca ag 342 <210> 6 <211> 114 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 6 Asp Ile Val Met Thr Gln Ser Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser             20 25 30 Gly Asn Gln Lys Thr Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn                 85 90 95 Asp Tyr Ser Tyr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu             100 105 110 Ile Lys          <210> 7 <211> 348 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 7 caggtgcagc tgcagcagtc cggcgccgag ctggtgaagc ccggcgcctc cgtgaagctg 60 tcctgcaagg cctccggcta caccttcacc tcctactaca tgtactgggt gaagcagcgc 120 cccggccagg gcctggagtg gatcggcgtg atcaaccccg gctccggcat caccaactac 180 aacgagaagt tcaagggcaa ggccaccctg accgccgaca agtcctccaa caccgcctac 240 atgcagctgt cctccctgtc ctccgacgac tccgccgtgt acttctgctc cggctccgcc 300 aactggttcg cctactgggg ccagggcacc ctggtgaccg tgtccgcc 348 <210> 8 <211> 116 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 8 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Ile Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ser Ser Asp Asp Ser Ala Val Tyr Phe Cys                 85 90 95 Ser Gly Ser Ala Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ala         115 <210> 9 <211> 339 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 9 gacatcgtga tgtcccagtc cccctcctcc ctggccgtgt ccgtgggcga gaaggtgacc 60 atgtcctgca agtcctccca gtccctgctg tactcctcca accagaagaa ctacctggcc 120 tggtaccagc agaagcccgg ccagtccccc aagctgctga tctactgggc ctccacccgc 180 gactccggcg tgcccgaccg cttcaccggc tccggctccg gcaccgactt caccctgacc 240 atctcctccg tgaaggccga ggacctggcc gtgtactact gccagcgcta ctacggctac 300 ccctggacct tcggcggcgg caccaagctg gagatcaag 339 <210> 10 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 10 Asp Ile Val Met Ser Gln Ser Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser             20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Asp Ser Gly Val     50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Arg                 85 90 95 Tyr Tyr Gly Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile             100 105 110 Lys      <210> 11 <211> 348 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 11 caggtgcagc tgcagcagtc cggcgccgag ctggtgcgcc ccggcacctc cgtgaaggtg 60 tcctgcaagg cctccggcaa cgccttcacc aactacctga tcgagtggat caagcagcgc 120 cccggccagg gcctggagtg gatcggcgtg atcaaccccg gctccggcat caccaactac 180 aacgagaagt tcaagggcaa ggccaccctg accgccgaca agtcctccaa caccgcctac 240 atgcagctgt cctccctgtc ctccgacgac tccgccgtgt acttctgctc cggctccgcc 300 aactggttcg cctactgggg ccagggcacc ctggtgaccg tgtccgcc 348 <210> 12 <211> 116 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 12 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn Ala Phe Thr Asn Tyr             20 25 30 Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Ile Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ser Ser Asp Asp Ser Ala Val Tyr Phe Cys                 85 90 95 Ser Gly Ser Ala Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ala         115 <210> 13 <211> 339 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 13 gacatcgtga tgtcccagtc cccctcctcc ctggccgtgt ccgtgggcga gaaggtgacc 60 atgtcctgca agtcctccca gtccctgctg tactcctcca accagaagaa ctacctggcc 120 tggtaccagc agaagcccgg ccagtccccc aagctgctga tctactgggc ctccacccgc 180 gactccggcg tgcccgaccg cttcaccggc tccggctccg gcaccgactt caccctgacc 240 atctcctccg tgaaggccga ggacctggcc gtgtactact gccagcgcta ctacggctac 300 ccctggacct tcggcggcgg caccaagctg gagatcaag 339 <210> 14 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 14 Asp Ile Val Met Ser Gln Ser Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser             20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Asp Ser Gly Val     50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Arg                 85 90 95 Tyr Tyr Gly Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile             100 105 110 Lys      <210> 15 <211> 114 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 15 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser             20 25 30 Gly Asn Gln Lys Thr Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn                 85 90 95 Asp Tyr Ser Tyr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu             100 105 110 Ile Lys          <210> 16 <211> 114 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 16 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser             20 25 30 Gly Asn Gln Lys Thr Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn                 85 90 95 Asp Tyr Ser Tyr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu             100 105 110 Ile Lys          <210> 17 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr             20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Leu Ile Trp Ala Gly Gly Ser Ile Asn Tyr Asn Ser Ala Leu Met     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Lys Gly His Met Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 18 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr             20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu         35 40 45 Gly Leu Ile Trp Ala Gly Gly Ser Ile Asn Tyr Asn Ser Ala Leu Met     50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Lys Gly His Met Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 19 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 19 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr             20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu         35 40 45 Gly Leu Ile Trp Ala Gly Gly Ser Ile Asn Tyr Asn Ser Ala Leu Met     50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Ser Ser Ser Ser Ser Ser 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Lys Gly His Met Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 20 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 20 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser             20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Asp Ser Gly Val     50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Arg                 85 90 95 Tyr Tyr Gly Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile             100 105 110 Lys      <210> 21 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 21 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser             20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Asp Ser Gly Val     50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Arg                 85 90 95 Tyr Tyr Gly Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile             100 105 110 Lys      <210> 22 <211> 116 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 22 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn Ala Phe Thr Asn Tyr             20 25 30 Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Ile Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ser Gly Ser Ala Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 23 <211> 116 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn Ala Phe Thr Asn Tyr             20 25 30 Leu Ile Glu Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Ile Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ser Gly Ser Ala Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 24 <211> 342 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 24 gacatcgtga tgacccagtc ccccgactcc ctggccgtgt ccctgggcga gcgcgccacc 60 atcaactgca agtcctccca gtccctgctg aactccggca accagaagac ctacctgacc 120 tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc ctccacccgc 180 gagtccggcg tgcccgaccg cttctccggc tccggctccg gcaccgactt caccctgacc 240 atctcctccc tgcaggccga ggacgtggcc gtgtactact gccagaacga ctactcctac 300 cccccctgga ccttcggcca gggcaccaag gtggagatca ag 342 <210> 25 <211> 357 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 25 caggtgcagc tgcaggagtc cggccccggc ctggtgaagc cctccgagac cctgtccctg 60 acctgcaccg tgtccggctt ctccctgacc tcctacggcg tgcactggat ccgccagccc 120 cccggcaagg gcctggagtg gctgggcctg atctgggccg gcggctccat caactacaac 180 tccgccctga tgtcccgcgt gaccatctcc aaggacacct ccaagaacca ggtgtccctg 240 aagctgtcct ccgtgaccgc cgccgacacc gccgtgtact actgcgcccg caagggccac 300 atgtactcct acgccatgga ctactggggc cagggcaccc tggtgaccgt gtcctcc 357 <210> 26 <211> 348 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 26 caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcaa cgccttcacc aactacctga tcgagtgggt gcgccaggcc 120 cccggccagg gcctggagtg gatgggcgtg atcaaccccg gctccggcat caccaactac 180 aacgagaagt tcaagggcaa ggccaccatc accgccgaca agtccacctc caccgcctac 240 atggagctgt cctccctgcg ctccgaggac accgccgtgt actactgctc cggctccgcc 300 aactggttcg cctactgggg ccagggcacc ctggtgaccg tgtcctcc 348 <210> 27 <211> 339 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 27 gacatcgtga tgacccagtc ccccgactcc ctggccgtgt ccctgggcga gcgcgccacc 60 atcaactgca agtcctccca gtccctgctg tactcctcca accagaagaa ctacctggcc 120 tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc ctccacccgc 180 gactccggcg tgcccgaccg cttctccggc tccggctccg gcaccgactt caccctgacc 240 atctcctccc tgcaggccga ggacgtggcc gtgtactact gccagcgcta ctacggctac 300 ccctggacct tcggcggcgg caccaaggtg gagatcaag 339 <210> 28 <211> 348 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 28 caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcaa cgccttcacc aactacctga tcgagtggat ccgccaggcc 120 cccggccagg gcctggagtg gatcggcgtg atcaaccccg gctccggcat caccaactac 180 aacgagaagt tcaagggcaa ggccaccctg accgccgaca agtccacctc caccgcctac 240 atggagctgt cctccctgcg ctccgaggac accgccgtgt actactgctc cggctccgcc 300 aactggttcg cctactgggg ccagggcacc ctggtgaccg tgtcctcc 348 <210> 29 <211> 339 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 29 gacatcgtga tgacccagtc ccccgactcc ctggccgtgt ccctgggcga gcgcgccacc 60 atcaactgca agtcctccca gtccctgctg tactcctcca accagaagaa ctacctggcc 120 tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc ctccacccgc 180 gactccggcg tgcccgaccg cttcaccggc tccggctccg gcaccgactt caccctgacc 240 atctcctccc tgcaggccga ggacgtggcc gtgtactact gccagcgcta ctacggctac 300 ccctggacct tcggcggcgg caccaaggtg gagatcaag 339 <210> 30 <211> 342 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 30 caggtgcagt tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60 acttgcactg tctctgggtt ttcattaacc acctatggtg tacactgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggagtg atgtggacta atggaatcac aaattataat 180 tcggctctca tgtccagact gagcatcagc aaagacaact ccaagagcca agttttctta 240 aaaatgaaca gtctgcaaac tgatgacaca gccatgtact actgtgccag aggaggacac 300 tacggtagta cctcctatgc tatggacttc tggagtcaag ga 342 <210> 31 <211> 114 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 31 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Ser Ser Gly Phe Ser Leu Thr Thr Tyr             20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu         35 40 45 Gly Val Met Trp Thr Asn Gly Ile Thr Asn Tyr Asn Ser Ala Leu Met     50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala                 85 90 95 Arg Gly Gly His Tyr Gly Ser Thr Ser Tyr Ala Met Asp Phe Trp Ser             100 105 110 Gln Gly          <210> 32 <211> 351 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 32 gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtgg gaatattcac aattatttaa catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctatact gcaaaaacct tagcagatgg tgtgccatca 180 aggttcagtg gcagtggatc aggaacacaa tattctctca agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat ttttggaata ctccttacac gttcggaggg 300 gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat c 351 <210> 33 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr             20 25 30 Leu Thr Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val         35 40 45 Tyr Thr Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Asn Thr Pro Tyr                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 34 <211> 342 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 34 caggtgcagc tggaggagtc aggacctggc ctggtggcga cctcacagag cctgtccatc 60 acttgcactg tctctgggtt ttcattaacc aactgtggtg tacactgggt tcgccagcct 120 caaggaaagg gtctagagtg gctgggagtg atatggccta atggaatcac aatttataat 180 tcgggtctca tgtccagact gagtatcagc aaagacaact ccaagagcca agttttctta 240 aaaaagaaca gtctgcaaac tgatgacaca gccatgtact actgtgccag aggaggacat 300 tacggtagta gctcctatgc tatggactac tggagtcaag ga 342 <210> 35 <211> 114 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 35 Gln Val Gln Leu Glu Glu Ser Gly Pro Gly Leu Val Ala Thr Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Cys             20 25 30 Gly Val His Trp Val Arg Gln Pro Gln Gly Lys Gly Leu Glu Trp Leu         35 40 45 Gly Val Ile Trp Pro Asn Gly Ile Thr Ile Tyr Asn Ser Gly Leu Met     50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Leu 65 70 75 80 Lys Lys Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala                 85 90 95 Arg Gly Gly His Tyr Gly Ser Ser Ser Tyr Ala Met Asp Tyr Trp Ser             100 105 110 Gln Gly          <210> 36 <211> 370 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polynucleotide " <400> 36 gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtgg gaatattcac aattatttaa catggtatca gcagaaaccg 120 ggaaaatctc ctcaactcct ggtctatact gcaaaaacct tagcagatgg tgtgccatca 180 aggttcagtg gcagtggatc aggaacacaa tattctctca agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat ttttggaata ctccttacac attcggaggg 300 gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 360 tccagtaagc 370 <210> 37 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr             20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Val         35 40 45 Tyr Thr Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Asn Thr Pro Tyr                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 38 <211> 113 <212> PRT <213> Homo sapiens <400> 38 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser             20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                 85 90 95 Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys      <210> 39 <211> 122 <212> PRT <213> Homo sapiens <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ser Ser Tyr             20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Arg His His Ser Ser Ser Trp Tyr Gly Arg Tyr Phe Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 40 <211> 121 <212> PRT <213> Homo sapiens <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ser Ser Tyr             20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg His His Ser Ser Ser Trp Tyr Gly Arg Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 41 <211> 43 <212> PRT <213> Homo sapiens <400> 41 Tyr Gly Asp Glu Ile Gly Leu Asp Ala Ala Leu Pro Gly Gln Pro 1 5 10 15 Met Glu Ala Pro Val Met Leu Trp Asp Glu Ser Ser Phe Pro Asp Ile             20 25 30 Pro Gly Ala Val Ser Ala Asn Met Thr Val Lys         35 40 <210> 42 <211> 7 <212> PRT <213> Homo sapiens <400> 42 Phe Pro Asp Ile Pro Gly Ala 1 5 <210> 43 <211> 4 <212> PRT <213> Homo sapiens <400> 43 Pro Gly Gln Pro One <210> 44 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 44 Ser Phe Pro Asp Ile Pro Gly Ala Val Ser Ala Asn Met Thr Val 1 5 10 15             5 10 15 <210> 45 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 45 Tyr Gly Asp Glu Ile Gly Leu Asp Ala Ala Ala 1 5 10 <210> 46 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 46 Gly Asp Glu Ile Gly Leu Asp Ala Ala Ala Leu 1 5 10 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 47 Asp Glu Ile Gly Leu Asp Ala Ala Ala Leu Pro 1 5 10 <210> 48 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 48 Glu Ile Gly Leu Asp Ala Ala Leu Pro Gly 1 5 10 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 49 Ile Gly Leu Asp Ala Ala Leu Pro Gly Gln 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 50 Gly Leu Asp Ala Ala Leu Pro Gly Gln Pro 1 5 10 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 51 Leu Asp Ala Ala Leu Pro Gly Gln Pro Met 1 5 10 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 52 Asp Ala Ala Leu Pro Gly Gln Pro Met Glu 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 53 Ala Ala Ala Leu Pro Gly 1 5 10 <210> 54 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 54 Ala Ala Leu Pro Gly Gln Pro 1 5 10 <210> 55 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 55 Ala Leu Pro Gly Gln Pro Met Glu Ala Pro Val 1 5 10 <210> 56 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 56 Leu Pro Gly Gln Pro Met Glu Ala Pro Val Met 1 5 10 <210> 57 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 57 Pro Gly Gln Pro Met Glu Ala Pro Val Met Leu 1 5 10 <210> 58 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 58 Gly Gln Pro Met Glu Ala Pro Val Met Leu Trp 1 5 10 <210> 59 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 59 Gln Pro Met Glu Ala Pro Val Met Leu Trp Asp 1 5 10 <210> 60 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 60 Ala Pro Val Met Leu Trp Asp Glu Ser Ser Phe 1 5 10 <210> 61 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 61 Pro Val Met Leu Trp Asp Glu Ser Ser Phe Pro 1 5 10 <210> 62 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 62 Val Met Leu Trp Asp Glu Ser Ser Phe Pro Asp 1 5 10 <210> 63 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 63 Met Leu Trp Asp Glu Ser Ser Phe Pro Asp Ile 1 5 10 <210> 64 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 64 Leu Trp Asp Glu Ser Ser Phe Pro Asp Ile Pro 1 5 10 <210> 65 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 65 Trp Asp Glu Ser Ser Phe Pro Asp Ile Pro Gly 1 5 10 <210> 66 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 66 Asp Glu Ser Ser Phe Pro Asp Ile Pro Gly Ala 1 5 10 <210> 67 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 67 Glu Ser Ser Phe Pro Asp Ile Pro Gly Ala Val 1 5 10 <210> 68 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 68 Ser Ser Phe Pro Asp Ile Pro Gly Ala Val Ser 1 5 10 <210> 69 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 69 Ser Phe Pro Asp Ile Pro Gly Ala Val Ser Ala 1 5 10 <210> 70 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 70 Phe Pro Asp Ile Pro Gly Ala Val Ser Ala Asn 1 5 10 <210> 71 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 71 Pro Asp Ile Pro Gly Ala Val Ser Ala Asn Met 1 5 10 <210> 72 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 72 Asp Ile Pro Gly Ala Val Ser Ala Asn Met Thr 1 5 10 <210> 73 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 73 Ile Pro Gly Ala Val Ser Ala Asn Met Thr Val 1 5 10 <210> 74 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 74 Pro Gly Ala Val Ser Ala Asn Met Thr Val Lys 1 5 10 <210> 75 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 75 Gly Leu Asp Ala Ala Ala Leu Pro Ala Gln Pro 1 5 10 <210> 76 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 76 Gly Leu Asp Ala Ala Ala Leu Ala Gly Gln Pro 1 5 10 <210> 77 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 77 Gly Leu Asp Ala Ala Leu Pro Gly Gln Pro 1 5 10 <210> 78 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 78 Gly Leu Asp Ala Gly Leu Pro Gly Gln Pro 1 5 10 <210> 79 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 79 Trp Asp Glu Ser Ser Phe Pro Ala Ile Pro Gly 1 5 10 <210> 80 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 80 Glu Ser Ser Phe Pro Asp Ala Pro Gly Ala Val 1 5 10 <210> 81 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 81 Trp Asp Glu Ser Ser Phe Pro Asp Ala Pro Gly 1 5 10 <210> 82 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 82 Pro Ala Ile Pro Gly Ala Val Ser Ala Asn Met 1 5 10 <210> 83 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 83 Glu Ser Ser Phe Pro Ala Ile Pro Gly Ala Val 1 5 10 <210> 84 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 84 Glu Ser Ser Phe Pro Asp Ile Pro Gly Gly Val 1 5 10 <210> 85 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 85 Ser Phe Pro Asp Ala Pro Gly Ala Val Ser Ala 1 5 10 <210> 86 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 86 Glu Ile Gly Leu Asp Ala Ala Ala Lea Ala Gly 1 5 10 <210> 87 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 87 Leu Pro Ala Gln Pro Met Glu Ala Pro Val Met 1 5 10 <210> 88 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 88 Leu Ala Gly Gln Pro Met Glu Ala Pro Val Met 1 5 10 <210> 89 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 89 Glu Ile Gly Leu Asp Ala Gly Leu Pro Gly 1 5 10 <210> 90 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 90 Ala Ala Leu Pro Ala Gln Pro 1 5 10 <210> 91 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 91 Asp Ala Ala Leu Pro Ala Gln Pro Met Glu 1 5 10 <210> 92 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 92 Glu Ile Gly Leu Asp Ala Ala Ala Leu Pro Ala 1 5 10 <210> 93 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 93 Glu Ile Gly Leu Asp Ala Ala Leu Pro Gly 1 5 10 <210> 94 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 94 Ser Phe Pro Ala Ile Pro Gly Ala Val Ser Ala 1 5 10 <210> 95 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       peptide " <400> 95 Pro Asp Ala Pro Gly Ala Val Ser Ala Asn Met 1 5 10 <210> 96 <211> 526 <212> PRT <213> Mus musculus <400> 96 Met Ser Gln Asp Thr Glu Val Asp Met Lys Asp Val Glu Leu Asn Glu 1 5 10 15 Leu Glu Pro Glu Lys Gln Pro Met Asn Ala Ala Asp Gly Ala Ala Ala             20 25 30 Gly Glu Lys Asn Gly Leu Val Lys Ile Lys Val Ala Glu Asp Glu Thr         35 40 45 Glu Ala Gly Val Lys Phe Thr Gly Leu Ser Lys Glu Glu Leu Leu Lys     50 55 60 Val Ala Gly Ser Pro Gly Trp Val Arg Thr Arg Trp Ala Leu Leu Leu 65 70 75 80 Leu Phe Trp Leu Gly Trp Leu Gly Met Leu Ala Gly Ala Val Val Ile                 85 90 95 Ile Val Arg Ala Pro Arg Cys Arg Glu Leu Pro Val Gln Arg Trp Trp             100 105 110 His Lys Gly Ala Leu Tyr Arg Ile Gly Asp Leu Gln Ala Phe Val Gly         115 120 125 Arg Asp Ala Gly Gly Ile Ala Gly Leu Lys Ser His Leu Glu Tyr Leu     130 135 140 Ser Thr Leu Lys Val Lys Gly Leu Val Leu Gly Pro Ile His Lys Asn 145 150 155 160 Gln Lys Asp Glu Ile Asn Glu Thr Asp Leu Lys Gln Ile Asn Pro Thr                 165 170 175 Leu Gly Ser Gln Glu Asp Phe Lys Asp Leu Leu Gln Ser Ala Lys Lys             180 185 190 Lys Ser Ile His Ile Ile Leu Asp Leu Thr Pro Asn Tyr Gln Gly Gln         195 200 205 Asn Ala Trp Phe Leu Pro Ala Gln Ala Asp Ile Val Ala Thr Lys Met     210 215 220 Lys Glu Ala Leu Ser Ser Trp Leu Gln Asp Gly Val Asp Gly Phe Gln 225 230 235 240 Phe Arg Asp Val Gly Lys Leu Met Asn Ala Pro Leu Tyr Leu Ala Glu                 245 250 255 Trp Gln Asn Ile Thr Lys Asn Leu Ser Glu Asp Arg Leu Leu Ile Ala             260 265 270 Gly Thr Glu Ser Ser Asp Leu Gln Gln Ile Val Asn Ile Leu Glu Ser         275 280 285 Thr Ser Asp Leu Leu Leu Thr Ser Ser Tyr Leu Ser Asn Ser Thr Phe     290 295 300 Thr Gly Glu Arg Thr Glu Ser Leu Val Thr Arg Phe Leu Asn Ala Thr 305 310 315 320 Gly Ser Gln Trp Cys Ser Trp Ser Val Ser Gln Ala Gly Leu Leu Ala                 325 330 335 Asp Phe Ile Pro Asp His Leu Leu Arg Leu Tyr Gln Leu Leu Leu Phe             340 345 350 Thr Leu Pro Gly Thr Pro Val Phe Ser Tyr Gly Asp Glu Leu Gly Leu         355 360 365 Gln Gly Ala Leu Pro Gly Gln Pro Ala Lys Ala Pro Leu Met Pro Trp     370 375 380 Asn Glu Ser Ser Ile Phe His Ile Pro Arg Pro Val Val Ser Leu Asn Met 385 390 395 400 Thr Val Lys Gly Gln Asn Glu Asp Pro Gly Ser Leu Leu Thr Gln Phe                 405 410 415 Arg Arg Leu Ser Asp Leu Arg Gly Lys Glu Arg Ser Leu Leu His Gly             420 425 430 Asp Phe His Ala Leu Ser Ser Ser Pro Asp Leu Phe Ser Tyr Ile Arg         435 440 445 His Trp Asp Gln Asn Glu Arg Tyr Leu Val Val Leu Asn Phe Arg Asp     450 455 460 Ser Gly Arg Ser Ala Arg Leu Gly Ala Ser Asn Leu Pro Ala Gly Ile 465 470 475 480 Ser Leu Pro Ala Ser Ala Lys Leu Leu Leu Ser Thr Asp Ser Ala Arg                 485 490 495 Gln Ser Arg Glu Glu Asp Thr Ser Leu Lys Leu Glu Asn Leu Ser Leu             500 505 510 Asn Pro Tyr Glu Gly Leu Leu Leu Gln Phe Pro Phe Val Ala         515 520 525 <210> 97 <211> 48 <212> PRT <213> Homo Sapiens <400> 97 Thr Leu Pro Gly Thr Pro Val Phe Ser Tyr Gly Asp Glu Ile Gly Leu 1 5 10 15 Asp Ala Ala Leu Pro Gly Gln Pro Met Glu Ala Pro Val Met Leu     20 25 30 Trp Asp Glu Ser Ser Phe Pro Asp Ile Pro Gly Ala Val Ser Ala Asn 35 40 45 <210> 98 <211> 48 <212> PRT <213> Mus Musculus <400> 98 Thr Leu Pro Gly Thr Pro Val Phe Ser Tyr Gly Asp Glu Leu Gly Leu 1 5 10 15 Gln Gly Ala Leu Pro Gly Gln Pro Ala Lys Ala Pro Leu Met Pro Trp     20 25 30 Asn Glu Ser Ser Ile Phe His Ile Pro Arg Pro Val Val Ser Leu Asn 35 40 45

Claims (70)

An isolated antibody or functional fragment thereof that binds to an epitope comprising residues A377, D397, I398, G400 and A401 of human CD98, as an isolated antibody or functional fragment thereof that specifically binds human CD98. The isolated antibody or functional fragment thereof of claim 1, wherein said epitope further comprises residues D374 and L378 of human CD98. 3. The isolated antibody or functional fragment thereof of claim 2, wherein the epitope further comprises residues P379 and G380 of human CD98. 4. The isolated antibody or functional fragment thereof of claim 3, wherein said epitope further comprises residues F395 and P396 of human CD98. 4. The isolated antibody or functional fragment thereof of claim 3, wherein said epitope further comprises residues Q381, P382 and P399 of human CD98. The isolated antibody of claim 1, wherein said epitope further comprises any one or more additional residues selected from the group consisting of D374, L378, P379, G380, Q381, P382, F395, P396 and P399 of human CD98 or Its functional fragment. An isolated antibody or functional fragment thereof that specifically binds human CD98, or an isolated fragment thereof, which binds to an epitope comprising residues P379, G380, D397 and I398 of human CD98. 8. The isolated antibody or functional fragment thereof of claim 7, wherein said epitope further comprises residues F395 and P396 of human CD98. 9. The isolated antibody or functional fragment thereof of claim 8, wherein said epitope further comprises residues Q381, P382, P399, G400 and A401 of human CD98. 9. The isolated antibody or functional fragment thereof of claim 8, wherein said epitope further comprises residues D374, A377 and L378 of human CD98. 8. The isolated polynucleotide of claim 7, wherein said epitope further comprises any one or more additional residues selected from the group consisting of D374, A377, L378, Q381, P382, F395, P396, P399, G400 and A401 of human CD98. Antibody or functional fragment thereof.  The isolated antibody or its functional fragment thereof according to claim 1, which binds to an epitope comprising residues D374, A377, L378, P379, G380, Q381, P382, F395, P396, D397, I398, P399, G400 and A401 of human CD98 snippet. An isolated antibody or functional fragment thereof, which specifically binds to human CD98, as an isolated antibody or functional fragment thereof, which binds to an epitope contained within amino acid residues 369 to 405 of human CD98. An isolated antibody or functional fragment thereof, which specifically binds to human CD98, as an isolated antibody or functional fragment thereof, which binds to an epitope consisting of amino acid residues 369 to 405 of human CD98. The antibody or the functional fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody. 16. The antibody of claim 15, wherein the monoclonal antibody is a humanized human or chimeric antibody, or a functional fragment thereof. 2. The antibody of claim 1, wherein the fragment is a Fab, F (ab ') 2, Fv or Sfv fragment, or a functional fragment thereof. All three heavy chain complementarity determining regions (CDRs) from the heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 12, and / or amino acid sequences selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: Wherein the light chain variable region comprises all three light chain CDRs from the light chain variable domain having the sequence. All three heavy chain CDRs from the heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 12, and a light chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 14 An isolated antibody comprising all three light chain CDRs or a functional fragment thereof. 20. The antibody of claim 19, comprising a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 20. The antibody of claim 19, comprising a light chain variable domain sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 14. 21. The antibody of claim 20, further comprising a light chain variable domain sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 14. 23. The antibody of claim 22 comprising the heavy chain variable domain sequence of SEQ ID NO: 8 and the light chain variable domain sequence of SEQ ID NO: 23. The antibody of claim 22 comprising the heavy chain variable domain sequence of SEQ ID NO: 12 and the light chain variable domain sequence of SEQ ID NO: 17. The humanized antibody of claim 16 comprising a heavy chain variable domain sequence selected from SEQ ID NO: 22 and SEQ ID NO: 19. The humanized antibody of claim 16 comprising a light chain variable domain sequence selected from SEQ ID NO: 20 and SEQ ID NO: 26. The humanized antibody of claim 25, further comprising a light chain variable domain sequence selected from SEQ ID NO: 20 and SEQ ID NO: 21. 28. The humanized antibody of claim 27, comprising the light chain variable domain sequence of SEQ ID NO: 20 and the heavy chain variable domain sequence of SEQ ID NO: 28. The humanized antibody of claim 27, comprising a light chain variable domain sequence of SEQ ID NO: 21 and a heavy chain variable domain sequence of SEQ ID NO: 28. The humanized antibody of claim 27, comprising the light chain variable domain sequence of SEQ ID NO: 21 and the heavy chain variable domain sequence of SEQ ID NO: 33. An antibody that binds to an essentially identical epitope as an epitope bound by the antibody of claim 30. 31. A binding agent which binds to essentially the same epitope as an epitope bound by the antibody of any one of claims 1 to 31. 33. The binding agent of claim 32, which inhibits the growth of tumors expressing CD98. 33. The binder of claim 32, wherein the binder is an antibody or functional fragment thereof. 33. The method of claim 32, wherein the anticalin, adnectin, affibody, DARPin, fynomer, affitin, affilin, avimer, A binder which is a cysteine-rich knottin peptide or a manipulated Kunitz type inhibitor. 31. A binding agent capable of binding to CD98, wherein the antibody of any one of claims 1 to 31 replaces said binding agent in a competitive binding assay. 31. A binding agent capable of binding to CD98, wherein the binding agent replaces the antibody of any one of claims 1 to 31 in a competitive binding assay. 37. The binder of claim 36, wherein the binder is an antibody or functional fragment thereof. 38. The binder of claim 37, wherein the binder is an antibody or functional fragment thereof. 40. An antibody or a functional fragment thereof according to any one of claims 1 to 31, 34, 38 and 39, attached to a cytotoxic agent. 41. The antibody of claim 40, wherein the cytotoxic agent is selected from a chemotherapeutic agent, a drug, a growth inhibitor, a toxin, and a radioactive isotope, or a functional fragment thereof. 39. The antibody or the functional fragment thereof according to any one of claims 1 to 31, 34, 38 and 39, which is bound to a detectable marker. 41. The antibody or functional fragment thereof of claim 40, wherein the detectable marker is selected from a radioactive isotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound. 16. A transgenic animal producing the monoclonal antibody of claim 15. 15. A hybridoma producing the monoclonal antibody of claim 15. A vector comprising a polynucleotide encoding an antibody of any one of claims 1 to 31, 34, 38 or 39 or a fragment thereof. 43. A pharmaceutical composition comprising an antibody of any one of claims 1 to 31, 34 and 38 to 43 or a functional fragment thereof and a pharmaceutically acceptable carrier. A method for inhibiting the growth of cancer cells expressing CD98, comprising exposing said cells to an antibody of any one of claims 1 to 31, 34 and 38 to 43 or a functional fragment thereof How to. 48. The method of claim 48, wherein the cancer cells are selected from the group consisting of bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, Or a transition of any one of these arms. 50. The method of claim 49, wherein the cancer cells are derived from acute myelogenous leukemia. 47. A method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition of claim 47. 53. The method of claim 51, wherein the cancer is selected from bladder cancer, breast cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma and leukemia Wherein one of the arms is a transition. 53. The method of claim 52, wherein the cancer is acute myelogenous leukemia. 52. The method of claim 51, wherein the subject has relapsed or refractory acute myelogenous leukemia. 52. The method of claim 51, wherein the subject is administered one or more chemotherapeutic compounds with the antibody or functional fragment, wherein the chemotherapeutic compound is selected from the group consisting of vendamustine hydrochloride, cyclophosphamide, Wherein the method is selected from the group consisting of lomycetin, lomycetin, lomex, lomycin, lomycetin, lomycin, lambin, gemcitabine, prednisone, prednisolone, methylprednisolone, paclitaxel, docked cell, vinorelbine, vincristine, etoposide, irinotecan, anthracycline, adriamycin, cisplatin, . 52. The method of claim 51, wherein the cancer is associated with increased expression of CD98 on the surface of the cell. 31. A method of detecting the presence of CD98 in a biological sample comprising contacting the biological sample with an antibody of any one of claims 1 to 31, 34 and 38 to 43 under conditions that allow binding of the antibody to CD98 And detecting whether a complex is formed between the antibody and CD98. 57. The method of claim 57, wherein the biological sample is selected from bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma and leukemia. , Or a biological sample of a mammal suspected of having or having a metastasis of any of these cancers. Contacting the test cell with the antibody of any one of claims 1 to 31, 34 and 38 to 43; Detecting the binding of the antibody to CD98 and measuring the level of expression of CD98; And comparing the level of expression of CD98 in the test cell with the level of expression of CD98 in the control cell, wherein the expression of CD98 in the test cell relative to the control cell Wherein the higher expression level of CD98 is indicative of the presence of cancer associated with increased expression of CD98. 59. The method of claim 59, wherein the test cells are selected from bladder cancer, breast cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, lung cancer, laryngeal cancer, kidney cancer, oral cancer, ovarian cancer, prostate cancer, sarcoma, melanoma, glioma, lymphoma and leukemia. , Or a patient suspected of having a metastasis of either of these cancers. 61. The method of claim 60, comprising measuring the level of expression of CD98 on the surface of the test cell, and comparing the level of expression of CD98 on the surface of the test cell with the level of expression of CD98 on the surface of the control cell Way. 63. The method of claim 61, wherein the test cell is a cancer cell and the control cell is a normal cell of the same tissue type. Use of an antibody or a functional fragment thereof according to any one of claims 1 to 31, 34 and 38 to 43 in the manufacture of a medicament, wherein said medicament inhibits the growth of cancer cells expressing CD98 To be used in a method that is to be used. An antibody or a functional fragment thereof according to any one of claims 1 to 31, 34 and 38 to 43 for use in inhibiting the growth of cancer cells expressing CD98. Use of the pharmaceutical composition of claim 47 in the manufacture of a medicament, wherein the medicament is for use in a method of treating cancer in a subject. Comprising an antibody of any one of claims 1 to 31, 34 and 38 to 43 for use in treating cancer in a subject or a functional fragment thereof and a pharmaceutically acceptable carrier, A pharmaceutical composition. Use of an antibody or a functional fragment thereof according to any one of claims 1 to 31, 34 and 38 to 43 in the manufacture of a medicament, wherein the agent detects the presence of CD98 in a biological sample Use for use in a method. An antibody or a functional fragment thereof according to any one of claims 1 to 31, 34 and 38 to 43 for use in a method for detecting the presence of CD98 in a biological sample. Use of an antibody or a functional fragment thereof according to any one of claims 1 to 31, 34 and 38 to 43 in the manufacture of a medicament, wherein the medicament is for the treatment of cancer associated with increased expression of CD98 Use for diagnostic methods. An antibody or a functional fragment thereof according to any one of claims 1 to 31, 34 and 38 to 43 for use in a method for diagnosing cancer associated with increased expression of CD98.

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