CN104302669A - Anti-CD98 antibodies and methods of use thereof - Google Patents

Abstract

The invention provides antibodies that bind CD98, and methods of use of the antibodies in the diagnosis and treatment of cancers.

Description

Anti-CD 98 antibody and using method thereof

Technical field

Present invention relates in general to the method for anti-CD 98 antibody and this antibody-like of use.

Background technology

CD98 (is also referred to as CD98 heavy chain; 42F heavy chain; SLC3A2) the II type transmembrane glycoprotein be made up of 529 amino-acid residues.This albumen comprises 75 amino acid whose N-terminal born of the same parents inner cell matter territories, single membrane-spanning domain and 426 amino acid whose C-terminal extracellular domains (people such as Parmacek, Nucleic Acids Res.17:1915-1931,1989).CD98 is covalently attached to one of several light chain (SLC7A5,6,7,8,10 or 11) by disulfide linkage, and these light chains are L-type amino acid transporters.This interaction is required for the cell surface expression of light chain and amino acid transport function.CD98 is also combined with integrin beta subunit, have adjusted integrin signaling people such as (, J.Cell Sci.118:889-899,2005) Cai controlling cell proliferation, survival, migration and Epithelial Cell Adhesion/polarity whereby.

CD98 was identified as the cell-surface antigens (people such as Haynes, J.Immunol.126:1409-1414,1981) relevant with lymphocyte activation originally.After this, in all cells type except thrombocyte, all identify CD98, and the expression level of CD98 in stomach and intestine (GI) road and uriniferous tubules the highest (people such as Verrey, Pflugers Arch.440:503-512,2000).The rise of CD98 has been observed in enteritis.Recently, intestines CD98 expresses to be presented at and controls to have vital role in homeostasis in intestines and innate immune response.Therefore, the adjustment that in intestinal epithelial cells, CD98 expresses is shown to be inflammatory bowel disease, as inflammatory bowel (IBD) and the treatment of colitis related cancer and the therapeutic strategy likely (people such as Nguyen of prevention, J.Clin.Invest.121:1733-1747,2011).Do not consider tissue-derived, CD98 is process LAN (people such as Itoh, Jpn.J.Cancer Res.92:13 13-1321,2001) on the cell surface of nearly all tumour cell also.

In polytype human cancer cell, also observed one of the light chain in conjunction with CD98, L-type amino acid transporter 1 (LAT 1; Also referred to as SLC7A5) expression increase, described cancer comprises mammary cancer, colorectal carcinoma, oral carcinoma, ovarian cancer, esophagus cancer, neurospongioma and leukemia people such as (, Biochem.Pharmacol.80:811-818,2010) Fan.May need to increase the Seedling height speed that cancer cells is supported in amino acid supply, this be amino acid building block by being provided for protein synthesis and by Mammals rapamycin target (mTOR) stimulating growth realize (people such as Fan, the same; The people such as Imai, Anticancer Res.30:4819-4828,2010).Compared with former site, in the transitivity site of human cancer, the expression of LAT1 and CD98 is significantly higher, and this shows that the process LAN of LAT1/CD98 is human cancer development and shifts necessary.Particularly, LAT1/CD98 process LAN seemingly metastases necessary (people such as Kaira, Cancer Sci.99:2380-2386,2008) in colorectal cancer patients.

CD98 and LAT1 and expression type and function show that these albumen are targets likely for the treatment of various human cancer.LAT1 activity inhibitor is in certain cancers type, comprise nonsmall-cell lung cancer (people such as Imai, the same), the colon cancer cell (people such as Oda, Cancer Sci.101:173-179,2010), cancer cell of oral cavity (people such as Kim, Biol.Pharm.Bull.33:1117-1121,2010), anti-tumor activity is demonstrated with in breast cancer cell (Shennan and Thomson, Oncol.Rep.20:885-889,2008).LAT1 also show be treatment ovarian cancer target people such as (, the same) Fan in demonstrate anti-tumor activity.

Find to differentiate that the mouse-anti CD98 monoclonal antibody for HBJ127 suppresses lymphopoiesis (Yagita and Hashimoto, J.Immunol.136:2062-2068,1986) and suppress the growth (people such as Yagita of sacculus tumour and lymphoma cell, Cancer Res.46:1478-1489,1986).Find that the epi-position of HJ127 antibody is residue 442AFS444 people such as (, 2007) Itoh of people CD98.Different mouse-anti CD98 monoclonal antibodies demonstrates remarkable inhibition tumor cell growth in vitro (Papetti and Herman, Am.J.Pathol.159:165-178,2001) to neurospongioma, prostate gland and colon cancer cell.Other anti-human CD98 monoclonal antibody is disclosed in U.S. Patent Publication No.20100143367.These monoclonal antibodies are bonded to the epi-position in amino acid district 372-530 and 104-371 of CD98.Find in these antibody the five kinds Amino Acid Absorptions suppressing in transitional cell bladder carcinoma cell lines, and in these antibody three kinds show Tumor suppression growth in mouse model.

As disclosed herein, the surface markers spectrotype of cell surface proteins group is used to differentiate transmembrane protein CD98 for exist with high-density on AML tumor cell surface to the analysis of patient Xin Fa primary acute myeloid leukaemia (AML) tumor sample.Therefore, CD98 is (such as) by using bonding agent, as specific binding to the antibody of CD98 treats the target of AML.The bonding agent special to CD98, as anti-CD 98 antibody, also demonstrate not only in the treatment of AML in heteroplastic transplantation model in multiple body, and at kinds cancer, in the treatment as sarcoma, lymphoma, nonsmall-cell lung cancer (NSCLC) and colorectal carcinoma, there is application.

The invention provides CD98 useful in the Diagnosis and Treat of polytype human cancer.

Summary of the invention

Use in the solution of complete AML tumor cell surface and mark, then CD98 is differentiated as compared with normal cell (comprising the hemocyte of growth) exist with high-density on the surface at most of AML cell subsets by the high-resolution Liquid Chromatography-Tandem Mass Spectrometry coupling (LC-MS/MS) based on solution.Therefore, the invention provides anti-CD 98 antibody and use this kind of Antybody therapy AML and other cancers, including, but is not limited to the method for lymphoma, sarcoma, nonsmall-cell lung cancer and colorectal carcinoma.

In one embodiment, the invention provides specific binding to the antibody of the separation of people CD98 or its functional fragment, wherein said antibody or functional fragment be bonded to comprise people CD98 residue A 377, D397,1398, the epi-position of G400 and A401.In some embodiments, described epi-position also comprises residue D374 and L378 of people CD98.In some embodiments, described epi-position also comprises residue P379 and G380 of people CD98.In some embodiments, described epi-position also comprises residue F395 and P396 of people CD98.In some embodiments, described epi-position also comprises residue Q381, P382 and P399 of people CD98.In some embodiments, described epi-position also comprises any one or other residues multiple in the group selecting D374, L378, P379, G380, Q381, P382, F395, P396 and P399 of freeman CD98 to form.

In one embodiment, the invention provides specific binding to the antibody of the separation of people CD98 or its functional fragment, wherein said antibodies is to the epi-position of residue P379, G380, D397 and 1398 of comprising people CD98.In some embodiments, described epi-position also comprises residue F395 and P396 of people CD98.In some embodiments, described epi-position also comprises residue Q381, P382, P399, G400 and A401 of people CD98.In some embodiments, described epi-position also comprises residue D374, A377 and L378 of people CD98.In some embodiments, described epi-position also comprises any one or other residues multiple in the group selecting D374, A377, L378, Q381, P382, F395, P396, P399, G400 and A401 of freeman CD98 to form.

In some embodiments, the invention provides antibody or its functional fragment of separation, wherein said antibody or functional fragment be bonded to comprise people CD98 residue D374, A377, L378, P379, G380, Q381, P382, F395, P396, D397,1398, the epi-position of P399, G400 and A401.

In some embodiments, the invention provides specific binding to the antibody of the separation of people CD98 or its functional fragment, wherein said antibody or functional fragment are bonded to the epi-position be included in the amino-acid residue 369-405 of people CD98.In some embodiments, the invention provides specific binding to the antibody of the separation of people CD98 or its functional fragment, wherein said antibody or functional fragment are bonded to the epi-position be made up of the amino-acid residue 369-405 of people CD98.

In some embodiments, monoclonal antibody of the present invention is humanized antibody, people's antibody or chimeric antibody.In some embodiments, antibody functional fragment of the present invention is Fab, F (ab') 2, Fv or scFv fragment.

In one embodiment, the invention provides antibody or its functional fragment of separation, it comprises from having whole three the heavy chain complementary determining regions (CDR) of heavy-chain variable domains of aminoacid sequence being selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:31 and SEQ ID NO:35, and/or from having whole three the light chain CDR of light variable domains of aminoacid sequence being selected from SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:33 and SEQ ID NO:37.

In one embodiment, the invention provides antibody or its functional fragment of separation, it comprises from having whole three the heavy chain CDR of heavy-chain variable domains of aminoacid sequence being selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:31 and SEQ ID NO:35, and from having whole three the light chain CDR of light variable domains of aminoacid sequence being selected from SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:33 and SEQ ID NO:37.In some embodiments, antibody or its functional fragment comprise whole heavy chain and light chain complementarity determining area (CDR), its from: (a) is called the antibody of 8-34B; B () is called the antibody of 18-2A2.2; C () is called the antibody of 18-2A7.1; D () is called the antibody of 1-47C; Or (e) is called the antibody of 1-115A.In some embodiments, described antibody or its functional fragment comprise to call oneself whole heavy chain of antibody of 8-34B and light chain CDR.In some embodiments, described antibody or its functional fragment comprise to call oneself whole heavy chain of antibody of 18-2A2.2 and light chain CDR.In some embodiments, described antibody or its functional fragment comprise to call oneself whole heavy chain of antibody of 18-2A7.1 and light chain CDR.In some embodiments, described antibody or its functional fragment comprise to call oneself whole heavy chain of antibody of 1-47C and light chain CDR.In some embodiments, described antibody or its functional fragment comprise to call oneself whole heavy chain of antibody of 1-115A and light chain CDR.

In some embodiments, described antibody comprises the heavy-chain variable domains sequence being selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:31 and SEQ ID NO:35.In some embodiments, described antibody comprises the light variable domains sequence being selected from SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:33 and SEQ ID NO:37.In some embodiments, described antibody comprises the heavy-chain variable domains sequence being selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:31 and SEQ ID NO:35, and comprises the light variable domains sequence being selected from SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:33 and SEQ ID NO:37.

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

In one embodiment, the invention provides humanized antibody.In some embodiments, described humanized antibody comprises the heavy-chain variable domains sequence being selected from SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:23.In some embodiments, described humanized antibody comprises the light variable domains sequence being selected from SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:21.In some embodiments, described humanized antibody comprises the heavy-chain variable domains sequence being 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 the light variable domains sequence being selected from SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:21.In some embodiments, described humanized antibody comprises the light variable domains sequence being selected from SEQ ID NO:15 and SEQ ID NO:16, and is selected from the heavy-chain variable domains sequence of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.In one embodiment, described humanized antibody comprises the light variable domains sequence of SEQ ID NO:15 and the heavy-chain variable domains sequence of SEQ ID NO:18.

In one embodiment, described humanized antibody comprises the light variable domains sequence of SEQ ID NO:20 and the heavy-chain variable domains sequence of SEQ ID NO:22.In one embodiment, described humanized antibody comprises light variable domains sequence and the heavy-chain variable domains sequence shown in SEQ ID NO:23 of SEQ ID NO:21.In one embodiment, described humanized antibody comprises the light variable domains sequence of SEQ ID NO:20 and the heavy-chain variable domains sequence of SEQ ID NO:23.In one embodiment, described humanized antibody comprises the light variable domains sequence of SEQ ID NO:21 and the heavy-chain variable domains sequence of SEQ ID NO:22.In one embodiment, the invention provides the antibody being bonded to the epi-position identical with humanized antibody, it comprises the light variable domains sequence of SEQ ID NO:21 and the heavy-chain variable domains sequence of SEQ ID NO:22.In substituting embodiment, the present invention comprises the bonding agent being bonded to the epi-position substantially identical with the antibody from bin1 or bin3-7, as shown in Figure 1.

In other embodiments, the present invention comprises the bonding agent being bonded to the epi-position substantially identical with above disclosed any antibody.In some embodiments, described bonding agent suppresses the growth of the tumour expressing CD98.In some embodiments, described bonding agent is antibody or its functional fragment.In other embodiments, described bonding agent is anticalin, adnectin, affine body (affibody), DARPin, fynomer, affitin, affilin, Avimers (avimer), kink rhzomorph peptide (knottin peptide) or the Kunitz type inhibitor of engineering design.

In one embodiment, the invention provides the bonding agent that can be bonded to CD98, disclosed any one antibody displacement bonding agent more than wherein in competitive binding assay.In some embodiments, described bonding agent is antibody or its functional fragment.In another embodiment, the invention provides the bonding agent that can be bonded to CD98, wherein more than bonding agent displacement any one antibody disclosed in competitive binding assay.In some embodiments, described bonding agent is antibody or its functional fragment.

In some embodiments, the invention provides the antibody being bonded to CD98, wherein said antibody comprises and is 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, the aminoacid sequence of SEQ ID NO:31 and SEQ ID NO:35 has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, the heavy-chain variable domains of the sequence iden of at least 98% or at least 99%.In some embodiments, described antibody comprise be selected from 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:33 and SEQ ID NO:37 aminoacid sequence there is the light variable domains of the sequence iden of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.In some embodiments, described antibody comprises and is 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, the aminoacid sequence of SEQ ID NO:31 and SEQ ID NO:35 has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, the heavy-chain variable domains of the sequence iden of at least 98% or at least 99%, and described antibody also comprises and is selected from 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, the aminoacid sequence of SEQ ID NO:33 and SEQ ID NO:37 has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, the light variable domains of the sequence iden of at least 98% or at least 99%.

In some embodiments, the invention provides the antibody of the variant as any above antibody, it has one or more aminoacid replacement, disappearance, insertion or modification, and it remains the biological function of described antibody.In some embodiments, the invention provides the CD98 being bonded to and expressing on cell surface and cytostatic antibody.In some embodiments, anti-CD 98 antibody is bonded to the CD98 and antiproliferative effect that express on cell surface.In some embodiments, anti-CD 98 antibody is bonded to the CD98 and inducing cell death that express on cell surface.In some embodiments, the invention provides the antibody of the variant as any one above-mentioned antibody, it has one or more character of improvement compared with the antibody of unmodified, as immunogenicity or the solubleness of binding affinity, specificity, thermostability, expression level, effector function, glycosylation, reduction.

In some embodiments, the invention provides any one in above-mentioned antibody or functional fragment, wherein said antibody or fragment are bonded to cytotoxic reagent.In multiple embodiment, described cytotoxic reagent is selected from chemotherapeutics, medicine, growth inhibitor, toxin or radio isotope.In some embodiments, the invention provides any one in above-mentioned antibody or functional fragment, wherein said antibody or fragment are bonded to detectable mark.In multiple embodiment, described detectable mark is selected from radio isotope, metal chelator, enzyme, fluorescent chemicals, bioluminescent compound and chemiluminescence compound.

In one embodiment, the invention provides the hybridoma producing monoclonal antibody of the present invention.In one embodiment, the invention provides the transgenic animal producing monoclonal antibody of the present invention.

In some embodiments, the polynucleotide of any above-mentioned antibody of encoding are provided.In one embodiment, the carrier comprising polynucleotide is provided.In one embodiment, the host cell comprising carrier is provided.In one embodiment, described host cell is protokaryon.In one embodiment, described host cell is intestinal bacteria (E.coli) cell.In another embodiment, described host cell is eukaryotic cell.In one embodiment, described host cell is Chinese Hamster Ovary (CHO) cell.In one embodiment, provide the method preparing anti-CD 98 antibody, wherein said method is included in and is suitable for cultivating host cell when expressing the polynucleotide of encoding said antibody, and is separated described antibody.

In one embodiment, the invention provides the pharmaceutical composition comprising any above-mentioned antibody of the present invention or its functional fragment, antibody conjugates or bonding agent.In other embodiments, the invention provides the method for growth of cancer cells suppressing to express CD98, described method comprises and described cell is exposed to any one or multiple above-mentioned antibody of the present invention or its functional fragment, antibody conjugates or bonding agent.In multiple embodiment, cancer cells comes from and is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or the cancer of these cancer metastasis cancers any.

In one embodiment, the invention provides the method for cancer in treatment experimenter, described method comprises uses to described experimenter the pharmaceutical composition comprising any above-mentioned antibody of the present invention or its functional fragment, antibody conjugates or bonding agent.In numerous embodiments, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.In some embodiments, described cancer is acute myelocytic leukemia.In some embodiments, described experimenter is recurrent or refractory AML.In some embodiments, described cancer increases relevant with the expression of CD98 on cell surface.

In some embodiments, described antibody or functional fragment and one or more chemotherapy compounds are combined and use to experimenter, wherein said chemotherapy compound is selected from bendamustine hydrochloride, endoxan, ifosfamide, fludarabine (fludurabine), cytosine arabinoside, gemcitabine, prednisone, prednisolone, methylprednisolone, taxol, docetaxel, vinorelbine, vincristine(VCR), Etoposide, irinotecan, anthracycline, Dx, cis-platinum, carboplatin and Rituximab.

In one embodiment, provide the method for the existence detecting CD98 in biological sample, described method is included in and allows by described biological sample and any above-mentioned antibody contacts under the condition that is combined with CD98 of antibody, and detects between antibody and CD98 whether form mixture.In some embodiments, described biological sample comes from the Mammals suffering from or suspect and suffer from cell or tissue cancer, described cancer comprises, but be not limited to, bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

In one embodiment, provide diagnosis and express with CD98 the method raising relevant cancer, described method comprises test cell and any above-mentioned antibody contacts; The expression level of CD98 is determined by the combination detecting described antibody and CD98; With by the CD98 expression level of test cell compared with the CD98 expression level of compared with control cells, wherein compared with compared with control cells, the CD98 expression level display existence of cancer that test cell is higher and CD98 express increase relevant.In some embodiments, described test cell is from suspecting the cell suffering from the patient of cancer, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.In one embodiment, described method comprises the expression level determining test cell CD98 on the surface, with compared with the expression level of described test cell expression level and the compared with control cells CD98 on the surface of CD98 on the surface.In some embodiments, described test cell is cancer cells, and described compared with control cells is the normal cell of homologue's type.

In one embodiment, the invention provides the purposes in any above-mentioned antibody or the preparation of functional fragment medicine, wherein said medicine is for suppressing in the method for the growth of the cancer cells of expressing CD98.In numerous embodiments, described cell comes from and is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or the cancer of these cancer metastasis cancers any.

In one embodiment, the invention provides any above-mentioned antibody or functional fragment for suppressing the purposes of the growth of cancer cells of expressing CD98.In numerous embodiments, described cell comes from and is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or the cancer of these cancer metastasis cancers any.

In one embodiment, the invention provides the purposes of pharmaceutical composition in medicine preparation comprising any above-mentioned antibody or functional fragment, wherein said medicament is used for the treatment of in the method for the cancer in experimenter.In numerous embodiments, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.In some embodiments, described cancer is acute myelocytic leukemia.In some embodiments, described experimenter is recurrent or refractory AML.In some embodiments, described cancer increases relevant with the expression of CD98 on cell surface.In some embodiments, this antibody or functional fragment combined to combine with one or more chemotherapy compounds and use to experimenter, wherein this chemotherapy compound is selected from bendamustine hydrochloride, endoxan, ifosfamide, fludarabine, cytosine arabinoside, gemcitabine, prednisone, prednisolone, methylprednisolone, taxol, docetaxel, vinorelbine, vincristine(VCR), Etoposide, irinotecan, anthracycline, Dx, cis-platinum, carboplatin and Rituximab.

In one embodiment, the invention provides the application of pharmaceutical composition in treatment experimenter in cancer comprising any above-mentioned antibody or functional fragment and pharmaceutically acceptable carrier.In numerous embodiments, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.In some embodiments, described cancer is acute myelocytic leukemia.In some embodiments, described experimenter is recurrent or refractory AML.In some embodiments, described cancer increases relevant with the expression of CD98 on cell surface.In some embodiments, described antibody or functional fragment combined to combine with one or more chemotherapy compounds and use to experimenter, wherein said chemotherapy compound is selected from bendamustine hydrochloride, endoxan, ifosfamide, fludarabine, cytosine arabinoside, gemcitabine, prednisone, prednisolone, methylprednisolone, taxol, docetaxel, vinorelbine, vincristine(VCR), Etoposide, irinotecan, anthracycline, Dx, cis-platinum, carboplatin and Rituximab.

In one embodiment, the invention provides any above-mentioned antibody or the purposes of functional fragment in medicine preparation, wherein said medicine is used in the method for the existence detecting CD98 in biological sample.In some embodiments, described method is included in and allows by described biological sample and any above-mentioned antibody contacts under the condition that is combined with CD98 of antibody, and detects between antibody and CD98 whether form mixture.In some embodiments, described biological sample comes from the Mammals suffering from or suspect and suffer from cell or tissue cancer, described cancer includes, but is not limited to bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

In one embodiment, the invention provides any above-mentioned antibody or the application of functional fragment in the method for existence detecting CD98 in biological sample.In some embodiments, described method is included in and allows by described biological sample and any above-mentioned antibody contacts under the condition that is combined with CD98 of antibody, and detects between antibody and CD98 whether form mixture.In some embodiments, described biological sample comes from the Mammals suffering from or suspect and suffer from cell or tissue cancer, described cancer includes, but is not limited to bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

In one embodiment, the invention provides any above-mentioned antibody or the functional fragment purposes in medicine preparation, wherein said medicine is used for expressing in the method increasing relevant cancer with CD98 in diagnosis using.In some embodiments, described method comprises test cell and any above-mentioned antibody contacts; The expression level of CD98 is determined by the combination detecting described antibody and CD98; With by the CD98 expression level of test cell compared with the CD98 expression level of compared with control cells, wherein compared with compared with control cells, the CD98 expression level display existence of cancer that test cell is higher and CD98 express raise relevant.In some embodiments, described test cell is from suspecting the cell suffering from the patient of cancer, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.In one embodiment, described method comprises the expression level determining test cell CD98 on the surface, with compared with the expression level of described test cell expression level and the compared with control cells CD98 on the surface of CD98 on the surface.In some embodiments, described test cell is cancer cells, and described compared with control cells is the normal cell of homologue's type.

In one embodiment, the invention provides any above-mentioned antibody or functional fragment and to express application in the method raising relevant cancer in diagnosis with CD98.In some embodiments, described method comprises test cell and any above-mentioned antibody contacts; The expression level of CD98 is determined by the combination detecting described antibody and CD98; With by the CD98 expression level of test cell compared with the CD98 expression level of compared with control cells, wherein compared with compared with control cells, the CD98 expression level display existence of cancer that test cell is higher and CD98 express increase relevant.In some embodiments, described test cell is from suspecting the cell suffering from the patient of cancer, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.In one embodiment, described method comprises the expression level determining test cell CD98 on the surface, with compared with the expression level of described test cell expression level and the compared with control cells CD98 on the surface of CD98 on the surface.In some embodiments, described test cell is cancer cells, and described compared with control cells is the normal cell of homologue's type.

In another embodiment of the invention, provide article of manufacture or " test kit ", it contains the material being used for the treatment of illness as above.Described article of manufacture comprises container and to be positioned on described container or label relevant with it or package insert.The container be applicable to comprises, such as, and bottle, bottle, syringe, Blister Package etc.Described container can by multiple material, as glass or plastics are formed.Described container contains the treatment effective antibody of the patient's condition or antibody-drug conjugates (ADC) composition, and aseptic hand-hole (such as, described container can be intravenous fluids bag or the bottle with the stopper that can be pierced through by hypodermic needle) can be had.In described composition, at least one promoting agent is antibody or ADC.Label or package insert show described composition and are used for the treatment of the selected patient's condition, as cancer.Alternatively or in addition, described article of manufacture can also comprise second (or 3rd) container, it comprises pharmaceutically acceptable damping fluid, as water for injection,bacteriostatic (BWFI), phosphate buffered saline (PBS), Ringer's solution and glucose solution.It can also comprise from the other materials desired by commercialization or user's angle, and it comprises other damping fluids, thinner, strainer, syringe needle and syringe.

Accompanying drawing explanation

Fig. 1 shows the protein expression level by the sTAg analysis and identification in AML, CLL, CRC sample and corresponding normal control and quantitative CD98.Line shows the mean value of the % normalization method spectrum abundance factor (NSAF) in positive.

Fig. 2 is the figure of the epi-position frame of display 39 anti-CD 98 antibodies also (epitope binning) result.

Fig. 3 shows the binding property of inosculating antibody CD98 monoclonal antibody 8-34B, 18-2A2.1,18-2A2.2 and 18-2A2.7.Fig. 3 A is the epi-position frame of display inosculating antibody CD98 monoclonal antibody and the figure of result.Four reference antibody as shown in Figure 1." homotype " is not in conjunction with the control antibodies of the identical homotype of CD98.Fig. 3 B shows the Kd of inosculating antibody CD98 monoclonal antibody, as determined in passed through with the facs analysis of colon carcinoma cell line DLD1.Fig. 3 C shows the facs analysis result with three kinds of AML primary tumor samples of inosculating antibody CD98 monoclonal antibody dyeing and the clone of expression cynomolgus monkey CD98 (cynCD98).

Fig. 4 shows the formation of humanization 8-34B antibody.Fig. 4 A shows the sequence with the mouse 8-34B light variable domains (IGN 34) of the sequence alignment of people's receptor sequence (AC) and humanization light chain L1 and L2.According to the CDR red display of Kabat numbering, and compared with L1, the replacement in L2 represents with underscore.Fig. 4 A according to appearance order individually disclose SEQ ID NO 6,38,15-16 and 38.Fig. 4 B shows the sequence with the mouse 8-34B heavy-chain variable domains (IGN 34) of the sequence alignment of people's receptor sequence (AC) and humanized heavy chain H1, H2 and H3.According to the CDR red display of KKabat numbering, and compared with H3, the replacement in H2 and H3 represents with underscore.Fig. 4 B according to appearance order individually disclose SEQ ID NO 4,39,17-19 and 40.

Fig. 5 shows anti-CD 98 antibody process and cause strong Tumor growth inhibition in the Ramos tumour set up.Gross tumor volume when treatment starts is from (A) about 75mm ³, (B) about 150mm ³be increased to (C) about 250mm ³.Stop antibody dosage using at the 29th day (A) or the 22nd day (B), and tumor regrowth duration of measuring research is long.By Rituximab (anti-CD20 antibodies) as positive treatment control antibody, and antibody HB121 (ATCC) is as IgG2a homotype negative control.

Fig. 6 shows the time of the handled RAMOS tumor development of anti-CD 98 antibody significant prolongation.Calculate above-mentioned tumor regrowth long data tumour doubling time (Fig. 4 A-C) and for predicting development time (TTP) further.Then, to every animal extrapolation TTP in treatment group, until will 2000mm be reached ³, and as Kaplan-Meier curve tracing.

Fig. 7 shows and compares with negative control IgG2a with rituxan (Mabthera), and anti-CD98 monoclonal antibody 18-2A is to the suppression of tumor growth in vivo in lymphoma xenotransplantation.Using of arrow display antibody treatment.

Fig. 8 shows compared with negative control IgG2a, and anti-CD98 monoclonal antibody 18-2A and 8-34B is to the suppression of tumor growth in vivo in acute myelocytic leukemia xenotransplantation.Using of arrow display antibody treatment.

Fig. 9 shows and compares with negative control IgG2a (second studies) with negative control IgG2a (first studies) and with DC101+CTX (endoxan) with Erbitux, and anti-CD98 monoclonal antibody 18-2A is to the suppression of tumor growth in vivo in colorectal carcinoma xenotransplantation.DC101 is rat anti-mouse VEGFR2/KDR IgG1 mAb (ATCC No.HB-11534) and is used as positive control.Using of arrow display antibody treatment.

Figure 10 shows and compares with negative control IgG2a with Erbitux (anti-EGFR), and anti-CD98 monoclonal antibody 18-2A is to the suppression of tumor growth in vivo in nonsmall-cell lung cancer xenotransplantation.Using of arrow display antibody treatment.

Figure 11 shows anti-CD98 monoclonal antibody to the mouse system with different immune deficiency background: (A) NSG mouse; (B) impact of the heteroplastic tumor growth in vivo of lymphoma in NOD.SCID mouse and (C) SCID mouse.

Figure 12 shows and compares with they parent's mouse monoclonal antibodies (18-2A with 8-34B), and inosculating antibody CD98 monoclonal antibody (18-2A-ch7.1 and 8-34B-ch) is on the comparison of the impact of the heteroplastic tumor growth in vivo of lymphoma.

Figure 13 shows and replaces to people CD98 sequence (SEQ ID NO:1) to form mouse CD98 sequence (the SEQ ID NO:96) district of 13 mouse-people CD98 mosaic component for drawing the people CD98 epi-position figure that Humanized monoclonal antibodies IGN523 combines.

Figure 14 shows the combination of Humanized monoclonal antibodies IGN523 and control antibodies and 13 mouse-people CD98 mosaic components, as determined by facs analysis.

Figure 15 shows the sequence in the people CD98 district that wherein IGN523 combines, as use mouse-people CD98 mosaic component differentiated, and the position of this sequence in the three-dimensional arrangement of CD98.Amino acid T358-G368 (underscore) to be embedded in crystalline structure and can not to be the part of bonding interface.The non-conservation residue between people and mouse sequence is shown with overstriking.

The target ring district that Figure 16 shows by the non-homology residue from mouse CD98 being introduced into human sequence causes IGN523 to be combined with four components.Component 4.1 comprises sudden change: the disappearance of I371L, D374Q, A375G and A376.Component 4.2 comprises sudden change: M383A and E384.Component 4.3 comprises sudden change: D391N, F395I, P396F and D397H.Component 4.4 comprises sudden change: G400R, A401P and A404L.Detected by the facs analysis of the Chinese hamster ovary celI with each component transfection and combine.

Figure 17 shows the combination of the single sudden change component of hydrophobic residue in IGN523 and target ring district.As directed, by each specified hydrophobic residue of highly charged aminoacid replacement.Detected by the facs analysis of the Chinese hamster ovary celI with each component transfection and combine.

Figure 18 shows IGN523 and the combination of component containing multiple residue mutations in target ring district, as by the facs analysis with the Chinese hamster ovary celI of each component transfection detect.M1 contains sudden change D374Q, D397H, G400R and A401P.M2 contains sudden change D374E and A375E.M3 contains sudden change D397S and I398T.

Figure 19 shows the result of the variable-length peptide screening of the epitope mapping for Humanized monoclonal antibodies IGN523.By the ELISA result of sea line display often kind of peptide.The starting point of line and terminal show the residue be included in described peptide.The Y value of line shows the ELISA result obtained this peptide.Result shows the main combination of 395FPDIPGA401 and the secondary combination to 379PGQP382 (shadow zone).

Figure 20 shows the result of the single position L-Ala replacement peptide group of best combination.Each residue is all replaced (or G, if Original amino is A) by A.The height replacing letter mapping in figure is the ELISA value obtained this mutant peptide.Center line and shade interval show with reference to ELISA value.

Figure 21 shows the thermal map representing the data deriving from the CLIPS conformation matrix structure two of people CD98 partial sequences (the SEQ ID NO45-59 shown in X-axis, and the SEQ ID NO60-74 shown in Y-axis) merged.

Figure 22 shows the screen mutation result of the strong binding peptide according to the matrix analysis shown in Figure 21.SEQ1 shows peptide sequence, and DIF1 shows the position suddenlyd change in peptide.Gray area shows the peptide with non-mutated sequence.Last row show the difference of ELISA value between wild-type and mutant peptide.Higher value display sudden change has strong side effect to combination.

Figure 23 shows the position determined on the sequence neutralization surface of the people CD98 in conjunction with important amino-acid residue of Humanized monoclonal antibodies IGN523.Figure 23 A shows by mosaic and mutation research (overstriking), analyzes (grey) by Pepscan, or the position in the residue sequence that both passing through, (shades) are determined.Figure 23 B shows the resi-dues determined by mosaic and mutation research (lead).Figure 23 C shows the resi-dues determined by Pepscan analysis (light gray).Figure 23 D shows the overlap (black) of two groups of residues.

Figure 24 shows and compares with negative control IgG with Rituximab, and Humanized monoclonal antibodies IGN523 is to the suppression of tumor growth in vivo in the xenotransplantation of RAMOS (RA.1) Burkitt lymphoma.At the 11st, 17 and 25 day, with dosage administration of antibodies between 10mg/kg peritonaeum.Using of arrow display antibody treatment.

Figure 25 shows and compares with negative control IgG with rituxan, and Humanized monoclonal antibodies IGN523 is to the suppression of tumor growth in vivo in the xenotransplantation of DAU Burkitt lymphoma.The 20th and 26 days, with dosage administration of antibodies between 10mg/kg peritonaeum.Using of arrow display antibody treatment.

Figure 26 A shows and compares with negative control IgG with carboplatin, and Humanized monoclonal antibodies IGN523 is to the suppression of tumor growth in vivo in IGN-LNG-12 lung tumor xenografts.At the 17th, 24 and 31 day, use IGN523 and carboplatin with dosage between 10mg/kg or 75mg/kg peritonaeum respectively.Using of arrow display process.Figure 26 B show corresponding in Figure 26 A with the measured body weight of the mouse of indicated agent treated.Use carboplatin with its maximum tolerated dose, it causes body weight loss in NOD-SCID mouse.

Figure 27 shows and compares with negative control IgG with rituxan, and Humanized monoclonal antibodies IGN523 is to the suppression of tumor growth in vivo in the xenotransplantation of KG-1 acute myelocytic leukemia.At the 21st, 28 and 34 day, with dosage administration of antibodies between 15mg/kg peritonaeum.Using of arrow display antibody treatment.

Figure 28 shows the dose-dependent inhibition of Humanized monoclonal antibodies IGN523 to tumor growth in vivo in lung tumor xenografts.The 12nd and 19 days, with indicated dosage intraperitoneal administration of antibodies.Using of arrow display antibody treatment.

Figure 29 shows the dyeing of people by Humanized monoclonal antibodies IGN523 and cynomolgus monkey frozen tissue section.Dye with the freezing microtome section of IGN523 to people and cynomolgus monkey kidney, brain and placenta of 10 μ g/mL.Amendment Tuson, Fung and Hierck being used for immunohistochemical method is used for eliminating to the needs of mark IGN523 and is used for getting rid of the anti-human igg of the second mark and treats inspection group and be woven to nonspecific reaction (Fung1992 between endogenous IgG, Hierck1994, Tuson1990).With about 5 μm of cutting sections.Start, evaluate the tissue elements of all slide glasss and the suitability of dyeing, then by research pathologist, evaluation is carried out and subjectively classification to staining power.Except human brain (20 ×), with the representative picture of 40 × amplification display.

Embodiment

general technology

The technology of described herein or reference and program are that those skilled in the art are generally known and use ordinary method to commonly use, as, such as, people such as Sambrook, Molecular Cloning:A Laboratory Manual, 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (people such as F.M.Ausubel chief editor, (2003)); Therapeutic Monoclonal Antibodies:From Bench to Clinic, Z.An edit, Wiley, Hoboken N J. (2009); Monoclonal Antibodies:Methods and Protocols, M.Albitar edit, Humana Press, Totawa, N.J. (2010); With Antibody Engineering, the 2nd edition, the 1st volume and the 2nd volume, Kontermann and Dubel edits, Springer-Verlag, Heidelberg, the widely used method described in 2010.

Definition and abbreviation

definition

For the object explaining this specification sheets, will be suitable for give a definition and when appropriate, also will comprise plural number with the term that odd number uses, and vice versa.If when described any definition conflicts with any document incorporated herein by reference, then should be as the criterion with definition what follows.

As used herein, unless otherwise indicated, otherwise term " CD98 " refers to any natural CD98 from any vertebrates source, described vertebrates comprises Mammals, as primates (such as, people, cynomolgus monkey (cyno)), dog and rodents (such as, Mouse and rat).Amino acid and the nucleic acid sequence encoding of people CD98 is provided below respectively as SEQ ID NO:1 and SEQ ID NO:2.

MSQDTEVDMKEVELNELEPEKQPMNAASGAAMSLAGAEKNGLVKIKVAEDEAEAAAAAKFTGLSKEELLKVAGSPGWVRTRWALLLLFWLGWLGMLAGAVVIIVRAPRCRELPAQKWWHTGALYRIGDLQAFQGHGAGNLAGLKGRLDYLSSLKVKGLVLGPIHKNQKDDVAQTDLLQIDPNFGSKEDFDSLLQSAKKKSIRVILDLTPNYRGENSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDIENLKDASSFLAEWQNITKGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGSTGEHTKSLVTQYLNATGNRWCSWSLSQARLLTSFLPAQLLRLYQLMLFTLPGTPVFSYGDEIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRLSDQRSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAGLQASDLPASASLPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA(SEQ?ID?NO：1)

ATGAGCCAGGACACCGAGGTGGATATGAAGGAGGTGGAGCTGAATGAGTTAGAGCCCGAGAAGCAGCCGATGAACGCGGCGTCTGGGGCGGCCATGTCCCTGGCGGGAGCCGAGAAGAATGGTCTGGTGAAGATCAAGGTGGCGGAAGACGAGGCGGAGGCGGCAGCCGCGGCTAAGTTCACGGGCCTGTCCAAGGAGGAGCTGCTGAAGGTGGCAGGCAGCCCCGGCTGGGTACGCACCCGCTGGGCACTGCTGCTGCTCTTCTGGCTCGGCTGGCTCGGCATGCTTGCTGGTGCCGTGGTCATAATCGTGCGAGCGCCGCGTTGTCGCGAGCTACCGGCGCAGAAGTGGTGGCACACGGGCGCCCTCTACCGCATCGGCGACCTTCAGGCCTTCCAGGGCCACGGCGCGGGCAACCTGGCGGGTCTGAAGGGGCGTCTCGATTACCTGAGCTCTCTGAAGGTGAAGGGCCTTGTGCTGGGTCCAATTCACAAGAACCAGAAGGATGATGTCGCTCAGACTGACTTGCTGCAGATCGACCCCAATTTTGGCTCCAAGGAAGATTTTGACAGTCTCTTGCAATCGGCTAAAAAAAAGAGCATCCGTGTCATTCTGGACCTTACTCCCAACTACCGGGGTGAGAACTCGTGGTTCTCCACTCAGGTTGACACTGTGGCCACCAAGGTGAAGGATGCTCTGGAGTTTTGGCTGCAAGCTGGCGTGGATGGGTTCCAGGTTCGGGACATAGAGAATCTGAAGGATGCATCCTCATTCTTGGCTGAGTGGCAAAATATCACCAAGGGCTTCAGTGAAGACAGGCTCTTGATTGCGGGGACTAACTCCTCCGACCTTCAGCAGATCCTGAGCCTACTCGAATCCAACAAAGACTTGCTGTTGACTAGCTCATACCTGTCTGATTCTGGTTCTACTGGGGAGCATACAAAATCCCTAGTCACACAGTATTTGAATGCCACTGGCAATCGCTGGTGCAGCTGGAGTTTGTCTCAGGCAAGGCTCCTGACTTCCTTCTTGCCGGCTCAACTTCTCCGACTCTACCAGCTGATGCTCTTCACCCTGCCAGGGACCCCTGTTTTCAGCTACGGGGATGAGATTGGCCTGGATGCAGCTGCCCTTCCTGGACAGCCTATGGAGGCTCCAGTCATGCTGTGGGATGAGTCCAGCTTCCCTGACATCCCAGGGGCTGTAAGTGCCAACATGACTGTGAAGGGCCAGAGTGAAGACCCTGGCTCCCTCCTTTCCTTGTTCCGGCGGCTGAGTGACCAGCGGAGTAAGGAGCGCTCCCTACTGCATGGGGACTTCCACGCGTTCTCCGCTGGGCCTGGACTCTTCTCCTATATCCGCCACTGGGACCAGAATGAGCGTTTTCTGGTAGTGCTTAACTTTGGGGATGTGGGCCTCTCGGCTGGACTGCAGGCCTCCGACCTGCCTGCCAGCGCCAGCCTGCCAGCCAAGGCTGACCTCCTGCTCAGCACCCAGCCAGGCCGTGAGGAGGGCTCCCCTCTTGAGCTGGAACGCCTGAAACTGGAGCCTCACGAAGGGCTGCTGCTCCGCTTCCCCTACGCGGCCTGA(SEQ?ID?NO：2)

Any type of CD98 that term " CD98 " covers " total length " untreated CD98 and produced by the process in cell.This term also covers naturally occurring variant or the sudden change of CD98, such as, shears variant, allelic variant, SNP variant and isoform.CD98 polypeptide as herein described can be separated from multiple source, as originated from people's organization type or from another kind, or by restructuring or synthetic method preparation." native sequences CD98 polypeptide " comprises the polypeptide with the aminoacid sequence identical with deriving from natural corresponding CD98 polypeptide.These natural sequence C D98 polypeptide can be separated from natural, or can be produced by restructuring or synthesis mode.Term " native sequences CD98 polypeptide " covers the specific C D98 polypeptide of naturally occurring brachymemma or secreted form (such as particularly, extracellular domain sequence), the naturally occurring variant form of described polypeptide (such as, alternatively, shear-form) and naturally occurring allelic variant.

Term " antibody " uses with implication the most widely and covers particularly, such as, single anti-CD98 monoclonal antibody (comprising agonist, antagonist, neutralizing antibody, total length or intact monoclonal antibodies), there is the specific anti-CD 98 antibody composition of multi-epitope, polyclonal antibody, multivalent antibody, Multispecific antibodies (such as, bispecific antibody, as long as they demonstrate required biological activity), it is formed by least two complete antibodies, the fragment of strand anti-CD 98 antibody and anti-CD 98 antibody, as defined hereinafter.In this article, term " immunoglobulin (Ig) " (Ig) and antibody use interchangeably.Antibody can be the antibody of people's antibody, humanized antibody and/or affinity maturation.

" antigen " is that antibody can the predetermined antigen of selective binding.Target antigen can be the compound of polypeptide, carbohydrate, nucleic acid, lipid, haptens or other natural existence or synthesis.Preferably, described target antigen is polypeptide.

The antibody of the antigen that " combination " is concerned about is with the antibody of enough avidity conjugated antigens, thus described antibody is useful as the therapeutical agent of the cell or tissue of antigen described in targeted expression, and with other albumen without significant cross reaction.In these embodiments, antibody and " non-target " protein bound degree will be less than antibody and its particular target protein bound about 10%, as pass through fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) determined.For the combination of antibody and target molecules, the epi-position that term " specific binding " or " specific binding to " specific polypeptide or specific polypeptide target are put on or its " special " is referred to the combination that can be different from non-specific interaction with measuring.Can by (such as) determine with contrast molecule combination compared with the incompatible measurement specific binding of molecular juction, described contrast molecule normally has similar structures but does not have the molecule of binding activities.Such as, can by be similar to target, such as, specific binding is determined in the competition of the contrast molecule of excessive unlabelled target.In this case, if the combination of mark target and probe is suppressed competitively by excessive unlabelled target, then specific binding is shown to be.As used herein, the epi-position that term " specific binding " or " specific binding to " specific polypeptide or specific polypeptide target are put on or can be had at least about 10 target by (such as) its " special " ^-4m, alternatively, at least about 10 ^-5m, alternatively, at least about 10 ^-6m, alternatively, at least about 10 ^-7m, alternatively, at least about 10 ^-8m, alternatively, at least about 10 ^-9m, alternatively, at least about 10 ^-10m, alternatively, at least about 10 ^-11m, alternatively, at least about 10 ^-12the molecule of the Kd of M or larger shows.In one embodiment, term " specific binding " refers to that wherein molecular juction is bonded to the epi-position on specific polypeptide or specific polypeptide, but the combination be not substantially combined with any other polypeptide or polypeptide epitope.

Term " anti-CD 98 antibody " or " being bonded to the antibody of CD98 " refer to and described antibody can be made in conjunction with CD98 as antibody useful when the diagnosis of target CD98 and/or therapeutical agent using enough avidities.Preferably, anti-CD 98 antibody and the protein bound degree of incoherent non-CD98 be less than that described antibody is combined with CD98 about 10%, such as, as passed through measured by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA)." specific binding to " CD98 or be as defined above to the antibody of CD98 " special ".In some embodiments, dissociation constant (Kd) the <1 μ Μ of the antibody of CD98 is bonded to, <100nM, <10nM, <1nM or <0.1nM.In some embodiments, anti-CD 98 antibody is bonded to CD98 epi-position conservative in from the CD98 of different plant species.

" antibody of separation " is the antibody differentiated and be separated and/or obtain from its physical environment composition.The contaminant component of its physical environment includes, but is not limited to the material therapeutic of interference antibody used, and described material can comprise enzyme, hormone and other protein or nonproteinaceous solute.In a preferred embodiment, purifying (1) is reached the antibody being greater than 95% by weight by described antibody, as by the Lowry method (people such as Lowry, J.Bio.Chem.193:265-275, 1951) determined, and be most preferably greater than 99% by weight, (2) at least 15 residues or the degree of internal amino acid sequence that are enough to by using spin cup sequenator (spinning cup sequenator) to obtain N end is reached, or (3) reach use coomassie brilliant blue staining, or preferably silver dye is in the homogeneity of reducing or determined by SDS-PAGE under non reducing conditions.The antibody be separated is included in the antibody iM situ in reconstitution cell, because at least one component of the natural surroundings of described antibody will not exist.But usually, the antibody of separation will be prepared by least one purification step.

Basic 4-chain antibody unit is the assorted four glycan albumen comprising two identical light (L) chains weight (H) chain identical with two.About 150 are generally, 000 dalton with regard to IgG, 4-chain unit.Each L chain is connected with H chain by a covalent disulfide bonds, and according to the homotype of H chain, two H chains are connected to each other by one or more disulfide linkage.Disulphide bridges in the chain that every bar H chain and L chain also have a regular interval.Every bar H chain has a variable domains (V at N-end _h), be then three (for often kind of α and γ chains) and four (for μ and ε homotype) constant domain (C _h).Every bar L chain has a variable domains (V at N-end _l), then at its other end, there is a constant domain (C _l).V _lwith V _harranged together, and C _lwith the first constant domain (C of heavy chain _h1) arranged together.Specific amino-acid residue is considered to form interface between light chain and heavy-chain variable domains.Paired V _hand V _lform single antigen binding site together.For the structures and characteristics of different classes of antibody, see, such as, Basic and Clinical Immunology, 8th edition, Daniel P.Stites, Abba I.Terr and Tristram G.Parslow (chief editor), Appleton & Lange, Norwalk, CT, the 1994,71st page and the 6th chapter.

" variable region " or " variable domains " or " V territory " of antibody refers to the heavy chain of antibody or the amino terminal domain of light chain.The variable domains of heavy chain can be called " VH ".The variable domains of light chain can be called " VL ".Term " variable " refers in antibody, the fact that some part of variable domains is significantly different in sequence.V territory regulates antigen combine and define the specificity of specific antibodies to its specific antigen.But mutability is uneven distribution in 110 amino acid intervals of variable domains.On the contrary, V district is made up of the shorter region being called the extreme variation of " hypervariable region " of 15-30 the amino acid whose section relatively do not made a variation being called framework region (FR) and each 9-12 the amino acid long separated by framework region.The variable domains of native heavy and light chain comprises four FR respectively, and they take beta sheet conformation mostly, by forming loop connecting and three hypervariable regions forming a part for beta sheet structure in some cases connect.Hypervariable region in every bar chain is closely linked by FR, and together with the hypervariable region from another chain the antigen binding site of enhancing antibody formation (see people such as Kabat, sequences of Proteins of Immunological Interest, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991).Constant domain does not participate in the combination of antibody and antigen directly, but demonstrates multiple effector function, as the participation in antibody dependent cellular cytotoxicity (ADCC) and the middle antibody of CDC (CDC).

" complete " antibody comprises antigen binding site and C _lat least heavy chain constant domain C _h1, C _h2 and C _hthe antibody of 3.Described constant domain can be native sequences constant domain (such as, naive sequence constant domains) or its amino acid sequence variation.Preferably, described complete antibody has one or more effector functions.

" antibody fragment " comprises a part for complete antibody, preferably, comprises antigen binding domain or the variable region of complete antibody.The example of antibody fragment unrestrictedly comprises Fab, Fab', F (ab') 2 and Fv fragment; Double antibody and two-double antibody (see, such as, the people such as Holliger, P., (1993) Proc.Natl.Acad.Sci.90:6444-8; The people such as Lu, D., (2005) J.Biol.Chem.280:19665-72; The people such as Hudson, Nat.Med.9:129-134 (2003); WO 93/11161; With U.S. Patent No. 5837242 and 6492123); Single-chain antibody molecules (see, such as, U.S. Patent No. 4946778; 5260203; 5482858 and 5476786); Two varied texture domain antibodies (see, such as, U.S. Patent No. 7612181); Single varied texture domain antibodies (SdAb) (see, such as, the people such as Woolven, Immunogenetics 50:98-101,1999; The people such as Streltsov, Proc Natl Acad Sci USA.101:12444-12449,2004); With the multi-specificity antibody formed by antibody fragment.

" functional fragment " of therapeutic antibodies will demonstrate the biological function of at least one owing to complete antibody, if not some or all biological functions, described function comprises at least to the specific binding of target antigen.

As used herein, term " monoclonal antibody " refers to the antibody deriving from substantially homogeneous antibody population, and namely except the possible naturally occurring sudden change that can exist on a small quantity, each antibody forming described colony is identical.Modifier " mono-clonal " should not regarded as and need to produce antibody by any ad hoc approach.Such as, can pass through first by people such as Kohier, Nature, the monoclonal antibody that the hybridoma preparation that 256:495 (1975) describes is useful in the present invention, or can bacterium, eucaryon animal or plant cell (see, such as, U.S. Patent No. 4816567) in use recombinant DNA method to carry out.Such as, the people such as Clackson are used, Nature, the people such as 352:624-628 (1991) and Marks, J.Mol.Biol., the technology described in 222:581-597 (1991), can also be separated " monoclonal antibody " from phage antibody library.

In this article, monoclonal antibody comprises wherein Partial heavy and/or light chain and to derive from the corresponding sequence of Special Thing species or genus in the antibody of specific antibodies kind or subclass equivalent or identical, and the rest part of described chain with derive from another species or the corresponding sequence belonged in the antibody of another kind of antibody type or subclass is equal to or identical " being fitted together to " antibody, and the fragment of these antibody, as long as they demonstrate required biological activity (see U.S. Patent No. 4816567; With people such as Morrison, Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984)).

" humanization " form of inhuman (such as, rodents) antibody refers to the chimeric antibody comprising the minimum sequence deriving from non-human antibody.Largely, humanized antibody refers to that the some hypervariable region residues of the acceptor in human normal immunoglobulin (receptor antibody) is had non-human species's (donor antibody) of desired antibodies specific, avidity and ability, as the human normal immunoglobulin of the some hypervariable region residues displacement of mouse, rat, rabbit or non-human primate.In some cases, framework region (FR) residue of human normal immunoglobulin is replaced with corresponding non-human residues.In addition, humanized antibody can be included in non-existent residue in receptor antibody or donor antibody.Carrying out these modifications is to improve antibody performance further.Usually, humanized antibody will comprise substantially whole at least one, and usual two variable domains, wherein whole or substantially whole Gao Bianhuan corresponds to those of non-human immunoglobulin, and whole or substantially whole FR is people's immunoglobulin sequences those.Humanized antibody optionally also will comprise constant region for immunoglobulin (Fc) at least partially, be generally the constant region of human normal immunoglobulin.Other details relevant, see people such as Jones, Nature 321:522-525 (1986); The people such as Riechmann, Nature 332:323-329 (1988); And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).Also see the following summary quoted in this article and patent and reference: 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. 5585089; 5693762; 6180370; With 6054297.

" people's antibody " refers to the antibody with such aminoacid sequence, and described aminoacid sequence corresponds to the aminoacid sequence of such antibody, and described antibody is produced by people and/or used as disclosed herein for the preparation of any technology preparation of people's antibody.This definition clear-cut of people's antibody gets rid of the humanized antibody comprising inhuman antigen binding residues.Multiple technologies as known in the art can be used to produce people's antibody, and it comprises phage display library (Hoogenboom and Winter, J.Mol.Biol, 227:381 (1991); The people such as Marks, J.Mol.Biol, 222:581 (1991)) and yeast display library (people such as Chao, Nature Protocols1:755-768 (2006)).The method described in the following documents also can be used for preparing human monoclonal antibodies: the people such as Cole, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, the 77th page (1985); The people such as Boerner, J.Immunol, 147 (l): 86-95 (1991).Also see van Dijk and van de Winkel, Curr.Opin.Pharmacol, 5:368-74 (2001).People's antibody can be prepared by antigen being applied to transgenic animal, described transgenic animal have been modified at when reacting to antigen stimulation can produce these antibody, but its endogenous loci loses function, such as, mouse (see, such as, Jakobovits, A., Curr.Opin, Biotechnol.1995,6 (5): 561-6; Briiggemann and Taussing, Curr.Opin.Biotechnol.1997,8 (4): 455-8; With relevant XENOMOUSE ^tMthe U.S. Patent No. 6075181 and 6150584 of technology).About the people's antibody produced by human B-lymphocyte hybridoma technology, see, such as, the people such as Li, Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006).

As use alpha nerein, term " hypervariable region ", " HVR " or " HV " refer to alterable height in sequence and/or form the region, antibody variable territory of the ring that structure defines.Usually, antibody comprises six hypervariable regions: three in VH (H1, H2, H3), three in VL (L1, L2, L3).Employ some hypervariable region schematic diagram and they are encompassed in herein.Kabat complementary determining region (CDR) is based on sequence variability, and be the most frequently used (people such as Kabat, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).On the contrary, Chothia refers to the position (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)) of structure ring.According to the length of ring, the end of Chothia CDR-H1 ring changes (this is because Kabat numbering plan is conceived to the insertion at H35A and H35B place between H32 and H34 when using Kabat coding rule numbering; If 35A and 35B does not all exist, then ring terminates at 32 places; If only 35A exists, then ring terminates at 33 places; If 35A and 35B all exists, then ring terminates at 34 places).AbM hypervariable region represents the compromise between Kabat CDR and Chothia structure ring, and is used by Oxford molecule AbM antibody model software (Oxford Molecular's AbM antibody modeling software)." contact " hypervariable region is based on the analysis of available complex crystal structure.Hereafter to have recorded in these hypervariable regions the residue of each.

Hypervariable region can comprise " hypervariable region of extension " as follows: 26-35 or 26-35A (H1) in 24-36 or 24-34 (L1) in VL, 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) and VH, 50-65 or 49-65 (H2) and 93-102,94-102 or 95-102 (H3).For each in these definition, variable domains residue is numbered according to the people such as Kabat (as above).As used herein, term " HVR " and " CDR " are used interchangeably.

" framework " or " FR " residue refers to those variable domains residues except some hypervariable region residues as defined herein.

Term " the variable domains residue according to Kabat is numbered " or " amino acid position number according to Kabat " and version thereof refer to the people such as Kabat, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, for the heavy-chain variable domains of antibody establishment or the numbering system of light variable domains in Bethesda, MD (1991).Use this numbering system, real linear amino acid sequence can contain the shortening of FR or CDR or the less of insertion or other amino acid that correspond to variable domains.Such as, heavy-chain variable domains can comprise single amino acids and insert (according to Kabat after the residue 52 of H2, residue 52a) and after heavy chain FR residue 82, comprise the residue (such as, according to Kabat, residue 82a, 82b and 82c etc.) of insertion.Can by comparing the Kabat residue numbering determining given antibody at antibody sequence homology region by " standard " Kabat numbered sequence.

When mentioning the residue in variable domains (being roughly the residue 1-107 of light chain and the residue 1-113 of heavy chain), usual use Kabat numbering system (such as, the people such as Kabat, Sequences of Immunological Interest. the 5th edition .Public Health Service, National Institutes of Health, Bethesda, Md. (1991))." EU numbering system " or " EU index " general use when mentioning the residue in immunoglobulin heavy chain constant region (such as, the EU index reported in the people such as Kabat, as above)." the EU index as in Kabat " refers to the residue numbering of human IgG1 EU antibody.Unless otherwise indicated herein, otherwise the residue numbering mentioning in antibody variable territory refer to and to number according to the residue of Kabat numbering system.Unless otherwise indicated herein, otherwise the residue numbering mentioning in antibody constant structural domain refer to and to number according to the residue of EU numbering system.

" avidity maturation " antibody refers to such antibody, in one or more HVR of this antibody, have one or more change, and this causes this antibody to increase compared with the parental antibody not having these to change to the avidity of antigen.The antibody of preferred avidity maturation will have the avidity of nmole or even picomole to target antigen.The ripe antibody of avidity is produced by program as known in the art.Relevant summary, see Hudson and Souriau, Nature Medicine9:129-134 (2003); Hoogenboom, Nature Biotechnol.23:1105-1116 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia4:39-51 (2010).

" blocking-up " antibody or " antagonist " antibody are the bioactive antibody suppressing or reduce the antigen that it combines.Preferred blocking antibody or antagonist antibodies suppress the biological activity of antigen substantially or completely.

As used herein, " agonist antibody " is the antibody of at least one functional activity of simulating the polypeptide be concerned about.

" binding affinity " generally refers to the intensity of the summation of noncovalent interaction between the single binding site of molecule (such as, antibody) and its binding partners (such as, antigen).Unless otherwise indicated, otherwise as used herein, and " binding affinity " refers to that reflection combines the interactional inherent binding affinity of 1:1 between member's (such as, antibody and antigen).Molecule X generally can pass through dissociation constant (KD) to the avidity of its companion Y and represent.Avidity can be measured by ordinary method as known in the art, comprise those methods as herein described.Low affinity antibodies usually is often easy to dissociate with antigen slow fixation, and high affinity antibody is combined more quickly with antigen usually and often keep combining for more time.The multiple method measuring binding affinity is well known in the art, and for purposes of the present invention, can use these methods any.The following describe illustrated embodiment specifically.

When in this article for representing binding affinity, " or better " refers to combination stronger between molecule (such as, antibody) and binding partners thereof, and it is by less Kd numeric representation.Such as, be " .6nM or better " antibody to the avidity of antigen, described antibody to the avidity <.6nM of antigen, i.e. .59nM .58nM .57nM etc., or is less than any value of .6nM.

In one embodiment, that radiolabeled antigen by using the Fab form of the antibody be concerned about and antigen thereof to carry out combines and measures (RIA) and measure according to " Kd " of the present invention or " Kd value ", as by measuring Fab solution to described by the following mensuration of the binding affinity of antigen, described mensuration be when there is the titration series of non-labeled antigen minimum concentration ( ¹²⁵i)-labelled antigen balance Fab, then with people such as (, (1999) J.Mol Biol 293:865-881) Chen that the plate that anti-Fab antibody applies catches that the antigen of combination carries out.According to another kind of embodiment, Kd or Kd value is by using (such as) BIAcore-2000 or TM BIAcore-3000 (BIAcore, Inc., Piscataway, NJ), the TM measured by measuring with surface plasma resonance.

According to the present invention, (such as) BIAcore-2000 can also be used ^tMor BIAcore-3000 ^tM(BIAcore Inc., Piscataway, NJ), uses surface plasma resonance technology identical as above to determine " on speed " or " association rate " or " kon ".

As used herein, phrase " substantially similar " or " substantially identical " represent two numerical value (general one relevant with antibody of the present invention, and another with reference antibody about) between the sufficiently high similarity of difference, thus the difference thought in the background by the biological property measured by described value between described two numerical value is not almost had biology and/or statistical significance by those skilled in the art.According to the value of reference antibody, the difference between described two values is preferably less than about 50%, is preferably less than about 40%, is preferably less than about 30%, be preferably less than about 20%, be preferably less than about 10%.

As used herein, phrase " basic reduce " or " substantially different " represent two numerical value (general one relevant with antibody of the present invention, and another with reference antibody about) between the sufficiently high similarity of difference, thus the difference thought in the background by the biological property (such as, Kd value, HAMA reaction) measured by described value between described two values is had statistical significance by those skilled in the art.According to the value of reference antibody, the difference between described two values is preferably more than about 10%, is preferably more than about 20%, is preferably more than about 30%, be preferably more than about 40%, be preferably more than about 50%.

The antibody " suppressing the growth of tumour cell of expressing CD98 polypeptide " or " growth inhibiting " antibody cause expressing or the cancer cells of CD98 polypeptide that process LAN is suitable produces measurable growth inhibiting antibody.CD98 polypeptide can be the transmembrane polypeptide of expressing on cancer cell surfaces, or can be the polypeptide being produced by cancer cells and secrete.Compared with suitable contrast, preferred growth inhibiting anti-CD 98 antibody will express the growth-inhibiting of the tumour cell of CD98 more than 20%, preferably inhibit about 20% to about 50%, and even more preferably, inhibit and be greater than 50% (such as, about 50% to about 100%), described contrast is not normally with the tumour cell of antibody treatment to be tested.In one embodiment, in cell culture, can measure growth-inhibiting with the antibody concentration of about 0.1 to 30g/ml or about 0.5nM to 200nM, wherein said growth-inhibiting is that 1-10 days after tumour cell is exposed to described antibody are determined.Can be suppressed by the tumor growth of various ways determination tumour cell as described below.If with about 1 μ g/kg to about 100mg/kg body weight use anti-CD 98 antibody can from the using first of described antibody about 5 days to 3 months, preferably cause tumor size or tumor cell proliferation to reduce in about 5 days to 30 days, then described antibody is that tumor growth suppresses.

The antibody of " cell death inducing " is the antibody of inducement of apoptosis, as formed determined by annexin V combination, DNA break, cell shrinkage, endoplasmic reticulum expansion, cytoclasis and/or membrane vesicle (being called apoptosis body).The cell of cell normally process LAN CD98 polypeptide.Preferably, described cell is tumour cell.Multiple method is available to evaluating with apoptosis-related cell event.Such as, measurement phosphatidylserine (PS) transposition can be combined by annexin; DNA break can be assessed by DNA ladder degree method (laddering); And core/Chromatin condensation and DNA break can be evaluated by any increase of hypodiploid cells.Preferably, the antibody of cell death inducing be annexin combine measure in cause about 2 to 50 times, the antibody of the annexin zygotic induction of preferably about 5 to 50 times, most preferably about 10 to 50 times relative to untreated cell.

The antibody of " inducing cell death " is the antibody causing viable cell death.Described cell is the cell type of specifically expressing or process LAN CD98 polypeptide.Described cell can be normal cell that is carcinous or particular cell types.CD98 polypeptide can be the transmembrane polypeptide of expressing on cancer cell surfaces, or can be the polypeptide being produced by cancer cells and secrete.The dead necrocytosis of inducing with the cytotoxicity (ADCC) or CDC (CDC) of distinguishing antibody dependent cellular mediation of cells in vitro can be determined when there is not complement and immune effector cell.Therefore, heat-inactivated sera (that is, when not having complement) can be used and carry out necrocytosis mensuration when there is no immune effector cell.In order to determine that antibody whether can inducing cell death, as passed through propidium iodide (PI), trypan blue (see people such as Moore, Cytotechnology 17:1-11 (1995)) or the absorption of 7AAD evaluate, can relative to the loss of untreated cell evaluated for film integrity.The antibody of preferred necrocytosis induction is those of inducing PI to absorb in the PI absorption measurement in BT474 cell.

Antibody " effector function " refers to that those are attributable to the biologic activity in antibody Fc district (native sequences Fc district or amino acid sequence variation Fc district), and it changes with antibody morphism.The example of antibody mediated effect subfunction comprises: C1q combines and CDC; Fc receptors bind; The cytotoxicity (ADCC) of antibody dependent cellular mediation; Phagolysis; The downward of cell surface receptor (such as, B-cell receptor); Activate with B cell.

In this article, term " Fc district " is for defining the C-terminal region of heavy chain immunoglobulin, and it comprises native sequences Fc district and variant Fc district.Although the border in the Fc district of heavy chain immunoglobulin can change, human IgG heavy chain Fc district usually defines from the amino-acid residue of position Cys226 or extends to its C-terminal from pro230.Such as, during the production or purifying of antibody or by the nucleic acid of recombined engineering design encoding said antibody heavy chain, the C-terminal Methionin (residue 447 according to EU numbering system) in Fc district can be removed.Therefore, the composition of complete antibody can comprise the removing of whole K447 residue antibody population, without the removing of K447 residue antibody population and there is K447 residue remove and the antibody population of not removed mixtures of antibodies.

" functional Fc district " has " effector function " in native sequences Fc district.Exemplary " effector function " comprises C1q and combines; CDC; Fc receptors bind; ADCC; Phagolysis; Cell surface receptor (such as, B-cell receptor; BCR) lower.These effector functions usually need Fc district to be combined (such as, antibody variable territory) with binding domain and can use such as (e.g.) many measure evaluation disclosed in definition in this article.

" native sequences Fc district " comprises the aminoacid sequence identical with the aminoacid sequence in the Fc district that occurring in nature exists.Native sequences people Fc district comprises native sequences human IgG1 Fc district (non-A and A allotype); Native sequences human IgG2 Fc district; Native sequences human IgG 3Fc district; With native sequences human IgG 4Fc district and naturally occurring variant thereof.

" variant Fc district " comprises because at least one is amino acid modified, preferably one or more aminoacid replacement and be different from the aminoacid sequence in native sequences Fc district.Preferably, with native sequences Fc district or compared with the Fc district of parental polypeptide, variant Fc district has at least one aminoacid replacement, such as, about 1 to about 10 aminoacid replacement in native sequences Fc district or the Fc district at parental polypeptide, and preferably about 1 to about 5 aminoacid replacement.In this article, variant Fc district preferably will have the homology at least about 80% with native sequences Fc district and/or with the Fc district of parental polypeptide, and most preferably has the homology at least about 90% with it, more preferably has the homology at least about 95% with it.

" cytotoxicity of antibody dependent cellular mediation " or " ADCC " refer to a kind of cytotoxic form, the Ig wherein secreted is bonded at some cytotoxic cell (such as, natural killer (NK) cell, neutrophil leucocyte and scavenger cell) the upper Fc acceptor (FcR) existed, thus these cytotoxic effect cells are combined with the target cell specificity with antigen and kill target cell by cytotoxin subsequently.Described antibody " arms " cytotoxic cell, and be definitely this kind of kill and wound necessary.The main cell of mediation ADCC only expresses Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.The FcR that table 3 in Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991) on the 464th page summarizes on hematopoietic cell expresses.In order to the ADCC evaluating be concerned about molecule is active, external ADCC mensuration can be carried out, as described in U.S. Patent No. 5500362 or 5821337.Useful effector cell is measured for these and comprises peripheral blood monouclear cell (PBMC) and natural killer (NK) cell.Alternatively or in addition, the ADCC that can evaluate be concerned about molecule is in vivo active, such as, in animal model, as disclosed in the people such as Clynes (USA) 95:652-656 (1998).

" Fc acceptor " or " FcR " describe the acceptor be combined with antibody Fc district.Preferred FcR is native sequences people FcR.In addition, preferred FcR is the FcR (γ acceptor) be combined with IgG antibody, and it comprises the acceptor of Fc γ RI, Fc γ RII and Fc γ RIII subclass, comprises the allelic variant and alternatively of these acceptors, the shear-form of these acceptors.Fc γ RII acceptor comprises Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" suppression acceptor "), they have similar aminoacid sequence, difference is mainly its cytoplasmic domain (see summary M.in Daeron, Annu.Rev.Immunol.15:203-234 (1997)).The summary of FcR is see Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991); The people such as Capel, Immunomethods4:25-34 (1994); With people such as de Haas, J.Lab.Clin.Med.126:330-41 (1995).Other FcR contained in this article in term " FcR ", comprises the FcR that will differentiate future.This term also comprises neonatal receptor (FcRn), it is responsible for the IgG of parent being transferred to the fetus (people such as Guyer, the people such as J.Immunol.117:587 (1976) and Kim, J.Immunol.24:249 (1994)).The antibody variants that the combination of FcR improves or weakens is described in, such as, WO2000/42072 and U.S. Patent No. 7183387; 7332581; With 7335742.Also see, such as, the people such as Shields, J.Biol.Chem.9 (2): 6591-6604 (2001).

" CDC " or " CDC " refers to the molten born of the same parents of the target cell when there is complement.The activation of CCP is that (the suitable subclass) antibody be combined with its isoantigen by the combination of complement system first component (C1q) is initial.In order to evaluate complement activation, can CDC mensuration be carried out, such as, the people such as Gazzano-Santoro, described in J.Immunol.Methods 202:163 (1996).There is the polypeptide variants (having the polypeptide in variant Fc district) of the Fc region amino acid sequence of change and the raising of C1q binding ability or reduction be described in, such as, U.S. Patent No. 6194551 Bl and WO1999/51642, also see, such as, the people such as Idusogie, J.Immunol.164:4178-4184 (2000).

" extracellular domain " or " ECD " of CD98 polypeptide refers to the form of the CD98 polypeptide substantially not containing cross-film and cytoplasmic domain.Usually, CD98 polypeptide ECD will have these cross-films and/or cytoplasmic domain of being less than 1%, and preferably, will have these territories being less than 0.5%.The membrane-spanning domain of CD98 comprises amino-acid residue 76-103 (people such as Parmacek, Nucleic Acids Res.17:1915-1931,1989).Membrane-spanning domain really trimming circle can change, but differentiates as initial, and the either end in described territory probably changes and is no more than about 5 amino acid.Therefore, optionally, the extracellular domain of CD98 polypeptide can comprise the amino acid of CD98 sequence from about 98-108 to 529, as people such as Parmacek, with upper disclosed.

" amino acid sequence identity per-cent (%) " relative to reference polypeptide sequence to be defined as at sequence alignment and (if desired) is introduced behind space to reach largest percentage sequence iden, but after not thinking that any conservative replacement is the part of sequence iden, the per-cent of the amino-acid residue in the candidate sequence identical with amino-acid residue in reference polypeptide sequence.The various ways in art technology can be used realize the comparison for determining aminoacid sequence percentage identities, such as, use openly available computer software, as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the suitable parameter of sequence alignment, and it comprises and realizes high specific to required any algorithm to the sequence that will compare.

As used herein, " modification " of amino-acid residue/position refers to compared with original amino acid sequence, the change of primary amino acid sequence, and wherein said change is produced by the sequence variation relating to described amino-acid residue/position.Such as, typical modification comprise with another amino-acid substitution residue (such as, guard or non-conservative displacement), be adjacent to one or more (being usually less than 5 or 3) amino acid whose insertion of described residue/position, and the disappearance of described residue/position.

" epi-position " is the site on the antigen molecule surface of monospecific antibody molecule combination.Usually, antigen there is several or multiple different epi-position and from multiple different antibody response.Term comprises linear epitope and conformational epitope particularly.

When two antibody recognition are identical or the epi-position of space overlap time, antibodies and reference antibody " substantially identical epi-position ".Determine whether two epi-positions are bonded to widely using most of the epi-position of identical or space overlap and method is competitive assay fast, its can applying marking antigen or traget antibody with multiple multi-form configuration.Usually, antigen is fixed on 96 orifice plates, and uses radioactivity or enzyme labelling to measure the ability of unmarked antibody prevention traget antibody combination.

" epitope mapping " differentiates the binding site of antibody to their target antigen or the method for epi-position.Antibody epitope can be linear epitope or conformational epitope.Linear epitope is formed by the continuous sequence of Amino Acids in Proteins.Conformational epitope is amino acids formed by what be interrupted in protein sequence, but merges together when protein folding becomes during its three-dimensional arrangement.

As herein defined, " epi-position frame is (epitope binning) also " is the method that the epi-position antagonist identified based on them divides into groups.More specifically, epi-position frame also comprises the method and system of epi-position recognition property distinguishing different antibodies, and combine based on they epi-position recognition property antagonist cluster and differentiate to have the computation process of the antibody of different binding specificity.

" illness " to benefit from any patient's condition or the disease of the treatment using substances/molecules of the present invention or method.This comprises chronic and acute disease, and it comprises makes Mammals to discussed illness those pathological conditions susceptible.In this article, the limiting examples of illness to be treated comprises cancerous condition, as bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, and these cancer metastasis cancers.

Term " cell proliferative disorders " refers to the illness relevant with abnormal cell proliferation to a certain degree with " proliferative disorders ".In one embodiment, described cell proliferative disorders is cancer.

As used herein, " tumour " refers to growth and the propagation of all neonatal cells, no matter is pernicious or optimum, and refers to all precancerous cells and cancer cells and tissue.As mentioned in this article, term " cancer ", " carcinous ", " cell proliferative disorders ", " proliferative disorders " and " tumour " are not mutually exclusive.

Term " cancer " and " carcinous " represent or the physiological conditions that Mammals is feature with unrestricted Growth of Cells are described.The example of cancer includes, but is not limited to cancer, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancy.These cancers more specifically example comprise squamous cell carcinoma (such as, epithelial cell squamous cell carcinoma), lung cancer, comprise small cell lung cancer, nonsmall-cell lung cancer, adenocarcinoma of lung and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, cancer of the stomach, comprise gastrointestinal cancer, pancreas cancer, glioblastoma, cervical cancer, ovarian cancer, oral carcinoma, liver cancer, bladder cancer, urethral carcinoma, liver cancer, mammary cancer, colorectal carcinoma, the rectum cancer, colorectal carcinoma, uterine endometrium or uterus carcinoma, salivary-gland carcinoma, kidney, prostate cancer, carcinoma vulvae, thyroid carcinoma, liver cancer, anus cancer, penile cancer, melanoma, multiple myeloma and B cell lymphoma, the cancer of the brain and head and neck cancer and associated transitions cancer.

" expressing the cell of CD98 " is endogenous at cell surface expression or the cell of the CD98 of transfection." cancer of expression CD98 " is the cancer of the cell with the CD98 albumen existed on cell surface." cancer of expression CD98 " produces the CD98 of enough levels on its cell surface, thus makes anti-CD 98 antibody to combine with it and to have result for the treatment of to cancer.The cancer of " process LAN " CD98 is compared with the non-cancerous cell of homologue's type, and its cell surface has the cancer of remarkable higher levels of CD98.These process LAN can transcribe or translate and cause by gene amplification or by what improve.(such as, Immunohistochemistry can be passed through by the raising evaluating the CD98 protein level that cell surface exists; Facs analysis), in diagnosis or prognosis measure, determine the process LAN of CD98.Alternatively or in addition, the level of CD98 coding nucleic acid or mRNA in cell can be measured, such as, pass through fluorescence in situ hybridization; (FISH; See WO 98/45479 disclosed in October, 1998), southern blotting technique, RNA trace or polymerase chain reaction (PCR) technology, as real-time quantitative PCR (RT-PCR).Except said determination, for skilled doctor, multiple in vivoassay is available.Such as, cell in patient body can be exposed to optionally uses detectable (such as, radio isotope) antibody that marks, and can by (such as) to radioactive external scan or the combination coming antibody and cell evaluate patient by analyzing the examination of living tissue gathered from the patient being previously exposed to described antibody.The cancer expressing CD98 includes, but is not limited to bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

As used herein, " treatment " (and grammatical variants) refers in the clinical intervention attempting changing in the natural course of individuality to be treated or cell, and can in order to prevent or carry out in clinical pathology process.Desired result for the treatment of comprises and prevents disease from occurring or recurrence, relaxes symptom, any direct or indirect pathological consequences eliminated a disease, with regard to cancer prevention transfer, reduces progression of disease speed, improves or palliate a disease state and remission or improve prognosis.In some embodiments, antibody of the present invention is for postponing the generation of disease or delaying the progress of disease.

By doctor the conventional procedure be familiar with, can easily measure for the above-mentioned parameter evaluating successful treatment and amelioration of disease.For cancer therapy, can by (such as) evaluation disease developing time (TTP) and/or by determining that response rate (RR) measures effect.Other terminals for measuring effect comprise, such as, and survival rate (RFS) that overall survival (OS), DFS (DFS) and nothing are fallen ill again (or without recurrence).Metastasis of cancer can be determined by testing by stages, and determine Bone tumour by the test of bone scanning and calcium level and other enzymes.CT scan can also be carried out shift and nodus lymphoideus transferring rate with the pelvis searched in this region.Chest X-ray and be respectively used to the transfer of searching lung and liver by the measurement of the liver enzyme levels of currently known methods.Other ordinary methods for monitoring of diseases comprise the scanning of TRUS ripple (TRUS) and per rectum needle biopsies art (TRNB).

" individuality " is vertebrates.In some embodiments, described vertebrates is Mammals.Mammals includes, but is not limited to farming animals (e.g., ox), sport animals, pet (as cat, dog and horse), primates, Mouse and rat.In some embodiments, described Mammals is people.

" significant quantity " refers to and effectively to measure for the required treatment of realization or prevention result with required dosage and time period." the treatment significant quantity " of substances/molecules of the present invention can change according to following factor, and as the disease condition of individuality, age, sex and body weight, and described substances/molecules causes the ability of required reaction in described individuality.Treatment significant quantity comprises wherein compared with treatment beneficial effect, and any toxicity of described substances/molecules or detrimental action are inappreciable amounts." prevention significant quantity " refers to and effectively to measure for realization required prevention result with required dosage and time period.Usually, but unrequired, due to before disease or disease in experimenter, use preventive dose in early days, therefore prevent significant quantity will be less than treatment significant quantity.With regard to cancer, the treatment significant quantity of medicine (such as) can reduce cancer cell number; Reduce tumor size; Suppress (that is, slow to a certain degree and preferably stop) cancer cell infiltration in peripheral organs; Suppress (that is, slow to a certain degree and preferably stop) metastases; In to a certain degree Tumor suppression growth; And/or alleviate one or more symptoms relevant to cancer to a certain extent.See the definition of above " treatment ".Can prevent from growing and/or kill the degree of existing cancer cells to medicine, it can be T suppression cell and/or Cytotoxic.

" for a long time " is used and is referred to that the continuous mode with contrary with acute form uses reagent, thus initial therapy effect (activity) is maintained for some time extended." intermittently " using is the treatment not having interruptedly to carry out continuously, but it is circulation in itself.

Use with one or more other treatment agent " combination " and comprise with any order (walking abreast) and continuous administration simultaneously.

As used herein " carrier " comprises the cell exposed with used dosage and concentration or nontoxic pharmaceutically acceptable carrier, vehicle or the stablizer of Mammals.Usually, physiology can carrier be pH aqueous buffer solution.Physiology can the example of carrier comprise damping fluid, as phosphoric acid salt, Citrate trianion and other organic acids; Antioxidant, comprises xitix; Lower molecular weight (being less than about 10 residues) polypeptide; Protein, as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, as polyvinylpyrrolidone; Amino acid, as glycine, glutamine, l-asparagine, arginine or Methionin; Monose, disaccharides and other carbohydrate, comprise glucose, seminose or dextrin; Sequestrant, as EDTA; Sugar alcohol, as mannitol or Sorbitol Powder; Salt-forming counterion, as sodium; And/or nonionic surface active agent, as TWEEN ^tM, polyoxyethylene glycol (PEG) and PLURONICS ^tM.

Term " pharmaceutical preparation " refers to be in and makes the effective form of the biological activity of activeconstituents and the preparation not comprising other components experimenter be applied to by described preparation to unacceptable toxicity.These preparations can be aseptic.

" aseptic " preparation is aseptic, and it is not containing microorganism and their spore of all work.As disclosed herein, " significant quantity " of antibody is the amount of the object be enough specifically described.Relative to illustrated object, can empirically and determine in a usual manner " significant quantity ".

Term " treatment significant quantity " refers to the amount to the disease in " treatment " experimenter or Mammals or the effective antibody of illness or other drug.With regard to cancer, the treatment significant quantity of medicine can reduce cancer cell number; Reduce tumor size; Suppress (that is, slow to a certain degree and preferably stop) cancer cell infiltration in peripheral organs; Suppress (that is, slow to a certain degree and preferably stop) metastases; In to a certain degree Tumor suppression growth; And/or alleviate one or more symptoms relevant to cancer to a certain extent.The definition of " treatment " see herein.Can prevent from growing and/or kill the degree of existing cancer cells to medicine, it can be T suppression cell and/or Cytotoxic." prevention significant quantity " refers to and effectively to measure for realization required prevention result with required dosage and time period.Usually, but unrequired, due to before disease or disease in experimenter, use preventive dose in early days, therefore prevent significant quantity will be less than treatment significant quantity.

As used herein term " cytotoxic agent " refers to and suppresses or place cell function and/or cause the material of cytoclasis.Term is intended to comprise radio isotope (such as, At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³²with the radio isotope of Lu), chemotherapeutics, such as methotrexate, Zorubicin, vinca alkaloids (vincristine(VCR), vinealeucoblastine(VLB), Etoposide), Dx, melphalan, ametycin, Chlorambucil, daunorubicin or other intercalating agent, enzyme and fragment thereof, as nucleic acid ferment enzyme, microbiotic and toxin, as the enzyme activity toxin of small molecule toxins or bacterium, fungi, plant or animal-origin, comprise its fragment and/or variant, and following disclosed multiple antitumor or carcinostatic agent.The following describe other cytotoxic reagents.Kill the destruction that tumor reagent causes tumour cell.

" toxin " is any material can with disadvantageous effect.

" chemotherapeutics " does not consider the mechanism of action, compound useful in cancer therapy.The compound that chemotherapeutics is included in " targeted therapies " and use in conventional chemotherapy.The example of chemotherapeutics includes, but is not limited to alkylating reagent, as Tespamin and CYTOXAN endoxan, alkyl sulfonic ester, as busulfan, improsulfan and piposulfan, ethylene imine class, as benzodepa, carboquone, meturedepa and urethimine, ethyleneimine class and methylmelamine class, comprise altretamine, Tretamine, triethylenephosphoramide, triethylene thiophosphoric acid amine and trimethylolmelamine, acetogenin class (particularly, bullatacin and bullatacin ketone), Δ-9-tetrahydrocannabinol (dronabinol, AR1NOL), β-lapachol, tecomin, colchicine, betulinic acid, camptothecine (comprising the analogue Hycamtin (HYCAMTIN) of synthesis, CPT-11 (irinotecan, CAMPTOSAR), acetyl camptothecine, scopolectintin and 9-aminocamptothecin), bryostatin, callystatin, CC-1065 (comprising its U 73975, U 80244 and U 77779 synthetic analogues), podophyllotoxin, Podophyllinic acid, teniposide, hidden algae element class (particularly, hidden algae element 1 and hidden algae element 8), dolastatin, times ganmycin (comprising synthetic analogues, KW-2189 and CB1-TM1), Ai Liusu, pancratistatin, to crawl a coral alcohol, Spongistatin, mustargen, as Chlorambucil, Chlornaphazine, courage phosphamide, estramustine, ifosfamide, mustargen, Nitromin hydrochloride, melphalan, Novoembichin, phenesterin, prednimustine, trofosfamide, uracil mustard, nitrosoureas, as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranomustine, microbiotic, as Enediyne Antibiotic (such as, calicheamicin, particularly, calicheamicin γ 1I and calicheamicin ω Il (see, such as, Angew, Chem.Intl.Ed.Engl.33:183-186 (1994)), enediyne anthracyclines microbiotic (dynemicin), comprises enediyne anthracyclines microbiotic A, Ai Sibo mycin, and neocarzinostatin chromophore and related colour fibroin Enediyne Antibiotic chromophore), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, sanarnycin, carabicin, carminomycin, cardinophyllin, Toyomycin, dactinomycin, daunorubicin, detorubicin, 6-diazonium-5-oxn-l-norieucin, ADRIAMYCIN, Dx (comprises morpholino-Dx, Cyanomorpholino-Dx, 2-pyrroline-Dx and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, as ametycin, Mycophenolic Acid, nogalamycin, Olivomycine, peplomycin, porphyromycin, tetracycline, triferricdoxorubicin, rodorubicin, Streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolic product, as methotrexate and 5 FU 5 fluorouracil (5-FU), folacin, as N10,9-dimethylfolic acid, Rheumatrex, talk endlessly sieve purine, trimetrexate, purine analogue, as fludarabine, 6-MP, ITG, Tioguanine, pyrimidine analogue, as Ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, two deoxyuridine, doxifluridine, enocitabine, floxuridine, androgens, as U-22550, dromostanolone propionate, Epitiostanol, mepitiostane, testolactone, antiadrenergic drug, as aminoglutethimide, mitotane, Win-24540, folic acid compensator, as folinic acid, aceglatone, aldol phosphamide glucosides, aminol evulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, Omaine, diaziquone, elfornithine, elliptinium acetate, esperamicin, Etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoid class, as maytenin and ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, promethazine, pirarubicin, losoxantrone, 2-ethyl hydrazides, Procarbazine, PSK polysaccharide compound (JHS Natural Products, Eugene, Oreg.), razoxane, agile new, sizofiran, Spirogermanium, tenuazonic acid, triaziquone, 2,2', 2 "-RA3s, trichothecene (particularly, T-2 toxin, verracurin A, Roridine A and Scirpenetriol4,15-diacetate), urethane, vindesine ( ), Dacarbazine, Mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, cytosine arabinoside (" Ara-C "), Tespamin, bearing taxanes, such as, taxol (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE ^tMnot containing taxol albumin engineering nanoparticle formulations (the American Pharmaceutical Partners of cremophor, Schaumberg, and TAXOTERE Taxotere (Rhone-Poulenc Rorer, Antony, France) Ill.), Chlorambucil, gemcitabine ( ), 6-Tioguanine, mercaptopurine, Rheumatrex, platinum analogs, as cis-platinum and carboplatin, vinealeucoblastine(VLB) ( ), platinum, Etoposide (VP-16), ifosfamide, mitoxantrone, vincristine(VCR) ( ), oxaliplatin, leucovorin, vinorelbine ( ), Novantrone, edatrexate, daunomycin, aminopterin-induced syndrome, ibandronate, topoisomerase enzyme inhibitor RFS 2000, α-difluorometylornithine (DMFO), retinoic acid-like class, as vitamin A acid, capecitabine ( ), the pharmacologically acceptable salt of any mentioned reagent, acid or derivative, and the combination of two or more mentioned reagent, as CHOP, i.e. the abbreviation of the combination treatment of endoxan, Dx, vincristine(VCR) and prednisolone, and FOLFOX, namely about the oxaliplatin (ELOXATIN combined with 5-FU and folinic acid ^tM) the abbreviation for the treatment of plan.Other chemotherapeutics comprise as the useful cytotoxic reagent of antibody drug binding substances, as maytansinoid (such as, DM1 and DM4) and auristatin (such as, MMAE and MMAF).

Also comprise in the definition of " chemotherapeutics ": (i) adjustment or inhibitory hormone are to the anti-hormonal agents of the effect of tumour, and as estrogen antagonist and selective estrogen receptor modulators (SERM), it comprises, and such as, tamoxifen (comprises citric acid Tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, raloxifene (keoxifene), LY117018, onapristone and (FC-1157a); (ii) aromatase inhibitor of inhibitory enzyme aromatase enzyme, it regulates estrogenic generation in suprarenal gland, e.g., such as, 4 (5)-imidazoles, aminoglutethimide, (megestrol), (Exemestane; Pfizer), Formestane, fadrozole, (vorozole), (letrozole; Novartis) and (Anastrozole; AstraZeneca); (iii) androgen antagonist, as flutamide, Nilutamide, bicalutamide, bright dried meat Li Te and goserelin; And troxacitabine (DOX nucleosides analogue of cytosine); (iv) kinases inhibitor, as ME inhibitor (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotide, suppresses those of genetic expression in the signal path relevant with abnormal cell proliferation particularly, such as, PKC-α, Raf and H-Ras, as Ao Limosen ( genta Inc.); (vii) ribozyme, as vegf expression inhibitor (such as, ) and HER2 expression inhibitor; (viii) vaccine, as gene therapy vaccine, such as, with rlL-2; Topoisomerase 1 inhibitor, as rmRH; (ix) anti-angiogenic original reagent, as rhuMAb-VEGF ( genentech); With the pharmaceutically useful salt of any mentioned reagent, acid and derivative.

As in the present patent application use, term " prodrug " refers to that the cytotoxin comparing cell with the parent compound of medicine can be lower and can by enzymolysis or the precursor or the derivative form that are hydrolyzed the compound of the present invention activating or be converted into active stronger parent fo.See, such as, Wilman, " Prodrugs in Cancer Chemotherapy " Biochemical Society Transactions, 14,375-382 page, the people such as 615th Meeting Belfast (1986) and Stella, " Prodrugs:A Chemical Approach to Targeted Drug Delivery; " Directed Drug Delivery, the people such as Borchardt (chief editor), 247-267 page, Humana Press (1985).Prodrug of the present invention includes, but is not limited to containing the amino acid modified prodrug of phosphatic prodrug, the prodrug containing thiophosphate, the prodrug containing vitriol, the prodrug containing peptide, D-, glycosylated prodrugs, the prodrug containing beta-lactam, the prodrug containing the phenoxy acetamide optionally replaced, prodrug, 5-flurocytosine and other 5-fluor-uracil prodrugs containing the phenylacetamide optionally replaced, and these prodrugs can change into active stronger no cytotoxicity medicine.The example changed into for the cytotoxic drug of the prodrug forms used in the present invention can be derived and include but not limited to compound of the present invention and chemotherapeutics as above.

In this article, " small molecules " is defined as to have is less than about 500 daltonian molecular weight.

" nucleic acid of separation " be substantially with natively with other genomic dna sequences of native sequences and protein or mixture, as the nucleic acid that rrna is separated with polysaccharase, such as, RNA, DNA or mixed polymer.This term covers the nucleotide sequence removed from its naturally occurring environment, and comprise restructuring or clone DNA isolate and chemosynthesis analogue or by the biosynthetic analogue of Heterologous System.Substantially pure molecule comprises the unpack format of described molecule.

" polynucleotide " or " nucleic acid " as being used interchangeably in this article refer to the nucleotide polymer of any length and comprise DNA and RNA.Described Nucleotide can be deoxyribonucleotide, ribonucleotide, the Nucleotide of modification or base and/or their analogue, or can by DNA or RNA polymerase or any matrix be incorporated into by building-up reactions in polymkeric substance.Polynucleotide can comprise the Nucleotide of modification, as methylated nucleotide and their analogue.As used herein, " oligonucleotide " generally refers to short, usual strand, normally synthetic polyribonucleotides, and the length of described synthetic polyribonucleotides usually (but not must) is less than about 200 Nucleotide.Term " oligonucleotide " and " polynucleotide " are not mutually exclusive.Above to the description of polynucleotide equally and be fully applicable to oligonucleotide.

The cell producing anti-CD 98 antibody of the present invention will comprise parents oncocyte, such as, be committed to the hybridoma of ATCC, and introduce the bacterium of nucleic acid and the eukaryotic host cell of encoding antibody.Disclosed below applicable host cell.

Term " package insert ", for representing the specification sheets in the commercial package being included in treatment product traditionally, it comprises that relevant these are treated the indication of the use of products, usage, dosage, used, the information of contraindication and/or warning.

The method of composition and the described composition of preparation

Provide the antibody being bonded to CD98.Provide the immune conjugate comprising anti-CD 98 antibody.Antibody of the present invention and immune conjugate are expressed for (such as) and CD98 and are changed, and such as, express the diagnosis of the relevant illness of rising or treat useful.In some embodiments, antibody of the present invention or immune conjugate for cell proliferative diseases, as cancer diagnosis or treat useful.

Anti-CD 98 antibody

In one embodiment, the invention provides can in this article as the anti-CD 98 antibody that therapeutical agent uses.Exemplary antibodies comprises polyclonal antibody, monoclonal antibody, humanized antibody, people's antibody, bispecific antibody and allos binding substances antibody and has the avidity of improvement or their variant of other character.

1. polyclonal antibody

Antibody of the present invention can comprise polyclonal antibody.The method preparing polyclonal antibody is known to the skilled.(such as) can be injected by the one or many of immunizing agent and (if needs) adjuvant, in Mammals, produce polyclonal antibody.Usually, will by repeatedly subcutaneous or peritoneal injection injecting immune agent and/or adjuvant in Mammals.Described immunizing agent can comprise CD98 polypeptide or its fusion rotein.Being bonded to by immunizing agent known in the Mammals wanting immunity be immunogenic albumen can be useful.The example of these immunogen proteins includes, but is not limited to keyhole keyhole limpet hemocyanin, serum albumin, ox thyroglobulin and Trypsin inhibitor SBTI.The example of operable adjuvant comprises Freund's incomplete adjuvant and MPL-TDM adjuvant (single phosphoramide A, trehalose synthesis two bacillus mycomycete acid esters).Those skilled in the art just can select immunization protocol without the need to undo experimentation.Then, by Mammals bloodletting, and the CD98 antibody titers of serum can be measured.If needed, Mammals can be promoted until antibody titers raises or reaches steady.

2. monoclonal antibody

Alternatively, antibody of the present invention can be monoclonal antibody.Can use the people such as Kohler, the hybridoma method that Nature, 256:495 (1975) first describe prepares monoclonal antibody, or can pass through recombinant DNA method (such as, see, U.S. Patent No. 4816567) preparation.

In hybridoma method, as mentioned above to mouse or other host animals be applicable to, as hamster carries out immunity to cause lymphocyte, described lymphocyte produces and maybe can produce specific binding to the antibody being used for immune described albumen.Alternatively, lymphocyte can ion vitro immunization.After immunity, be separated lymphocyte, then use applicable fusion reagent, as polyoxyethylene glycol, with myeloma cell line merge with formed hybridoma ( goding, Monoclonal Antibodies:Principles? and Practice,59-103 page (Academic Press, 1986)).

Therefore inoculated by prepared hybridoma and grow in the substratum be applicable to, described substratum is preferably containing the growth of Parent Myeloma Cell suppressing not merge or one or more materials (also referred to as fusion partner) of survival.Such as, if Parent Myeloma Cell lacks enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), selective medium then for hybridoma will comprise xanthoglobulin, aminopterin and thymidine (HAT substratum) usually, and described material stops the growth of HGPRT deficient cell.

Preferred fusion partner myeloma cell is merged effectively, supports that selected antibody produced cell is stably high-level and produces antibody and do not merge those of the Selective agar medium sensitivity of parental cell to selection opposing.Preferred myeloma cell line is rat bone marrow tumour system, as derived from from Salk Institute Cell Distribution Center, San Diego, those of available MOPC-21 and the MPC-11 mouse tumor of California USA, such as, with SP-2 and derivative, from American Type Culture Collection (Manassas, Virginia, USA) available X63-Ag8-653 cell.Have also been described the human myeloma and mouse-people's heteromyeloma cell lines (Kozbor, J.Immunol., 133:3001 (1984) that produce for human monoclonal antibodies; With people such as Brodeur, Monoclonal Antibody Production Techniques and Applications, 51-63 page (Marcel Dekker, Inc., New York, 1987)).

The substratum grown wherein hybridoma determines the generation of the monoclonal antibody for described antigen.Preferably, measured, as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) determine the binding specificity of the monoclonal antibody produced by hybridoma by immunoprecipitation or external combination.Can (such as) by people such as Munson, the binding affinity of the Scatchard analysis determination monoclonal antibody that Anal.Biochem., 107:220 (1980) describe.

Once identify produce there is the hybridoma of the antibody of desired specificity, avidity and/or activity after, can by limiting dilution procedures by this clone's subclone and by standard method cultivate (Goding, monoclonal Antibodies:Principles and Practice, 59-103 page (Academic Press, 1986)).For this purpose, the substratum be applicable to comprises (such as) D-MEM or RPMI-1640 substratum.In addition, can as ascites tumour culturing in vivo hybridoma in animal, such as, by cell i.p. is injected to mouse.

The monoclonal antibody of being secreted by subclone by conventional antibody purifying procedure is suitably separated with substratum, ascites fluid or serum, described purifying procedure is such as (e.g.) affinity chromatography (such as, using albumin A or Protein G-Sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis etc.

Use conventional procedure (such as, by use can specific binding to the oligonucleotide probe of coding murine antibody heavy chain with light chain gene) DNA of encodes monoclonal antibody is easily separated and checks order.Hybridoma is used as this DNA and preferably originates.Once be separated, DNA can be placed in expression vector, then described carrier is transfected in host cell, as intestinal bacteria (E.coli) cell, ape COS cell, Chinese hamster ovary (CHO) cell or the myeloma cell not producing antibody protein, thus in recombinant host cell, obtain the synthesis of monoclonal antibody.Summary recombinant expressed in the bacterium of the DNA of antibody described in relevant code comprises the people such as Skerra, Curr.Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol.Revs.130:151-188 (1992).

In other embodiments, monoclonal antibody or antibody fragment can be separated from using (such as) Antibody Phage Display:Methods and Protocols, P.M.O ' Brien and R.Aitken edits, Humana Press, Totawa N.J., the antibody phage libraries that the technology described in 2002 produces.Substantially, by merging the screening phage library selection synthetic antibody clone to the phage of multiple fragments of the antibody variable region (Fv) of bacteriophage coat protein containing displaying.For for desired antigen, screen these phage libraries.Be adsorbed to described antigen by expressing the clone that can be bonded to the Fv fragment of desired antigen, and therefore with the non-binding clone and separate in described library.Then, from described antigen elution of bound clone, and enrichment can be carried out by the extra circulation of Antigen adsorption/wash-out.

Functionally variable domains can be shown on phage or as scFv (scFv) fragment, wherein VH and VL is covalently bound by short flexible peptide, or functionally show variable domains as Fab fragment, wherein they merge respectively to constant domain, and non covalent bond ground interacts, as people such as Winter, described in Ann.Rev.Immunol., 12:433-455 (1994).

The set that polymerase chain reaction (PCR) clones separately VH and VL gene can be passed through, and recombinate at random in phage library, then can find antigen to it and combine clone, as people such as Winter, described in the same.Library from immune origin provides antibody immunogen to high affinity, and without the need to constructing hybridoma.Alternatively, natural set (naive repertoire) can be cloned to provide the people's antibody for the single source of non-autologous and self antigen widely, and without the need to any immunity, as by people such as Griffiths, described in EMBO J, 12:725-734 (1993).Finally, also can by the V constant gene segment C do not reset from stem cell clone, and use the variable CDR3 district of the PCR primer code level containing stochastic sequence and realize rearrangement in vitro to synthesize preparing naive libraries, as by Hoogenboom and Winter, J.Mol.Biol., described by 227:381-388 (1992).

The screening in library can be completed by multiple technologies as known in the art.Such as, CD98 may be used for the hole applying adsorption plate, can express on the host cell being fixed to adsorption plate or can use in cell sorting, or vitamin H can be bonded to catch for the pearl applied with streptavidin, or can use in any other method of elutriation display libraries.By using long-time cleaning and monovalent phage display (as people such as Bass, Proteins, described in 8:309-314 (1990) and WO 92/09690) and the low coating density of antigen (as people such as Marks, Biotechnol., described in 10:779-783 (1992)), the selection of the antibody with slow Dissociation (with good combination avidity) can be promoted.

The antigen screening procedure that can be applicable to by design is to select the phage clone be concerned about, then the Fv sequence from be concerned about phage clone is used to clone (as people such as Kabat with constant region (Fc) the sequence construct total length anti-CD 98 antibody be applicable to, Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, described in Bethesda MD (1991), 1-3 volume) obtain any anti-CD 98 antibody of the present invention.

3. antibody fragment

Present invention encompasses antibody fragment.In some cases, there is the advantage using antibody fragment, instead of whole antibody.The size that fragment is less allows to remove fast, and can cause the improvement to solid tumor accessibility.For the summary of some antibody fragment, see people such as Hudson, (2003) Nat.Med.9:129-134.

Develop the multiple technology for the production of antibody fragment.Traditionally, these fragments be by the proteolytic digestion of complete antibody obtain (see, such as, the people such as Morimoto, Journal of Biochemical and Biophysical Methods24:107-117 (1992); With people such as Brennan, Science, 229:81 (1985)).But, directly can produce these fragments by recombinant host cell now.Fab, Fv and scFv antibody fragment all can be expressed and secrete from them in intestinal bacteria (E.coli) or yeast cell, therefore makes it possible to easily produce these fragments a large amount of.Antibody fragment can be separated from antibody phage libraries discussed above.Alternatively, can directly from intestinal bacteria (E.coli) reclaim Fab'-SH fragment and chemical coupling to form F (ab') 2 fragment (people such as Carter, Bio/Technology 10:163-167 (1992)).According to another kind of method, F (ab') 2 fragment can directly be separated from recombinant host cell is cultivated.Fab and F (ab') 2 fragment of the Half-life in vivo raising comprising salvage receptor binding epitope residue is described in U.S. Patent No. 5869046.For skilled doctor, the other technologies for generation of antibody fragment will be apparent.In some embodiments, antibody is Single-Chain Fv Fragment of Murine (scFv).See WO 93/16185; U.S. Patent No. 5571894; With 5587458.Fv and scFv onlyly has the complete haptophoric kind lacking constant region; Therefore, in vivo between the usage period, they can be suitable for the non-specific binding reduced.ScFv fusion rotein can be constructed to produce the fusion of the effector albumen at arbitrary place in the amino or C-terminal of scFv.See Antibody Engineering, ed.Borrebaeck, the same.Described antibody fragment also can be " linear antibodies ", such as, described in the reference quoted as former.These linear antibodies can be monospecific or multiple specific, as dual specificity.

The integrated structure of minimum antibody sources is the independent variable domains (V territory) being also called single varied texture domain antibodies (SdAb).The biology of some type, camellid and selachian, have and be positioned at high affinity list V sample territory on Fc domain structure of equal value (people such as Woolven, Immunogenetics50:98-101,1999 as they immune parts; The people such as Streltsov, Proc Natl Acad Sci USA.101:12444-12449,2004).V sample territory (be called VhH in camellid, and be called V-NAR in shark) generally show long surperficial ring, and it allows the infiltration in target antigen chamber.They also stablize by sheltering hydrophobic surface section the VH territory be separated.These VhH and V-NAR territories are for engineering design sdAb.Use and devised people V territory variant from the selection of phage library and the additive method that produces the territory that stable, height is originated in conjunction with VL-and VH-.

4. humanized antibody

Present invention encompasses humanized antibody.For humanized for non-human antibody multiple method is well known in the art.Such as, humanized antibody can have one or more amino-acid residue of introducing from inhuman source.These non-human amino acid residues so-called " input " residue, described residue takes from " input " variable region usually.Humanization can carry out (people (1986) Nature321:522-525 such as Jones according to the method for Winter and colleague thereof by the corresponding sequence replacing people's antibody with hypervariable region sequence; The people such as Riechmann (1988) Nature332:323-327; The people such as Verhoeyen (1988) Science239:1534-1536).

In some cases, transplanted by CDR and build humanized antibody, wherein the aminoacid sequence of 6 of parent's rodent antibodies complementary determining regions (CDR) is transplanted on people's antibody framework.The people such as Padlan (FASEB J.9:133-139,1995) determine in fact only to have an appointment in CDR 1/3rd contact residues antigen, and these are called " specificity determining residue " or SDR.In SDR implantation technique, only SDR residue is transplanted to (people such as Kashmiri, Methods36:25-34,2005) on people's antibody framework.

Can be important in the selection preparing the people's variable domains (light chain and heavy chain) used in humanized antibody concerning reduction antigenicity.According to so-called " best-fit (best-fit) " method, for the whole library screening variable domain sequence of rodent antibodies of known people's variable domain sequence.Then, accept as humanized antibody people's framework (people (1993) J.Immunol.151:2296 such as Sims using with the immediate human sequence of rodents; The people such as Chothia (1987) J.Mol.Biol.196:901).Another kind method employs the specific frame of the consensus sequence of everyone antibody of the specific subgroup deriving from light chain or heavy chain.Identical frames may be used for several different humanized antibody (people (1992) Proc.Natl.Acad.Sci.USA, the 89:4285 such as Carter; The people such as Presta (1993) J.Immunol., 151:2623).In some cases, described framework derives from people's subclass (V of maximum _lκ subgroup I (V _lκ I) and V _hsubgroup III (V _hiII) consensus sequence).In another approach, at framework region source end user's germ line genes.

In the substituting example (being called super humanized) compared based on CDR, FR homology is incoherent.Described method comprises comparing of nonhuman sequence and the set of functional human germ line genes.Then, those genes that are identical with mouse sequence or closely-related standard construction (canonical structure) of encoding are selected.Then, having in the gene of described standard construction with non-human antibody, those selecting to have higher homology in CDR are as FR donor.Finally, described inhuman CDR is transplanted to (people such as Tan, J.Immunol.169:119-1125,2002) on these FR.

Usually, also expect that the reservation of humanized antibody is to the high affinity of antigen and other good biological properties.In order to realize this target, according to a kind of method, prepare humanized antibody by the parental array analytical procedure and multiple conceptual humanized products using the stereoscopic model of parent and humanized sequence.Three-dimensional immunoglobulin models be generally available and be those skilled in the art be familiar with.The computer program of the roughly steric configuration structure illustrating and show selected candidate immunoglobulin sequences sequence is available.These comprise (such as) WAM (Whitelegg and Rees, Protein Eng.13:819-824,2000), Modeller (Sali and Blundell, J.Mol.Biol.234:779-815,1993) and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-2713,1997).Make it possible to analyze described residue possibility role in the function of candidate immunoglobulin sequences sequence, namely on affecting the analysis of candidate immunoglobulin sequences in conjunction with the residue of the ability of its antigen to the inspection of these display patterns.By this way, can FR residue be selected from recipient and list entries and merge, thus the antibody characteristic needed for realizing, as the avidity improved target antigen.Usually, some hypervariable region residues participates in directly and the most significantly affect antigen combination.

Another kind of method for antibody humanization is measuring based on the antibody human being called as human sequence's content (HSC).The set of mouse sequence and human germline gene compares by this method, and difference is marked as HSC.Then, by making its HSC maximum, instead of use and produce the overall identity method of multiple diversity humanization variant and make target sequence humanization people such as (, Mol.Immunol.44:1986-1998,2007) Lazar.

Contrary with aforesaid method, empirical method may be used for producing and selecting humanized antibody.These methods are based on the selection using the generation of the large humanization Mutant libraries of beneficiation technologies or High Throughput Screening Assay and best clone.Antibody variants can be separated from phage, rrna and yeast display library and by bacterium colony screening and separating (Hoogenboom, Nat.Biotechnol.23:1105-1116,2005; The people such as Dufner, Trends Biotechnol.24:523-529,2006; The people such as Feldhaus, Nat.Biotechnol.21:163-70,2003; The people such as Schlapschy, Protein Eng.Des.Sel.17:847-60,2004).

In FR library approach, series of residues variant is incorporated into the specific position in FR, then selects library to select the FR supported the CDR the best of transplanting.The residue replaced can comprise some or all (Foote and Winter of " vernier " residue differentiated as contributing to CDR structure, J.Mol.Biol.224:487-499,1992), or from the more limited group of the target residue that the people such as Baca (J.Biol.Chem.272:10678-10684,1997) differentiate.

In FR reorganization, whole FR is combined with inhuman CDR, instead of creates combinatorial library people such as (, Methods 36:43-60,2005) Dall'Acqua of selected residue variant.Can combine library screening in two step chosen processs (first humanization VL, then VH).Alternatively, a step FR Shuffling Method can be used.The method has proved more effective than two step screenings, this is because the antibody of gained demonstrates biochemical property and the physico-chemical property of improvement, it comprises the expression of raising, the avidity of raising and thermostability (people such as Damschroder, Mol.Immunol.44:3049-60,2007).

" people's chemical industry journey (humaneering) " method based on substantially minimum specificity determinants (MSD) experiment differentiate and based on non-human fragments in people FR library sequence replace and to combine evaluation.It from the CDR-3 district of inhuman VH and VL chain and gradually by the displacement of other districts (comprising CDR-l and CDR-2 of VH and VL) of non-human antibody in people FR.The method causes epi-position to retain and from the discriminating of antibody of multiple subclass with different people V fragment CDR usually.Humaneering makes it possible to be separated the antibody (Alfenito with human germline gene's antibody 91-96% homology, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

5. people's antibody

Variable domain sequence can be cloned by the Fv merging the phage display library being selected from the people source with people's constant domain sequence known as above and construct people's anti-CD 98 antibody of the present invention.Alternatively, human monoclonal anti-CD 98 antibody of the present invention can be prepared by hybridoma method.By following document description for generation of the human myeloma of human monoclonal antibodies and mouse-human heteromyeloma's clone, such as, Kozbor J.Immunol., 133:3001 (1984); The people such as Brodeur, Monoclonal Antibody Production Techniques and Applications, 51-63 page (Marcel Dekker, Inc., New York, 1987); With people such as Boerner, J.Immunol., 147:86 (1991).

Also may produce once immunity, the transgenic animal (such as, mouse) of whole set of people's antibody can be produced when there is not endogenous immunoglobulin and producing.Express the transgenic mice of people's collection of antibodies for generation of the high affinity human sequence monoclonal antibody of anti-multiple potential drug target.See, such as, Jakobovits, A., Curr.Opin.Biotechnol.1995,6 (5): 561-6; Briiggemann and Taussing, Curr.Opin.Biotechnol.1997,8 (4): 455-8; U.S. Patent No. 6075181 and 6150584; With people such as Lonberg, Nature Biotechnol.23:1117-1125,2005.

Alternatively, can by produce for the antibody of target antigen human B lymphocyte (these bone-marrow-derived lymphocytes can from individuality results or can ion vitro immunization) immortalization prepare people's antibody.See, such as, the people such as Cole, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, the 77th page (1985); The people such as Boerner, J.Immunol., 147 (l): 86-95 (1991); With U.S. Patent No. 5750373.

Gene shuffling can also be used for from inhuman, and such as rodent antibodies obtains people's antibody, and wherein said people's antibody has the avidity similar with starting non-human antibody and specificity.According to the method, it is also called as " the epi-position marking " or " guided selection ", the set of employment V domain gene substitutes heavy chain or the variable region of light chain of the non-human antibody fragment obtained by display technique of bacteriophage as described herein, thus produces the non-chimeric colony of human chain/human chain scFv or Fab.Carry out selecting to cause non-human chain/human chain to be fitted together to the separation of scFv or Fab with antigen, wherein human chain makes the antigen binding site of destruction recover eliminate corresponding non-human chain in one-level phage display clone after, i.e. the selection of epi-position guiding (marking) human chain companion.When repeating the method to replace remaining non-human chain, obtain people's antibody (see people such as PCT WO93/06213 and Osbourn, Methods., 36,61-68,2005).From traditional to transplant the humanization of the non-human antibody carried out by CDR different, this technology provides fully human antibodies, they are containing FR or the CDR residue in inhuman source.FBP (the people such as Figini example of mouse antibodies to the humanized guided selection of cell-surface antigens being comprised ovarian cancer cell exists, Cancer Res., 58,991-996,1998) and CD147, it is the high expression level (people such as Bao in hepatocellular carcinoma, Cancer Biol.Ther., 4,1374-1380,2005).

The defect that guided selection method is possible is an antibody chain reorganization and keeps another constant epi-position that may cause to drift about.In order to maintain the epi-position that non-human antibody identifies, CDR can be applied and retain (people such as Klimka, Br.J.Cancer., 83,252-260,2000; The people such as Beiboer, J.Mol.Biol., 296,833-49,2000).In the method, inhuman CDR-H3 retains usually, this is because CDR is positioned at the center of antigen binding site and the most important region of antibody proved for antigen recognition.But, in some cases, CDR-H3 and CDR-L3 and CDR-H3, CDR-L3 and CDR-L2 of non-human antibody can be retained.

6. bispecific antibody

Bi-specific antibody be at least two not synantigen there is the monoclonal antibody of binding specificity.In some embodiments, bispecific antibody is people or humanized antibody.In some embodiments, wherein a kind of binding specificity is to CD98, and another kind is to any other antigen.In some embodiments, bispecific antibody may be bonded to two different CD98 epi-positions.Bispecific antibody can also be used for cytotoxic reagent being positioned to the cell of expressing CD98.These antibody have CD98 brachium conjunctivum and such as, arm in conjunction with cytotoxic reagent (e.g., saporin, anti-interferon-α, vinca alkaloids, ricin A chain, methotrexate or radioactive isotope hapten).Bispecific antibody can be prepared into full length antibody or antibody fragment (such as, F (ab') 2 bispecific antibody).

The method preparing bispecific antibody is well known in the art, as, such as, by the coexpression that two kinds of heavy chain immunoglobulin-light chains are right, wherein said two heavy chains have different specificity (Milstein and Cuello, Nature, 305:537 (1983)).About producing the details of bispecific antibody, see, such as, Bispecific Antibodies, Kontermann edit, Springer-Verlag, Hiedelberg (2011).

7. multivalent antibody

Multivalent antibody can be subject to the internalization (and/or katabolism) of the cell of expressing this antibody institute conjugated antigen faster than bivalent antibody.Antibody of the present invention can be have three or more antigen binding site (such as, tetravalent antibody) multivalent antibody (they are except IgM class), it can easily be produced by the recombinant expressed of the nucleic acid of encoding said antibody polypeptide chain.Multivalent antibody can comprise dimerisation domain and three or more antigen binding sites.In certain embodiments, described dimerisation domain comprises (or consisting of) Fc district or hinge area.In this case, antibody will comprise aminoterminal three or more the antigen binding sites in Fc district and Fc district.In some embodiments, multivalent antibody comprises (or consisting of) three to about eight antigen binding sites.In a this embodiment, multivalent antibody comprises (or consisting of) four antigen binding sites.Multivalent antibody comprises at least one polypeptide chain (such as, two polypeptide chains), and wherein said polypeptide chain comprises two or more variable domains.Such as, polypeptide chain can comprise VD1-(X1) n-VD2-(X2) n-Fc, and wherein VD1 is the first variable domains, and VD2 is the second variable domains, and Fc is a polypeptide chain in Fc district, X1 and X2 represented amino acid or polypeptide, and n is 0 or 1.Such as, polypeptide chain can comprise: VH-CH1-flexible joint-VH-CH1-Fc district chain; Or VH-CH1-VH-CH1-Fc district chain.Multivalent antibody herein can also comprise at least two (such as, four) light variable domains polypeptide.Multivalent antibody herein (such as) can comprise about 2 to about 8 light variable domains polypeptide.The light variable domains polypeptide considered herein comprises light variable domains, and optionally also comprises CL territory.

8. effector function is engineered

Can expect for effector function to modify antibody of the present invention, such as, thus improve cytotoxicity (ADCC) or the CDC (CDC) of the antigen dependent cell mediation of described antibody.This can introduce one or more aminoacid replacement to realize by the Fc district at described antibody.See, such as, the people such as Lazar, Proc.Natl.Acad.Sci.USA 2006,103 (11): 4005-4010; Presta, L.G., Curr.Opin.Immunol.2008,20 (4): 460-70; With U.S. Patent No. 7183387; 7332581; With 7335742.

Alternatively or in addition, cysteine residues can be introduced Fc district, allow whereby to form interchain disulfide bond in this region.Therefore the cell that produced equal diabodies can have the internalization capability of improvement and/or the complement-mediated of raising kills and antibody dependent cellular cytotoxicity (ADCC).See people such as Caron, J.Exp Med.176:1191-1195 (1992) and Shopes, B.J.Immunol.148:2918-2922 (1992).Allos bi-functional cross-linking agent can also be used to prepare the equal diabodies with the anti-tumor activity of raising, as people such as Wolff, described in Cancer Research 53:2560-2565 (1993).Alternatively, engineeredly can have two Fc district and therefore there is the molten born of the same parents of complement of raising and the antibody of ADCC ability.See people such as Stevenson, Anti-Cancer Drug Design 3:219-230 (1989).In order to improve the serum half-life of antibody, a kind of method in described antibody (particularly antibody fragment), mixes salvage receptor binding epitope, such as, as described in United States Patent (USP) 5739277.As used herein, term " salvage receptor binding epitope " refers to the epi-position of serum half-life in the responsible body improving described IgG molecule in IgG molecule (such as, IgG1, IgG2, IgG3 or IgG4) Fc district.

9. substitute bonding agent

Present invention encompasses the NIg bonding agent of specific binding to the epi-position identical with anti-CD 98 antibody disclosed herein.In some embodiments, bonding agent is the differentiated reagent of replacing anti-CD 98 antibody of the present invention or being replaced by anti-CD 98 antibody of the present invention in competitive binding assay.These alternative bonding agents can comprise (such as) any engineered peptide backbone as known in the art.These skeletons comprise, and such as, anticalins, it is based on lipocalin skeleton, and the feature of its protein structure is the rigidity beta sheet bucket supporting four Gao Bianhuan forming ligand binding site.By target random mutation in ring region and combined function show and guided selection engineered novel binding specificity (Skerra (2008) FEBS J.275:2677-2683).Other skeletons be applicable to can comprise such as adnectin or monomer (monobody), based on the tenth people's fi-bronectin type III extracellular domain (Koide and Koide (2007) Methods Mol.Biol.352:95-109); Affine body (affibody), based on the Z structural domain (people such as Nygren, (2008) FEBS J.275:2668-2676) of SP; DARPin, based on ankyrin repeat protein (people such as Stumpp, (2008) Drug.Discov.Today13:695-701); Fynomers, based on the SH3 territory (people such as Grabulovski, (2007) J.Biol.Chem.282:3196-3204) of people Fyn protein kinase; Affitins, based on the Sac7d (people such as Krehenbrink, (2008) J.Mol.Biol.383:1058-1068) from Sulfolobus acidolarius; Affilins, based on people y-B-crystallin (people such as Ebersbach, (2007) J.Mol.Biol.372:172-185); Avimers (avimer), based on the A territory (people such as Silverman, (2005) Biotechnol.23:1556-1561) of membrane receptor protein; Be rich in the knot peptide (Kolmar (2008) FEBS J.275:2684-2690) of halfcystine; With engineered Kunitz type inhibitor (Nixon and Wood (2006) Curr.Opin.Drug.Discov.Dev.9:261-268).Relevant summary, see Gebauer and Skerra (2009) Curr.Opin.Chem.Biol.13:245-255.

antibody variants

In some embodiments, the amino acid sequence modifications of antibody as herein described is considered.Such as, can expect binding affinity and/or the other biological character of improving antibody, it includes, but is not limited to specificity, thermostability, expression level, effector function, glycosylation, the immunogenicity of reduction or solubleness.Except anti-CD 98 antibody as herein described, consideration can prepare anti-CD 98 antibody variant.Anti-CD 98 antibody variant can be prepared by the change of suitable Nucleotide being incorporated in coding DNA and/or by the synthesis of required antibody or polypeptide.It will be appreciated by those skilled in the art that amino acid change can change process after the translation of anti-CD 98 antibody, as changed number or the position of glycosylation site or changing film anchoring features.

Change can be the replacement of one or more codons of encoding said antibody or polypeptide, disappearance or insertion, and it causes compared with the antibody of native sequences or polypeptide, the change of aminoacid sequence.Aminoacid replacement can be with there is similar structure and/or a kind of amino acid whose result of another kind of amino acid replacement of chemical property, as substituted leucine with Serine, namely conserved amino acid substitutes.Inserting or lack can optionally in about 1 to 5 amino acid whose scope.Can by systematically carrying out amino acid whose insertion, disappearance or replacement in the sequence and the activity of testing shown by the total length of gained variant or ripe native sequences determines allowed change.

Aminoacid sequence inserts and comprises the amino-of length in the scope of 1 residue to the polypeptide containing 100 or more residues and/or carboxy-terminal fusion, and inserts between the sequence of single or multiple amino-acid residue.The example that end inserts comprises the antibody with N-terminal methionyl residue.Other of described antibody molecule insert enzyme (such as, for antibody directed enzyme pro-drug therapy) or the peptide that variants N-or C-terminal fusion comprised to antibody improves the described antibody serum transformation period.

Remarkable change in antibody biological property is along with selective cementation, they are for the structure maintaining polypeptide backbone in (a) replacement areas, such as, as folding or helical conformation, (b) target site punishment son electric charge or hydrophobicity, or (c) whole side chain affect significant difference.According to the similarity of side chain properties, amino acid can divide into groups (A.L.Lehninger, in Biochemistry, the 2nd edition, 73-75 page, Worth Publishers, New York (1975)):

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

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

(3) acid: Asp (D), Glu (E)

(4) alkalescence: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residue can be grouped into based on common side chain properties:

(1) hydrophobic: nor-leucine, Met, Ala, Val, Leu, Ile;

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

(3) acid: Asp, Glu;

(4) alkalescence: His, Lys, Arg;

(5) residue of chain orientation is affected: Gly, Pro;

(6) fragrance: Trp, Tyr, Phe.

Non-conservative displacement needs the member of in these classifications and another kind of exchange.The residue of described displacement can also be introduced into conservative substitution site, or is introduced into all the other (non-conservative) sites.

Use method as known in the art can prepare described change, as oligonucleotide mediated (orientation) mutagenesis, Alanine-scanning and PCR mutagenesis.Rite-directed mutagenesis (people such as Carter, Nucl.Acids Res., 13:4331 (1986) can be carried out to the DNA of clone; The people such as Zoller, Nucl.Acids Res., 10:6487 (1987)), the cassette mutagenesis (people such as Wells, Gene, 34:315 (1985)), restriction-select the sudden change (people such as Wells, Philos.Trans.R.Soc.London SerA, 317:415 (1986)) or other known technology to produce anti-CD 98 antibody modification D NA.

Any cysteine residues of the correct conformation of maintenance anti-CD 98 antibody can also be had neither part nor lot in improve the oxidative stability of described molecule and to prevent abnormal crosslinked by usual Serine.On the contrary, halfcystine key can be joined in anti-CD 98 antibody to improve its stability (particularly, wherein said antibody is antibody fragment, as Fv fragment).Describe in (such as) WO 2006/034488 and may be used for producing the engineered antibody of the halfcystine of antibody-drug conjugates.

In one embodiment, anti-CD 98 antibody molecule of the present invention is " going immunity " antibody.Anti-CD 98 antibody is the antibody deriving from humanization or chimeric anti-CD 98 antibody " to go immunity ", and it has one or more change in its aminoacid sequence, thus causes non-ly going compared with immune antibody with original separately, and the immunogenicity of described antibody reduces.One of program for generation of these antibody mutants relates to discriminating and the removing of antibody molecule t cell epitope.In a first step, the immunogenicity of described antibody molecule can be determined by several method, such as, be determined or the prediction of these epi-positions on silicon chip by the external of t cell epitope as known in the art.Once authenticated the Key residues of t cell epitope function, then can carry out suddenling change to remove immunogenicity and retaining antibody activity.For summary, see, such as, the people such as Jones, Methods in Molecular Biology 525:405-423,2009.

External avidity is ripe

In one embodiment, compared with parental generation antibody, the antibody variants with the character (as avidity, stability or expression level) of improvement is that external avidity is ripe.With natural prototype class seemingly, external avidity is ripe based on sudden change and the principle selected.Antibody library is shown as at biological (such as, phage, bacterium or yeast) on the surface or be combined Fab, scFv or V territory fragment of (covalent linkage or non covalent bond) with their coding mRNA or DNA.Show that the avidity of antibody is selected to allow the biology of the genetic information of carrying encoding said antibody or the separation of mixture.Use methods of exhibiting (as phage display), 2 or 3 take turns sudden change and select usually to cause the avidity of antibody fragment in low nanomolar range.The antibody of preferred Affinity maturation will have the avidity of nmole or even picomole to target antigen.

Phage display is the method the most widely of antibody display and selection.Antibody is shown on Fd or M13 phage surface as the fusion on bacteriophage coat protein.Select to relate to be exposed to antigen and to be combined with their target to allow the antibody of phage display, this process is called " elutriation ".To the phage results of antigen be bonded to and in bacterium, infect to produce the phage being used for other and selecting.For summary, see Hoogenboom, Methods.Mol.Biol.178:1-37,2002; Bradbury and Marks, J.Immuno.Methods 290:29-49,2004).

In yeast display systems (people such as Boder, Nat.Biotech.15:553-57,1997; The people such as Chao, Nat.Protocols 1:755-768,2006), antibody is shown as the variable fusion of strand (scfv), and wherein heavy chain is connected by flexible joint with light chain.ScFv merges in the attachment subunit of yeast agglutinant protein Aga2p, and it is by being connected to yeast cells wall with the disulfide linkage of Aga1p.Show described protein away from cell surface by the protein exhibiting expression of Aga2p, thus the interaction possible with other molecules on yeast cells wall is minimized.Magneticseparation and flow cytometry are used for screen library to select that there is the avidity of improvement and the antibody of stability.By mark yeast to determine with biotinylated antigen and the second reagent (as being bonded to the streptavidin of fluorophore) to the combination of soluble antigen be concerned about.Can by the immunofluorescence label of the c-Myc Epitope tag of homo agglutinin or side joint scFv measure antibody surface express in change.Express the stability shown with institute display protein interrelated, and the antibody that stability and avidity therefore can be selected to improve people such as (, J.Mol.Biol.292:949-956,1999) Shusta.Other advantages of yeast display are that shown albumen is folding in the endoplasmic reticulum of eucaryon yeast cell, thus make use of endoplasmic reticulum chaperone and quality control mechanism.Once ripe completely, then " titration " antibody affinity easily while can showing on yeast surface, thus eliminate the needs to each clonal expression and purifying.The theory restriction of yeast surface display is functional library size that may be less compared with other methods of exhibiting; But nearest method employs yeast cell breeding system to produce the combination diversity (U.S. Patent Publication 2003/0186374 being expected to be 1014 sizes; The people such as Blaise, Gene 342:211-218,2004).

In ribosomal display, create antibody-ribosomal-mRNA (ARM) mixture and select in cell-free system.By the DNA library genetic fusion in encoding particular antibodies library to the intervening sequence lacking terminator codon.During translation, this intervening sequence is still connected to peptidyl tRNA and occupies rrna passage, and therefore allows the albumen be concerned about stretch out from rrna and fold.The mixture of mRNA, rrna and protein gained can be bonded to the part of surface bonding, thus allows to be separated described antibody and the mRNA that encodes by catching with the avidity of described part simultaneously.Then, the mRNA reverse transcription combined by rrna is in cDNA, and then it can experience sudden change and use people such as (, Nucleic Acids Res.34, e127,2006) Fukuda in next round is selected.In mRNA shows, puromycin is used between antibody and mRNA, to set up covalent linkage people such as (, Proc.Natl.Acad.Sci.USA 98,3750-3755,2001) Wilson as adaptor molecule.

Carry out because these methods are completely external, therefore they provide two main advantages being better than other selection technique.First, the diversity in library not by the restriction of bacterial cell conversion efficiency, but is only subject to the restriction of the number of rrna and the different mRNA molecule existed in test tube.The second, easily can introduce random mutation after often taking turns selection, such as, by the polysaccharase without proofreading activity, this is because need not library be transformed after any diversification step.

Can with target mode or by introduce at random diversity is incorporated into antibody library CDR or whole V gene in.A kind of front method comprises the focus (people such as Ho of the separation of the whole CDR of the sudden change targeting antibodies that passes sequentially through high or low level or target somatic hypermutation, J.Biol.Chem.280:607-617,2005) or on experiment basis or suspect the residual sequence affecting avidity for reasons in structure.The strain of intestinal bacteria (E.coli) mutator gene can be used, use the fallibility of archaeal dna polymerase people such as (, J.Mol.Biol.226:889-896,1992) Hawkins or rna replicon enzyme to copy random mutation is incorporated in whole V gene.Can also to be reorganized by DNA or diversity ((people such as Lu, J.Biol.Chem 278:43496-43507,2003 are introduced in similar techniques displacement natural diversities region; U.S. Patent No. 5565332; U.S. Patent No. 6989250).Alternative technologies target extend into the Gao Bianhuan (people such as Bond in framework residues, J.Mol.Biol.348:699-709,2005), use the ring deletion and insertion in CDR or use the variation (U.S. Patent Publication No.2004/0005709) based on hybridization.Disclose in U.S. Patent No. 7985840 and produce multifarious additive method in CDR.The screening in library can be completed by multiple technologies as known in the art.Such as, CD98 can be fixed on solid carrier, post, pin or Mierocrystalline cellulose/poly-(vinylidene fluoride) film/other strainers, the host cell being fixed to adsorption plate is expressed or uses in cell sorting, or be bonded to vitamin H for using catching of streptavidin coating pearl, or use in for any other method of elutriation display libraries.

About the summary of external avidity maturation, see Hoogenboom, Nature Biotechnology23:1105-1116,2005 and Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51,2010 and reference wherein.

the modification of anti-CD 98 antibody

The covalent modification of anti-CD 98 antibody comprises within the scope of the invention.Covalent modification comprises and the Target amino acid Residue of anti-CD 98 antibody and organic derivatization reagent that can react with the side chain selected by anti-CD 98 antibody or N-or C-terminal residue being reacted.Other are modified and comprise glutaminyl and the asparaginyl deacylated tRNA amine respectively to corresponding glutamyl and aspartyl residue, the hydroxylation of proline(Pro) and Methionin, the phosphorylation of the hydroxyl of seryl or threonyl residues, Methionin, (the T.E.Creighton that methylates of the α amino of arginine and histidine side chains, Proteins:Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, pp.79-86 (1983)), the acetylizing of N-terminal amine and the amidation of any C-terminal carboxyl.

The covalent modification comprising the anti-CD 98 antibody of other types within the scope of the present invention comprises and changes described antibody or the natural type of glycosylation of polypeptide (people such as Beck, Curr.Pharm.Biotechnol.9:482-501,2008; Walsh, Drug Discov.Today 15:773-780,2010), and with U.S. Patent No. 4640835; 4496689; 4301144; 4670417; Mode described in 4791192 or 4179337, is connected to one of multiple charged non-protein polymer by described antibody, such as, and polyoxyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.

Can also modify anti-CD 98 antibody of the present invention merges to another kind of heterologous polypeptide or aminoacid sequence to be formed to comprise, such as, Epitope tag (Terpe, Appl.Microbiol.Biotechnol.60:523-533,2003) or the chimeric molecule (Aruffo of the anti-CD 98 antibody in the Fc district of IgG molecule, " Immunoglobulin fusion proteins " in Antibody Fusion Proteins, S.M.Chamow and A.Ashkenazi edits, Wiley-Liss, New York, 1999,221-242 page).

the preparation of anti-CD 98 antibody

Anti-CD 98 antibody can be produced with containing the vector of anti-CD 98 antibody coding nucleic acid or the cell of transfection by cultivating.Standard recombinant techniques can be used to obtain the polynucleotide sequence of the polypeptide composition of coding antibody of the present invention.Required polynucleotide sequence can be separated and check order from antibody produced cell, as hybridoma.Alternatively, nucleotide synthesizer or round pcr synthetic polyribonucleotides can be used.Once obtain, the sequence of coding said polypeptide is inserted into and can copies in host cell with in the recombinant vectors of expressing heterologous polynucleotide.For purposes of the present invention, can use and can use in the art and known variety carrier.The selection of suitable carrier by depend primarily on the nucleic acid that will be inserted in carrier size and will with the concrete host cell of described vector.The host cell being suitable for expressing antibody of the present invention comprises prokaryotic organism, as archeobacteria and eubacterium, it comprises Gram-negative or gram-positive organism, eukaryotic microorganisms, as filamentous fungus or yeast, invertebral zooblast, as insect or vegetable cell and vertebrate cells, as mammalian host cell line.With above-mentioned expression vector transformed host cell also according to being used for evoked promoter, selecting the situation of the gene of sequence needed for transformant or amplification coding, in the conventional medium of appropriate change, cultivate host cell.Use the antibody that standard protein purification method purifying as known in the art is produced by described host cell.

Comprise vector construction, the antibody production method of expression and purification is also described in the people such as Pluckthun, (1996) in Antibody Engineering:Producing antibodies in Escherichia coli:From PCR to fermentation (McCafferty, J., Hoogenboom, H.R., and Chiswell, D.J. edit), the 1st edition, 203-252 page, IRL Press, Oxford; Kwong, K. & Rader, the people such as C.E.coli expression and purification of Fab antibody fragments.Current protocols in protein science editorial board John E Coligan, 6th chapter, Unit 6.10 (2009); Tachibana and Takekoshi, " Production of Antibody Fab Fragments in Escherischia coli, " in Antibody Expression and Production, M.Al-Rubeai edit, Springer, New York, 2011; Therapeutic Monoclonal Antibodies:From Bench to Clinic (Z.An chief editor), John Wiley & Sons, Inc., Hoboken, NJ, USA.

Certainly, consider to use additive method well known in the art to prepare anti-CD 98 antibody.Such as, can by use solid phase technique direct peptide synthesis produce be applicable to aminoacid sequence or its part (see, such as, the people such as Stewart, Solid-Phase Peptide Synthesis, W.H.Freeman Co., San Francisco, CA (1969); Merrifield, J.Am.Chem.Soc, 85:2149-2154 (1963)).Manual technique can be used or carry out protein synthesis in vitro by automatization.Can multiple parts of chemosynthesis anti-CD 98 antibody individually, and use chemistry or enzymatic means to merge to produce required anti-CD 98 antibody.Alternatively, can from engineered with the cell of the transgenic animal of expressing described antibody or body fluid, as antibody purification in milk, such as disclosed in U.S. Patent No. 5545807 and U.S. Patent No. 5827690.

immune conjugate

Present invention also offers immune conjugate (to be called interchangeably " antibody drug binding substances ", or " ADC "), its comprise by synthetic linker be covalently bond in the anti-CD 98 antibody of the present invention of one or more cytotoxic reagents any one.The high specific of monoclonal antibody is combined with the pharmacological efficacy of cytotoxic molecule by ADC, thus allows specifically cytotoxic reagent targets neoplastic cells to be avoided the non-specific toxicity of most of cancer therapy drug.For summary, see, such as, Carter and Senter, Cancer is (2008) J.14:154-169; Ducry and Stump, B ioconjugate Chem.21:5-13 (2010); The people such as Beck, Discov.Med.10:329-339 (2010).

The cytotoxic reagent used in immune conjugate of the present invention can comprise chemotherapeutics as above, medicine or growth inhibitor, toxin (such as, the enzyme activity toxin of bacterium, fungi, plant or animal-origin or its fragment) or radio isotope.In some embodiments, described immune conjugate comprises DNA bonding agent (such as, calicheamycin) or tubulin depolymerizing agent (such as, statin (auristatin) in class maytansine or Austria).Invention also contemplates that (such as, rnase or DNA endonuclease, as deoxyribonuclease with having nucleic acid enzyme activity at antibody; DNA enzymatic) compound between the immune conjugate that formed.

The enzyme activity toxin that can use in immune conjugate of the present invention and fragment thereof comprise diphtheria toxin A chain, the nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa (Pseudomonas aeruginosa)), ricin (ricin) A chain, toxalbumin (abrin) A chain, capsule lotus root toxalbumin (modeccin) A chain, α-broom aspergillin (sarcin), tung oil tree (Aleurites fordii) albumen, Dianthus caryophyllus L. toxalbumin (dianthin proteins), dyers' grapes (Phytolaca americana) albumen (PAPI, PAPII and PAP-S), balsam pear (Momordica charantia) inhibitor, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin (gelonin), NSC-69529 (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).See, such as, WO 93/21232.

The generation of multiple radio isotope to radioactivity binding antibody is available.Example comprises At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³², Pb ²¹²with the radio isotope of Lu.When described binding substances is for detecting, it can comprise the radioactive atom for scintiscanning research, such as, tc99m or I123, or for nucleus magnetic resonance (NMR) imaging (also referred to as magnetic resonance imaging MRI, MRI) spin label, as iodo-123, iodine-131, indium-111, fluoro-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Can in a known way radio isotope be incorporated in binding substances, such as, as Reilly, " The radiochemistry of monoclonal antibodies and peptides; " in Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer, R.M.Reilly edit, Wiley, Hoboken N.J., described in 2010.

Described joint can be conducive to " can cut joint " that cytotoxic drug discharges in cell, but also contemplates in this article and non-ly cut joint.The joint used in immune conjugate of the present invention unrestrictedly comprises the unstable joint of acid (such as, hydrazone joint), joint containing disulphide, peptidase-sensitive joint (such as, peptide linker, as citrulline-α-amino-isovaleric acid or Phe-Lys), the joint of light shakiness, dimethyl linker (people such as Chari, Cancer Research52:127-131 (1992); U.S. Patent No. 5208020), thioether linker or be designed for the hydrophilic linker (people such as Kovtun, Cancer Res.70:2528-2537,2010) of the tolerance avoiding multidrug transporter to mediate.

Can use the binding substances of multiple bifunctional protein coupling agent Dispersal risk and cytotoxic reagent, described coupling agent is as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo group-EMCS, sulfo group-GMBS, sulfo group-KMUS, sulfo group-MBS, sulfo group-SIAB, sulfo group-SMCC and sulfo group-SMPB and SVSB (succinimido-(4-vinyl sulphone) benzoic ether)).Such as, can as people such as Vitetta, Science238:1098 prepares ricin immunotoxin described in (1987).The present invention also considers to use the binding substances of any applicable method Dispersal risk as disclosed in the art and cytotoxic reagent, such as, and Bioconjugate Techniques, 2nd edition, G.T.Hermanson edits, Elsevier, San Francisco, 2008.

Conventional antibody-drug combines strategy based on the random incorporation chemistry of the thiol group of the epsilon-amino or Cys residue that relate to Lys residue, and it causes creating allos binding substances.The technology of immediate development allows to be combined with the locus specificity of antibody, thus result in homogeneous medicine loading and avoid the ADC subgroup of antigen combination or the pharmacokinetics with change.These positions be included on heavy chain and light chain comprise the engineered of " sulfo-monoclonal antibody (thiomab) " of halfcystine replacement, which provide reactive thiol group and do not destroy immunoglobulin folding and assembling or change antigen and combine (people such as Junutula, J.Immunol.Meth.332:41-52 (2008); The people such as Junutula, Nat.Biotechnol.26:925-932,2008).In another approach, by inserting recodification terminator codon UGA from end to seleno-cysteine, be inserted in antibody sequence while seleno-cysteine is translated altogether, thus allow when there is another kind of natural amino acid to carry out locus specificity covalent attachment (people such as Hofer, Proc.Natl.Acad.Sci.USA105:12451-12456 (2008) at the nucleophilic selenol group place of seleno-cysteine; The people such as Hofer, Biochemistry 48 (50): 12047-12057,2009).

pharmaceutical preparation

Antibody of the present invention or antibody-drug conjugates (ADC) can be used by any approach being suitable for the patient's condition that will treat.Usually, by parenteral (i.e. infusion), subcutaneous, intramuscular, intravenously, intracutaneous, sheath and epidural administration of antibodies or ADC.

For Therapeutic cancer, in one embodiment, described antibody or antibody-drug conjugates is used by intravenous infusion.The dosage used by infusion is at each dosage about 1 μ g/m ²to about 10000 μ g/m ²scope in, usually weekly dosage, amounts to 1,2,3 or 4 dosage.Alternatively, described dosage range is about 1 μ g/m ²to about 1000 μ g/m ², about 1 μ g/m ²to about 800 μ g/m ², about 1 μ g/m ²to about 600 μ g/m ², about 1 μ g/m ²to about 400 μ g/m ², about 10 to about 500 μ g/m ², about 10 μ g/m ²to about 300 μ g/m ², about 10 μ g/m ²to about 200g/m ²about 1 μ g/m ²to about 200 μ g/m ².Described dosage can daily once, use weekly once, use weekly repeatedly but be less than daily once, monthly use repeatedly but be less than daily once, monthly use repeatedly but be less than and use weekly once, monthly to use once or interval uses to alleviate or palliate a disease symptom.Can continue to use at any disclosed interval, until tumour to be treated or cancer symptoms are alleviated.Realizing remission or after alleviating, can continuing to use, wherein extend this alleviation by using of this continuation or alleviate.

In one aspect, present invention also offers pharmaceutical preparation, it comprises at least one anti-CD 98 antibody of the present invention and/or its at least one immune conjugate and/or at least one anti-CD 98 antibody-drug conjugates of the present invention.In some embodiments, pharmaceutical preparation comprises 1) anti-CD 98 antibody and/or anti-CD 98 antibody-drug conjugates and/or its immune conjugate, and 2) pharmaceutically acceptable carrier.In some embodiments, pharmaceutical preparation comprises 1) anti-CD 98 antibody and/or its immune conjugate, and optionally, 2) agent of at least one other treatment.

By by have the antibody of required purity or antibody-drug conjugates and optional physiology can carrier, vehicle or stablizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. edit (1980)) be mixed with the pharmaceutical preparation comprising antibody of the present invention or immune conjugate or antibody-drug conjugates of the present invention of aqueous solution form for storing or lyophilized form or other dried forms.Preparation herein can also comprise the necessary more than a kind of active compound of concrete indication to be treated, and preferably, having of can not adversely affecting each other supplements active those.Such as, except anti-CD 98 antibody, can be desirably in a kind of preparation and comprise other antibody, such as, in conjunction with the second anti-CD 98 antibody of epi-position different on CD98 polypeptide, or for affecting some other targets of particular cancers growth, as the antibody of somatomedin.Alternatively or in addition, described composition can also comprise chemotherapeutics, cytotoxic reagent, cytokine, growth inhibitor, antihormone agent and/or heart protective agent.These molecules are effectively to measure intended purpose compatibly to combine existence.

Antibody of the present invention or immune conjugate can be mixed with any form being suitable for being delivered to target cell/tissue, such as, as microcapsule or huge emulsion (Remington's Pharmaceutical Sciences, the 16th edition, Osol, A. edit (1980); The people such as Park, Molecules 10:146-161 (2005); The people such as Malik, Curr.Drug.Deliv.4:141-151 (2007)); (people such as Maclean, Int.J.Oncol.11:235-332 (1997) as sustained release preparation (Putney and Burke, Nature Biotechnol.16:153-157, (1998)) or in liposome; Kontermann, Curr.Opin.Mol.Ther.8:39-45 (2006)).

methods for the treatment of

Antibody of the present invention or immune conjugate can use in external (such as), in vitro and interior therapeutic method.In one aspect, the invention provides in body or the method for vitro inhibition Growth of Cells or propagation, described method comprises cell is exposed to anti-CD 98 antibody or its immune conjugate under permission immune conjugate is bonded to the condition of CD98." cell growth inhibiting or propagation " represents the growth of cell or propagation is reduced at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, and comprises and cause necrocytosis.In some embodiments, described cell is tumour cell.In some embodiments, described cell is tumor of bladder, breast tumor, colon tumor, rectal neoplasm, gastric tumor, esophageal tumor, lung tumor, laryngeal neoplasm, tumor of kidney, mouth neoplasm, ovarian tumor or prostate tumor cells, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia cell.

In one aspect, antibody of the present invention or immune conjugate are used for the treatment of or prevent cell proliferative diseases, as cancer.In some embodiments, cell proliferative diseases is relevant with the expression that CD98 improves and/or activity.Such as, in some embodiments, described cell proliferative diseases is relevant with the expression of the raising of the CD98 on cancer cell surfaces.The example of the cell proliferative diseases of being treated by antibody of the present invention or immune conjugate includes, but is not limited to bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

In one aspect, the invention provides the method for the treatment of cell proliferative diseases, it comprises anti-CD 98 antibody from significant quantity to individuality or its immune conjugate of using.In some embodiments, the method being used for the treatment of cell proliferative diseases comprises the pharmaceutical preparation using significant quantity to individuality, described pharmaceutical preparation comprises anti-CD 98 antibody or anti-CD98 immune conjugate, and optionally at least one other treatment agent, as provided below those.In one embodiment, anti-CD 98 antibody or immune conjugate can by contacting to form antibody by described antibody or immune conjugate with CD98 or immune conjugate-antigenic compound carrys out the CD98 on target cancer cell, thus the cytotoxic agent that immune conjugate combines enters into cell interior.In one embodiment, in conjunction with antibody or immune conjugate internalization to express CD98 cancer cells in.

For therapeutic purpose, anti-CD 98 antibody or immune conjugate can be applied to people.In addition, anti-CD 98 antibody or immune conjugate can be applied to the non-human mammal of expressing CD98, and for animal doctor's object or as the animal model antibody of human disease and described non-human mammal cross reaction (such as, primates, pig, rat or mouse).For the latter, these animal models may be used for the treatment effect (such as, testing application dosage and time course) evaluating antibody of the present invention or immune conjugate.

Antibody of the present invention or immune conjugate can be used alone or are combined with other compositions in therapy.Such as, antibody of the present invention or immune conjugate can be used altogether with the agent of at least one other treatment and/or adjuvant.In some embodiments, other treatment agent is cytotoxic agent, chemotherapeutics or growth inhibitor.In some embodiments, chemotherapeutics is reagent or agent combination, as alkylating agent (such as, bendamustine hydrochloride, endoxan or ifosfamide), nucleoside analog (such as, fludarabine, cytosine arabinoside or gemcitabine), reflunomide (such as, prednisone, prednisolone or methylprednisolone), antimitotic agent (such as, taxol, docetaxel or vinorelbine), vinca alkaloids (such as, vincristine(VCR) or Etoposide), topoisomerase inhibitors (such as, irinotecan), microbiotic (such as, anthracycline or Dx), platinum analogs (such as, cis-platinum or carboplatin), therapeutic antibodies (such as, Rituximab) or reagent combination (such as, CHOP or CVP), wherein said combination treatment is useful in the treatment of cancer.In some embodiments, other compounds are the therapeutic antibodies (such as, Rituximab) except anti-CD 98 antibody.

These combination treatments above-mentioned comprise mixing and use (wherein two or more therapeutical agents are included in identical or independent preparation) and use separately, in this case, antibody of the present invention or immune conjugate use can before the using of other treatment agent and/or adjuvant, simultaneously and/or occur afterwards.Antibody of the present invention or immune conjugate can also use with other treatment scheme combination, and described other treatment scheme unrestrictedly comprises radiotherapy and/or marrow and peripheral blood and transplants.

Antibody of the present invention or immune conjugate (with any other therapeutical agent or adjuvant) can be used by any applicable method, it comprises parenteral, subcutaneous, intraperitoneal, lung interior and intranasal administration, and if need for topical therapeutic, also comprise in infringement and using.Parenteral infusions comprises intramuscular, intravenously, intra-arterial, intraperitoneal or subcutaneous administration.In addition, described antibody or immune conjugate are compatibly used by pulse infusion, the antibody of using dosage reduction particularly or immune conjugate.Part is used of short duration or long-term based on described, and dosage is used can by any applicable approach, such as, by injection, as intravenously or subcutaneous injection.

Antibody of the present invention or immune conjugate are prepared in the mode according to good medical practice, dose distribution and using.For consideration within this context, factor comprises the concrete illness that will treat, the concrete Mammals that will treat, the clinical setting of individual patient, causes other factors that the reason of illness, agent delivery site, application process, time of application table and practitioner are known.Described antibody or immune conjugate do not need, but optionally prepare together with current one or more reagent for preventing or treat discussed illness.The significant quantity of these other reagent depends on the amount of antibody or the immune conjugate existed in described preparation, illness or treatment type and other factors discussed above.These are usually with identical dosage by route of administration as described herein, or the dosage of as herein described about 1 to 99%, or any dosage determining to be applicable to experience/clinical and being used by any approach.

In order to prevent or disease therapy, antibody of the present invention or the suitable dosage of immune conjugate (when be used alone or with one or more other extra therapeutical agent, as chemotherapeutic agent combination use time) by depend on to treat the type of disease type, antibody or immune conjugate, the severity of disease and process, as described in antibody or immune conjugate be use for prevention or therapeutic purpose, the medical history of above-mentioned therapy, patient and to described antibody or the reaction of immune conjugate and the judgement of attending doctor.Described antibody or immune conjugate once or in a series for the treatment of are compatibly applied to patient.According to type and the seriousness of disease, no matter be that (such as) is used separately by one or many or pass through continuous infusion, the antibody of about 1 μ g/kg to 100mg/kg (such as, 0.1mg/kg-20mg/kg) or immune conjugate can be the initial candidate dosage for being applied to patient.According to above-mentioned factor, a typical per daily dose can in the scope of about 1 μ g/kg to 100mg/kg or more.For repeatedly using in several days or longer time, according to circumstances, described treatment will continue usually until the disease symptoms needed for occurring suppresses.An exemplary dose of described antibody or immune conjugate is by the scope of about 0.05mg/kg to about 10mg/kg.Therefore, antibody or the immune conjugate of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or their arbitrary combination) of one or more dosage can be used to patient.These dosage can intermittently be used, such as, weekly or every three weeks (such as, thus described patient accepts about 2 to about 20, or such as, the described antibody of about 6 dosage or immune conjugate).Can use initial higher loading dose, be then one or more lower dosage.Exemplary dose application program comprises the initial loading dose using about 4mg/kg, is then the maintenance dose weekly of about 2mg/kg antibody.But other dosages can be useful.By routine techniques and mensuration, easily monitor the development of this therapy.

diagnostic method and detection method

In one aspect, anti-CD 98 antibody of the present invention and immune conjugate are useful for the existence of CD98 in detection biological sample.As used herein, term " detection " comprises quantitatively or qualitative detection.In some embodiments, biological sample comprises cell or tissue.In some embodiments, these tissues comprise relative to its hetero-organization, the normal of CD98 and/or cancerous tissue is expressed with higher level, such as, bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

In one aspect, the invention provides the method for the existence detecting CD98 in biological sample.In some embodiments, described biological sample contacts with anti-CD 98 antibody under being included in the condition allowing anti-CD 98 antibody and CD98 to combine by described method, and detects between anti-CD 98 antibody and CD98 whether form mixture.

In one aspect, the invention provides diagnosis and express with CD98 the method raising relevant illness.In some embodiments, described method comprises and test cell being contacted with anti-CD 98 antibody; By detecting the CD98 expression level in conjunction with determination test cell (quantitative or qualitative) of anti-CD 98 antibody and CD98; With by the CD98 expression level of test cell and compared with control cells (such as, the cell of the normal cell of originating with test cell homologue or the horizontal expression CD98 suitable with this normal cell) CD98 expression level compared with, wherein compared with compared with control cells, the CD98 of test cell higher level shows that existence is expressed with CD98 and raises relevant illness.In some embodiments, as compared with normal cell, the expression of raising corresponds to the CD98 expression of higher density on tumor cell surface.In some embodiments, test cell derives from and suspects to suffer from the individuality raising relevant illness with the expression of CD98.In some embodiments, described illness is cell proliferative diseases, as cancer or tumour.The exemplary cells proliferative disorders that antibody of the present invention can be used to make a definite diagnosis comprises bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

In some embodiments, as previously discussed those, diagnosis or detection method comprise the CD98 that detects and anti-CD 98 antibody and cell surface are expressed or the combination with the CD98 derived from the film preparation of the cell of expressing CD98 in its surface.In some embodiments, cell contacts with anti-CD 98 antibody under being included in the condition allowing anti-CD 98 antibody and CD98 to combine by described method, and detects between the CD98 on anti-CD 98 antibody and cell surface whether form mixture.That " FACS " measures for detecting the exemplary mensuration of the combination of the CD98 that anti-CD 98 antibody and cell surface are expressed.

Some additive method may be used for the combination detecting anti-CD 98 antibody and CD98.These methods include, but is not limited to antigen well known in the art-combination and measure, as immunoblotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immune precipitation determination, fluorescence immunoassay, albumin A immunoassay and immunohistochemistry (IHC).

In some embodiments, marked anti-CD 98 antibody.Mark includes, but is not limited to mark or the part (as fluorescence, color development, electron density, chemoluminescence and radio-labeling) of direct-detection, and (such as) is by the part of enzymatic reaction or molecular interaction indirect detection, as enzyme or part.Exemplary indicia includes but not limited to radio isotope ³²p, ¹⁴c, ¹²⁵i, ³h and ¹³¹i, fluorophore is as Rare Earth Chelate or fluorescein and derivative thereof, rhodamine and derivative thereof, dansyl, Umbelliferone, luciferase, such as, fluorescence Luci and bacterial luciferase (U.S. Patent No. 4737456), luciferin, 2, 3-dihydro naphthyridine diketone, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase enzymes, glucoamylase, N,O-Diacetylmuramidase, carbohydrate oxidase, such as, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD), Heterocyclic oxidases is as uriKoxidase and XOD, it is combined with using the enzyme of hydrogen peroxide oxidation dyestuff former, as HRP, lactoperoxidase or micro-peroxidase, vitamin H/avidin, spin label, bacteriophage labels thing, stable free radical etc.

In some embodiments, anti-CD 98 antibody is fixed on insoluble matrix.Immobilization needs anti-CD 98 antibody to be separated with keeping free any CD98 in solution.This makes anti-CD 98 antibody insoluble along with before mensuration program usually, as by being adsorbed to water-fast matrix or the surface (people such as Bennich, U.S.3720760), or by covalent coupling (such as, use glutaraldehyde cross-linking), or make anti-CD 98 antibody insoluble after forming mixture by (such as) immunoprecipitation between anti-CD 98 antibody and CD98.

Immune conjugate of the present invention can be used to replace anti-CD 98 antibody or except anti-CD 98 antibody, use immune conjugate of the present invention carry out any above-mentioned embodiment diagnosed or detect.

measure

Physical/chemical and/or the biological activity of anti-CD 98 antibody of the present invention and immune conjugate can be identified by many measure as known in the art.

determination of activity

In one aspect, the mensuration differentiating there is bioactive anti-CD 98 antibody or its immune conjugate is provided.Biological activity can comprise the ability (such as, " cell kills " is active) of (such as) cell growth inhibiting or propagation, or the ability of inducing cell death, comprises apoptosis (apoptosis).To additionally provide in body and/or external to there is this bioactive antibody or immune conjugate.

In some embodiments, the ability of anti-CD 98 antibody or its immune conjugate vitro inhibition Growth of Cells or propagation is tested.The suppression of Growth of Cells or propagation measures to be known in the art.Some being measured illustrational cell proliferation by " cell kills " as herein described measures cell viability.A this mensuration is CellTiter-Glo ^tMphotogenic cell viability measures, and it is commercially available from Promega (Madison, WI).This mensuration is based on the number quantitatively determining viable cell in cultivation of existing ATP (it is the instruction of metabolic active cells).See the people such as Crouch (1993) J.Immunol.Meth.160:81-88, U.S. Patent No. 6602677.Described mensuration can be carried out in 96 or 384 well format, can carry out automatic high flux screening (HTS).See the people such as Cree (1995) Anticancer Drugs6:398-404.

It is that " MTT " measures that the another kind of cell proliferation measures, and it measures 3-(4,5-dimethylthiazole-2-base)-2,5-hexichol tetrazolium bromide are oxidized to first a ceremonial jade-ladle, used in libation colorimetric estimation by mitochondrial reductases.Picture CellTiter-Glo ^tMmeasure the same, this mensuration shows the number of the metabolic active cells existed in cell cultures.See, such as, people (2005) the Cancer Res.65:3877-3882 such as Mosmann (1983) J.Immunol.Meth.65:55-63 and Zhang.

In one aspect, the ability of the external death of anti-CD 98 antibody inducing cell is tested.The mensuration of inducing cell death is known in the art.In some embodiments, these measure the loss of measuring (such as) film integrality, indicated by the absorption by propidium iodide (PI), trypan blue (see the people such as Moore (1995) Cytotechnology, 17:1-11) or 7AAD.In exemplary PI absorption measurement, cultivate in the Eagle's medium (D-MEM) that cell is improved at Dulbecco: be supplemented with the FBS (Hyclone) of 10% heat inactivation and the Han Mushi F-12 (50:50) of 2mM L-glutaminate.Therefore, described mensuration is carried out when not having complement and immune effector cell.Cell is with 3 × 10 ⁶the density of/ware is inoculated and is made it adhere to and spends the night in 100 × 20mm culture dish.Removing substratum is also only replaced with the substratum of fresh culture or the antibody containing different concns or immune conjugate.By the cell culture time period of 3 days.After process, clean individual layer with PBS and be separated by trysinization.Then, with 1200rpm, cell is centrifugal 5 minutes at 4 DEG C, by particle settling flux in the cold Ca of 3ml ²⁺binding buffer liquid (10mM Hepes, pH7.4,140mM NaCl, 2.5mM CaCl ₂) and be distributed to 35mm and be stamped in 12 × 75mm pipe of filter screen (strainer-capped) (often pipe lml, each treatment group 3 is managed) to remove cell mass.Then, Xiang Guanzhong adds PI (10 μ g/ml).Use FACSCAN ^tMflow cytometer and FACSCONVERT ^tMcellQuest software (Becton Dickinson) analytic sample.Therefore, authenticated antibody or the immune conjugate of the necrocytosis as absorbed determined induction statistical significant level by PI.

In one aspect, the ability of anti-CD 98 antibody or immune conjugate vitro induction of apoptosis (apoptosis) is tested.The antibody of cell death inducing or the exemplary mensuration of immune conjugate are that annexin combines mensuration, such as, as described in the people such as Zhang (BioTechniques 23:525-531,1997).The antibody of cell death inducing or another exemplary mensuration of immune conjugate detect the histone DNA ELISA colorimetric estimation of degrading between the nucleosome of genomic dna.(such as) necrocytosis can be used to detect ELISA kit (Roche, Palo Alto, CA) and to carry out this mensuration.

The cell used in any above-mentioned external test comprises cell or the clone of natural expression CD98 or engineered expression CD98.These cells comprise the tumour cell of the normal cell process LAN CD98 relative to homologue source.These cells also comprise the clone (comprising tumor cell line) and abnormal expression CD98 of expressing CD98 but by the clone of nucleic acid transfection of the CD98 that encodes.

In one aspect, the ability of cell growth inhibiting or propagation in anti-CD 98 antibody or its immune conjugate body is tested.In some embodiments, the ability of anti-CD 98 antibody or its immune conjugate Tumor suppression tumor growth is tested.In vivo model system, as heteroplastic transplantation model may be used for these tests.In exemplary Xenograft system, human tumor cells is incorporated into the non-human animal of suitable immunocompromised host, such as, in SCID mouse.Antibody of the present invention or immune conjugate are applied to animal.Measure the ability of antibody or immune conjugate suppression or reduction tumor growth.In some embodiment of above-mentioned Xenograft system, human tumor cells is the tumour cell from people patient.These cells for the preparation of heteroplastic transplantation model unrestrictedly comprise expresses the cell of external source CD98 and the cell of natural expression CD98.In some embodiments, by subcutaneous injection or by being transplanted to applicable site (as mammary fat pad), human tumor cells is incorporated in the non-human animal of suitable immunocompromised host.

measure in conjunction with mensuration and other

In one aspect, the antigen-binding activity of anti-CD 98 antibody is tested.Such as, in some embodiments, the ability that anti-CD 98 antibody is bonded to external source or the endogenous CD98 expressed on cell surface is tested.FACS measures and may be used for these tests.

The monoclonal antibody of one group of anti-CD98 is divided into groups by the epi-position that can identify based on them, and the method is called epi-position frame also.Usual use competition assay carries out epi-position frame also, and it to have rated when there is another kind of antibody antibodies to the ability of antigen.In exemplary competition assay, to the solution of the second unmarked antibody of the ability of CD98, cultivate immobilization CD98 with described first antibody competition binding at the antibody and test comprising the first mark being bonded to CD98.Described second antibody may reside in doma supernatant.In contrast, do not comprise in the solution of described second unmarked antibody and cultivate immobilization CD98 at the antibody comprising described first mark.When allow described first antibody and CD98 in conjunction with cultivate after, remove excessive unconjugated antibody, and the amount of the measurement marker relevant with immobilization CD98.If the amount of marker relevant with immobilization CD98 relative to control sample in the test sample significantly reduces, so it shows that described second antibody and described first antibody competition binding are to CD98.In some embodiments, immobilization CD98 is present on cell surface or in the film preparation deriving from the cell of expressing CD98 in its surface.

Epi-position frame high throughput method also is also as known in the art.See, such as, the people such as Jia, J.Immunol.Methods 2004,288 (1-2): 91-98, which depict the multichannel competitive antibody frame method also for Identification of Monoclonal Antibodies; With the people such as Miller, J.Immunol.Methods 2011,365 (1-2): 118-25, which depict the epi-position frame of the mouse monoclonal antibody measured by multichannel pairing also.

Epitope mapping

" epitope mapping " differentiates the binding site of antibody on its target proteins antigen or the method for epi-position.Antibody epitope can be linear epitope or conformational epitope.Linear epitope is formed by the continuous sequence of Amino Acids in Proteins.Conformational epitope is amino acids formed by what be interrupted in protein sequence, but merges together when protein folding becomes during its three-dimensional arrangement.

On positioning target proteantigen, the multiple method of antibody epitope is well known in the art.These comprise sudden change method, pepscan method, displaying method, relate to mass spectrographic method and structure determines.

Fixed-point mutation method relates to target rite-directed mutagenesis, wherein replacing by introducing along protein sequence system, then determining that the impact that each replacement antagonist combines authenticated key amino acid.This can be undertaken by some other forms of amino-acid residue point mutation in " Alanine scanning mutagenesis " as described in Cunningham and Wells (1989) Science 244:1081-1085 or people CD98.But mutation research can also show the integral vertical body structure of CD98 to be crucial but not to participate in the amino-acid residue of antibody-antigene contact directly, and therefore additive method to confirming that the functional epitope using the method to determine can be required.

Air gun sudden change location utilizes target gene plasmid-mutated library widely, and in its Chinese library, each clone has unique amino acid mutation and whole library covers each amino acid in target proteins.The clone forming mutated library is arranged respectively in microwell plate, expresses in mammalian cell alive, and test the immunoreactivity with the antibody be concerned about.Be authenticated the amino acid of antagonist epi-position key by reactive loss, then locate to show epi-position on protein structure.By automatic analysis, new epi-position figure can be obtained within a couple of days to several weeks.Owing to it using the natural structure of mammalian cell internal protein, therefore described technology allows linear and conformational epitope structure to draw in complex proteins (people such as Paes, J.Am.Chem.Soc.131 (20): 6952-6954 (2009); Banik and Doranz, Genetic Engineering and Biotechnology News 3 (2): 25-28 (2010)).

The epi-position that pepscan method determination anti-CD 98 antibody combines can also be used.In pepscan, the short peptide sequence library tested from the overlapping fragments of target proteins matter CD98 combines the ability of the antibody be concerned about.As " pepscan " method (WO 84/03564; WO 93/09872) described in, synthesize described peptide, and such as use ELISA or BIACORE, or by any multiple solid-phase screening method (people such as Reineke, Curr.Opin.Biotechnol.12:59-64,2001) on chip, screen the combination of described peptide.These peptide screening methods may not detect some functional epitope be interrupted, and namely relate to the functional epitope of the amino-acid residue that the primary sequence along CD98 polypeptide chain does not adjoin.

The technology being called as CLIPS (the chemistry of peptides key on support) of latest developments may be used for locating conformational epitope.The loose end (loose end) of described peptide is fixed to synthesis support, thus described support peptide may can adopt the three-dimensional arrangement identical with corresponding sequence in intact proteins.CLIPS technology is used for linear peptides to be fixed to ring texture (" monocycle " form), and gather together the protein binding site of different piece (form such as " dicyclo ", " three rings "), thus create the conformational epitope (U.S. Patent No. 7972993) that can measure antibodies.

Display technique can also be used to be located through the epi-position of antibodies of the present invention, and described display technique comprises, and such as, phage display as above, Microorganism display and rrna/mRNA shows.In these methods, phage or cell surface illustrate peptide fragment library.Then, use selective binding to measure, locate epi-position by the mAb screening these fragments anti-.Developed some computational tools, its peptide making it possible to select based on the linear avidity using phage display to obtain predicts conformational epitope people such as (, Bioinformatics 23:3244-3246,2007) Mayrose.Method also can be used for detecting conformational epitope by phage display.Microorganism display system can also for expressing correct folding antigen fragment for discriminating conformational epitope (people such as Cochran, J.Immunol.Meth.287:147-158,2004 on cell surface; The people such as Rockberg, Nature Methods5:1039-1045,2008).

Relate to proteolysis and mass spectrographic method also may be used for determining antibody epitope (people such as Baerga-Ortiz, Protein Sci.2002June; 11 (6): 1300-1308).In limited proteolysis, when there is and do not exist described antibody by different proteolytic enzyme cutting antigen, and differentiate fragment by mass spectroscopy.Epi-position is once binding antibody, becomes protection by proteoclastic antigenic domains people such as (, Proc.Natl.Acad.Sci.USA 87:9848-9852,1990) Suckau.Other comprise based on proteoclastic method, such as, selective chemical modification (the people such as Fiedler, Bioconjugate Chemistry 1998,9 (2): 236-234,1998), the epitope excision (people such as Van de Water, Clin.Immunol.Immunopathol.1997,85 (3): 229-235,1997) and hydrogen-deuterium (H/D) exchange process (Flanagan of latest developments, N., Genetic Engineering and Biotechnology News3 (2): 25-28,2010).

The epi-position of antibodies of the present invention can also be determined by structural approach, as X-ray structure is determined (such as, WO 2005/044853), molecular model and nucleus magnetic resonance (NMR) spectroscopy, it to comprise when free and when be concerned about antibody in the composite in conjunction with time, the NMR of the H-D exchange rate of acylamino hydrogen active in IL-23R determines (people such as Zinn-Justin, (1992) Biochemistry31:11335-11347; The people such as Zinn-Justin, (1993) Biochemistry 32:6884-6891).

Can by (such as) anti-CD 98 antibody to the screening being bonded to described epi-position, by making animal immune with the peptide of the people CD98 fragment comprised containing epitope sequences, or by using phage display to select the antibody being bonded to epitope sequences can obtain other antibody being bonded to the epi-position identical with antibody of the present invention.The antibody that it is expected to be bonded to same functionality epi-position demonstrates similar biological activity, as blocked the biological activity of CD98, and can measure by antibody functional these activity confirmed.

Other determinations of activity

In one embodiment, anti-CD 98 antibody of the present invention suppresses the bioactive antagonist antibodies of CD98.Anti-CD 98 antibody of the present invention can be measured to determine whether they suppress the biological activity of CD98, such as, with the combination of light chain.In order to determine whether CD98 antibody of the present invention suppresses the combination with light chain, according to people such as Kim, Biochim.Biophys.Acta 1565:112-122, the method described in 2002, determines the ability of Amino Acid Absorption in CD98 antibody suppression cancerous cell line.

In one aspect, can also be carried out the anti-CD 98 antibody of purification Identification by a series of mensuration, described mensuration includes, but is not limited to N-terminal order-checking, amino acid analysis, non denatured size exclusion high pressure liquid phase chromatography (HPLC), mass spectroscopy, ion exchange chromatography and papain digestion.

In one embodiment, contemplated by the invention and there are some but the antibody of the change of not all effector function, this Half-life in vivo becoming antibody be wherein important but some effect because of function (as complement and ADCC) be candidate desired in unrequired or harmful multiple application.In some embodiments, the Fc measuring antibody is active in guarantee only to maintain required character.External and/or in vivo cytotoxicity can be introduced measure with the reduction/elimination confirming CDC and/or ADCC activity.Such as, Fc acceptor (FcR) can be introduced and combine mensuration to guarantee that described antibody lacks Fc γ R and combines (therefore probably lacking ADCC activity), but remain FcRn bonding properties.The example of the vitro test of the ADCC activity of the molecule that evaluation is concerned about is described in U.S. Patent No. 5500362 or 5821337.Useful effector cell is measured for these and comprises peripheral blood monouclear cell (PBMC) and natural killer (NK) cell.Alternatively or in addition, the ADCC that can evaluate be concerned about molecule is in vivo active, such as, in animal model, as people such as Clynes, disclosed in PNAS (USA) 95:652-656 (1998).Clq can also be carried out and combine mensuration to confirm that therefore antibody in conjunction with Clq, and can not lack CDC activity.In order to evaluate complement activation, can CDC mensuration be carried out, such as, as people such as Gazzano-Santoro, described in J.Immunol.Methods202:163 (1996).Method as known in the art can also be used to carry out the removing/transformation period in FcRn combination and body determine.

Although for making understanding, clearly object is by illustrations and example in detail foregoing invention, and described explanation and example should not be considered as limitation of the scope of the invention.The all patents quoted in this article and the disclosure of scientific literature are incorporated to as a reference with its full content clearly.

Embodiment

It is below the example of method and composition of the present invention.Should understand and the above general remark provided is provided, other embodiments multiple can be put into practice.

Embodiment 1: the qualification of CD98 on acute myeloid leukaemia (AML) tumor cell surface

Amount to 16 primary AML samples and derive from Fred Hutchinson DKFZ (FHCRC).Analyze 11 samples from healthy donors.In order to monitor the quality of each AML sample, carry out the paotoblastic Hematorylin eosin stains of AML.Only analyze the sample containing at least 75% tumour cell.In addition, 23 primary chronic Lymphocytic leukemia (CLL) samples deriving from Billings clinic or University of Florida (University of Florida) and 27 primary colorectal carcinoma (CRC) and 22 normal adjacent colon control samples deriving from cooperation people organization network (Cooperative Human Tissue Network) or national disease research exchanging meeting (the National Disease Research Interchange, NDRI) are analyzed.Optimize sample preparation with the cell viability during at utmost maintaining sample separation.Determine the optimum mark time of AML, CLL and CRC sample, thus make it possible to significant notation and can not cell integrity be damaged.

By the spectrotype (sTAg) of surface markers for the also quantitative rendered surface albumen set spectrum of identification of cell upper surface albumen set in 16 core AML samples, 5 myelomonocyte (BMMC) contrasts and 6 peripheral blood monouclear cell (PBMC) control samples, 20 core CLL samples, 27 CRC samples and 22 normal adjacent colonic specimen samples.The protein ectodomain that chemical labeling is relevant with the AML tumor cell membrane of complete primary tumor cell, then uses the protein that solid phase affinity resin is marked by chromatography enrichment.As described below, before mass spectroscopy by the albumen of wash-out-80 DEG C of storages.

Differentiate by the albumen of sTAg method enrichment and use high resolving power shotgun Liquid Chromatography-Tandem Mass Spectrometry (MS) quantitative.To the susceptibility of linear ion hydrazine be combined and rotate the mixing TherrnoFisher LTQ-Orbitrap Velos mass spectrograph (people such as Olsen of high resolving power that orbitrap mass analyzer provides and mass accuracy, Mol.Cell Proteomics 8:2759-2769, 2009) be coupled to and receive stream (nanoflow) liquid-chromatography apparatus, and for the proteomics from bottom to top based on shotgun, to determine (the people such as Yates of the identity of protein and quantitative abundance measurement in AML cell surface enrichment part, Annu.Rev.Biomed.Eng.11:49-79, 2009).By receiving stream liquid chromatography online, be separated the tryptic digestive juice of the surface protein of self enrichment according to hydrophobicity, simultaneously by quality and the fracture mode of mass spectrograph dynamically recording peptide.In order to determine the identity of peptides and proteins, be used in the original MS data of the SEQUEST algorithm process that the fast processing Sorcerer2 platform performs (people such as Lundgren, Curr.Protoc.Bioinformatics, 13rd chapter: Unit 13.3,2009), to determine to test fracture mode and human protein group determined best-fit coupling between those on silicon chip.Use the PeptideProphet (people such as Keller, Anal.Chem.74:5383-5392,2002) and the ProteinProphet (people such as Nesvizhskii, Anal.Chem.75:4646-4658,2003) Software tool statistically verifies that the coupling of gained is to guarantee minimum possible false discovery rate (FDR) and therefore comprise the protein only roughly differentiated in candidate compound.

Spectrum counting process is used to determine relative quantification level people such as (, Proteomics 11:535-553,2011) Neilson of the protein differentiated in sTAg sample.Spectrometer base is in empirical real example: the closely related (people such as Liu of the relative abundance of protein in the number of the distribution relevant with the peptide from often kind of protein (positive differentiate) spectrum and original stock, Anal.Chem.76:4193-4201,2004).The spectrum counting of the peptide differentiated derives from proteome analysis software platform, it comprises Scaffold (proteomics software) and ProteoIQ (NuSep), their display, sorting and filtered SEQUEST search mass-spectrometric data result.Original spectrum counting is converted into the per-cent normalized spectrum abundance factor (%NASF) value people such as (, J.Proteome Res.5:2339-2347,2006) Zybailov so that the difference of protein length difference and sample size to be described.Independent, the outside confirmatory using quantitative FACS to compose as the proteomics of the primary tumors cells surface expression based on sTAg mass spectroscopy is measured, and selected monoclonal antibody is used for verifying that proteomics is measured.

Use sTAg, the heterodimer II type transmembrane glycoprotein CD98 with the aminoacid sequence shown in SEQ ID NO:1 is differentiated as to exist with high-density on AML, CLL and CRC tumor cell surface.As shown in Figure 1, use sTAg, identified CD98 in 7 in 16 primary AML samples, its average %NSAF is 0.11, has identified CD98 in 20 in 20 primary CLL samples, and its average %NSAF is 0.15.Identified CD98 in 5 in 5 in 5 myelomonocyte (BMMC) samples and 6 peripheral blood monouclear cell (PBMC) samples, its average %NSAF is respectively 0.05 and 0.06.In addition, identified CD98 in 11 in 27 primary CRC samples, its average %NSAF is 0.10, and in 1 normal adjacent colonic specimen samples, only identified CD98, and its average %NSAF is less than 0.01.Based on this analysis, relative to corresponding normal control, CD98 is enriched on the signal portion of AML, CLL and CRC primary tumor sample in patient source in a large number.

Embodiment 2: the discriminating of CD98 in tumour

Anti-CD98 monoclonal antibody 8-34B (see embodiment 3) and isotype control Ab HB-121 is used to carry out antigen titration experiment to set up the dilution that will minimum background and peak signal caused to detect.Use based on the antigen retrieval (citrate buffer solution of pH6.0) of steam, formalin is fixed, the tissue of paraffin embedding (FFPE) or with 1:50,1:100,1:200 and 1:400, serial dilution is carried out to fresh freezing tissue.Thered is provided by Igenica and prepare the fixing compared with control cells system (F244, RM and F244-P) of freezing compared with control cells system (F244 and F244-P) and formalin by LifeSpan.Select the dilution of the 8-34B of 1:20 and 1:50 to carry out formalin to fix, the research of paraffin-embedded tissue, and the 8-34B selecting 1:400 to dilute carries out the research of fresh frozen tissue.

Main detection system is by carrier anti-mouse second antibody (BA-2000) and Vector ABC-AP kit (AK-5000) and form for generation of red substrate test kit (SK-5100) of carrier that red-purple precipitates.Also use positive control antibodies (for CD31 and vimentin) to tissue staining, to guarantee to maintain tissue antigen and it is spendable to immunohistochemical analysis.Only selecting CD31 and vimentin dyeing is the research that positive tissue is left.

In 15 routine malignant melanomas 10 example and 18 routine lung cancer in 4 examples in, with 1:20 and 1:50 dilution antibody 8-34B formalin is fixed, paraffin-embedded tissue demonstrates positive staining.In addition, with 1:400 dilution, in 2 examples in 6 examples of antibody 8-34B in 6 routine freezing lung cancer samples and in 2 routine freezing melanoma samples, positive staining is demonstrated.

Table 1: the frequency of lung cancer and the positives CD98 dyeing of melanoma

Cancer	Frequency
		Nonsmall-cell lung cancer	4/18 (FFPE); 6/6 (freezing)
Melanoma	10/15 (FFPE); 2/2 (freezing)

Embodiment 3: the preparation of anti-CD98 monoclonal antibody

Monoclonal antibody is prepared according to the general method described in " Antibodies A Laboratory Manual " (Harlow and Lane1988CSHPress).Male 129S6/SvEv mouse purchased from Taconic farm is used for immunity.With 10 ⁶individual's CD98 (huCD98) expressing tumor cell is by side, subcutaneous injection makes mouse immune.After immunity the 39th day, strengthen mouse immunes with 5,000,000 huCD98 expressing tumor cell intraperitoneal.At the 42nd day results spleen.As described in as in " Antibodies A Laboratory Manual " (Harlow and Lane1988CSH Press), use the method based on PEG to prepare single splenocyte and merge to set up hybridoma with CRL-2016 myeloma cell (ATCC).

Hybridoma is grown in 384 hole tissue culturing plates, and is screened the generation of the antibody identifying huCD98 in the supernatant liquor in each hole by ELISA.Then, positive hole is transferred in 48 orifice plates, increase and gather supernatant liquor for the huCD98 connection confirming by ELISA.By by single hybridoma bed board in the hole of 96 orifice plates, the single hybridoma producing anti-huCD98 antibody is established as the Unique clones of the generation monoclonal anti huCD98 antibody of confirmation.These Growth of Cells become colony, and by ELISA screening from the supernatant liquor of these single colonies to confirm to be bonded to the monoclonal antibody of huCD98.Clone hybridization knurl is expelled in the Balb/C mouse of isooctadecane process to produce ascites.Collect ascites and the general antibody purification code published according to Thermo Scientific (product description #21001), use Gammabind agarose (GE Healthcare product code 17-0885-01), albumin A IgG binding buffer liquid (Thermo Scientific parts number 21001) and IgG elution buffer (Thermo Scientific parts number 21004) carry out purifying.

Below show the heavy chain of antibody 8-34B and the nucleic acid of variable region of light chain and aminoacid sequence:

8-34B variable region of heavy chain

CAGGTGCAGCTGAAGGAGTCCGGCCCCGGCCTGGTGGCCCCCTCCCAGTCCCTGTCCATCACCTGCACCGTGTCCGGCTTCTCCCTGACCTCCTACGGCGTGCACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCCTGATCTGGGCCGGCGGCTCCATCAACTACAACTCCGCCCTGATGTCCCGCCTGTCCATCTCCAAGGACAACTCCAAGTCCCAGGTGTTCCTGAAGATGAACTCCCTGGAGACCGAGGACACCGCCATGTACTACTGCGCCCGCAAGGGCCACATGTACTCCTACGCCATGGACTACTGGGGCCAGGGCACCTCCGTGACCGTGTCCTCC(SEQ?ID?NO：3)

QVQLKESGPGLVAPSQSLSITCTVS gFSLTSYGVHwIRQPPGKGLEWLG lIWAGGSINYNSALMSrLSISKDNSKSQVFLKMNSLETEDTAMYYCAR kGHMYSYAMDYwGQGTSVTVSS (SEQ ID NO:4; CDR underscore represents).

8-34B variable region of light chain

GACATCGTGATGACCCAGTCCCCCTCCTCCCTGACCGTGACCGCCGGCGAGAAGGTGACCATGTCCTGCAAGTCCTCCCAGTCCCTGCTGAACTCCGGCAACCAGAAGACCTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCGAGTCCGGCGTGCCCGACCGCTTCACCGGCTCCGGCTCCGGCACCGAGTTCACCCTGACCATCTCCTCCGTGCAGGCCGAGGACCTGGCCGTGTACTACTGCCAGAACGACTACTCCTACCCCCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG(SEQ?ID?NO：5)

DIVMTQSPSSLTVTAGEKVTMSC kSSQSLLNSGNQKTYLTwYQQKPGQPPKLLIY wASTRESgVPDRFTGSGSGTEFTLTISSVQAEDLAVYYC q nDYSYPPWTfGGGTKLEIK (SEQ ID NO:6; CDR underscore represents).

Below show the heavy chain of antibody 18-2A2.2 and the nucleic acid of variable region of light chain and aminoacid sequence:

18-2A2.2 variable region of heavy chain

CAGGTGCAGCTGCAGCAGTCCGGCGCCGAGCTGGTGAAGCCCGGCGCCTCCGTGAAGCTGTCCTGCAAGGCCTCCGGCTACACCTTCACCTCCTACTACATGTACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCGTGATCAACCCCGGCTCCGGCATCACCAACTACAACGAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCTCCAACACCGCCTACATGCAGCTGTCCTCCCTGTCCTCCGACGACTCCGCCGTGTACTTCTGCTCCGGCTCCGCCAACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCGCC(SEQ?ID?NO：7)

QVQLQQSGAELVKPGASVKLSCKAS gYTFTSYYMYwVKQRPGQGLEWIG vINPGSGITNYNEKFKGkATLTADKSSNTAYMQLSSLSSDDSAVYFCSG sANWFAYwGQGTLVTVSA (SEQ ID NO:8; CDR underscore represents).

18-2A2.2 variable region of light chain

GACATCGTGATGTCCCAGTCCCCCTCCTCCCTGGCCGTGTCCGTGGGCGAGAAGGTGACCATGTCCTGCAAGTCCTCCCAGTCCCTGCTGTACTCCTCCAACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGTCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCGACTCCGGCGTGCCCGACCGCTTCACCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCGTGAAGGCCGAGGACCTGGCCGTGTACTACTGCCAGCGCTACTACGGCTACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG(SEQ?ID?NO：9)

DIVMSQSPSSLAVSVGEKVTMSCKSS qSLLYSSNQKNYLAwYQQKPGQSPKLLIY wASTRDSgVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC q rYYGYPWTfGGGTKLEIK (SEQ ID NO:10; CDR underscore represents).

Below show the heavy chain of antibody 18-2A7.1 and the nucleic acid of variable region of light chain and aminoacid sequence:

18-2A7.1 variable region of heavy chain

CAGGTGCAGCTGCAGCAGTCCGGCGCCGAGCTGGTGCGCCCCGGCACCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCAACGCCTTCACCAACTACCTGATCGAGTGGATCAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCGTGATCAACCCCGGCTCCGGCATCACCAACTACAACGAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCTCCAACACCGCCTACATGCAGCTGTCCTCCCTGTCCTCCGACGACTCCGCCGTGTACTTCTGCTCCGGCTCCGCCAACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCGCC(SEQ?ID?NO：11)

QVQLQQSGAELVRPGTSVKVSCKAS gNAFTNYLIEwIKQRPGQGLEWIG vINPGSGITNYNEKFKGkATLTADKSSNTAYMQLSSLSSDDSAVYFCSG sANWFAYwGQGTLVTVSA (SEQ ID NO:12; CDR underscore represents).

18-2A7.1 variable region of light chain

GACATCGTGATGTCCCAGTCCCCCTCCTCCCTGGCCGTGTCCGTGGGCGAGAAGGTGACCATGTCCTGCAAGTCCTCCCAGTCCCTGCTGTACTCCTCCAACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGTCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCGACTCCGGCGTGCCCGACCGCTTCACCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCGTGAAGGCCGAGGACCTGGCCGTGTACTACTGCCAGCGCTACTACGGCTACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG(SEQ?ID?NO：13)

DIVMSQSPSSLAVSVGEKVTMSCKSS qSLLYSSNQKNYLAwYQQKPGQSPKLLIY wASTRDSgVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC q rYYGYPWTfGGGTKLEIK (SEQ ID NO:14; CDR underscore represents)

Below show the heavy chain of antibody 1-47C and the nucleic acid of variable region of light chain and aminoacid sequence:

1-47C variable region of heavy chain

CAGGTGCAGTTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCACCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATGTGGACTAATGGAATCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGGAGGACACTACGGTAGTACCTCCTATGCTATGGACTTCTGGAGTCAAGGA(SEQ?ID?NO：30)

QVQLKESGPGLVAPSQSLSITCTVS gFSLTTYGVHwVRQPPGKGLEWLG vMWTNGITNYNSALMSrLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR gGHYGSTSYAMDFwSQG (SEQ ID NO:31; CDR underscore represents)

1-47C variable region of light chain

GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAACATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATACTGCAAAAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAATACTCCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATC(SEQ?ID?NO：32)

DIQMTQSPASLSASVGETVTITC rASGNIHNYLTwYQQKQGKSPQLLVY tAKTLADgVPSRFSGSGSGTQYSLKINSLQPEDFGSYYC qHFWNTP yTFgGGTKLEIK (SEQ ID NO:33; CDR underscore represents)

Below show the heavy chain of antibody 1-115A and the nucleic acid of variable region of light chain and aminoacid sequence:

1-115A variable region of heavy chain

CAGGTGCAGCTGGAGGAGTCAGGACCTGGCCTGGTGGCGACCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAACTGTGGTGTACACTGGGTTCGCCAGCCTCAAGGAAAGGGTCTAGAGTGGCTGGGAGTGATATGGCCTAATGGAATCACAATTTATAATTCGGGTCTCATGTCCAGACTGAGTATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAAAGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGGAGGACATTACGGTAGTAGCTCCTATGCTATGGACTACTGGAGTCAAGGA(SEQ?ID?NO：34)

QVQLEESGPGLVATSQSLSITCTVS gFSLTNCGVHwVRQPQGKGLEWLG vIWPNGITIYNSGLMSrLSISKDNSKSQVFLKKNSLQTDDTAMYYCAR gGHYGSSSYAMDYwSQG (SEQ ID NO:35; CDR underscore represents)

1-115A variable region of light chain

GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAACATGGTATCAGCAGAAACCGGGAAAATCTCCTCAACTCCTGGTCTATACTGCAAAAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAATACTCCTTACACATTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAAGC(SEQ?ID?NO：36)

DIQMTQSPASLSASVGETVTITC rASGNIHNYLTwYQQKPGKSPQLLVY tAKTLADgVPSRFSGSGSGTQYSLKINSLQPEDFGSYYC qHFWNTP yTfGGGTKLEIK (SEQ ID NO:37; CDR underscore represents)

Embodiment 4: the somatotype of monoclonal antibody and frame are also

By using the checking and appraising of isotypic specificity second antibody on ELISA purchased from Jackson Immunologicals (goat x IgG1 HRP-Product#115-035-206, goat x IgG2a HRP-Product#115-035-206, goat x IgG2b HRP-Product#115-035-207, goat x IgG3 HRP-Product#115-035-209) containing the antibody typing of single doma supernatant of antibody identifying huCD98.

Be at war with ELISA to set up competitive binding frame also.In the single hole of elisa plate, allow the single anti-huCD98 Isotype antibody containing doma supernatant in conjunction with huCD98.After 1 hour, wash-out hole also uses 4% paraformaldehyde to fix.Then, in the single hole of elisa plate, to allow containing the single anti-huCD98 Isotype antibody (different homotype) of doma supernatant huCD98 in conjunction with 1 hour.After cleaning, described hole and specific second antibody (Jackson Immunologicals, goat x IgG2a HRP-Product#115-035-206) are cultivated and used Supersignal ELISA Pico chemical luminous substrate (Thermo Scientific-Product#37069) to detect.The single IgG2a Isotype antibody that can combine when there is IgG1 is considered to be in the distinct epitopes frame of specific IgG1.The single IgG2a Isotype antibody that can not combine when there is IgG1 is considered to be in the epi-position frame identical with specific IgG1.By this way, multiple epi-position frame is defined to huCD98 binding antibody, as shown in Figure 2.

Embodiment 5: binding affinity

By the people such as Carderelli (2002) Cancer Immunol Immunother 51; The general method that 15-24 delivers, tests the avidity of the anti-CD98 monoclonal antibody of purifying.Briefly, cultivated from the anti-CD98 monoclonal antibody of different amount by CD98 express cell and spend the night, the second antibody (Jackson Immunologicals) then using Goat anti human's Fc specificity or anti-mouse Fc specificity PE to combine is evaluated by FACS.Use Graphpad Prism software analysis FACS data, and use Graphpad Prism Kd computational tool to determine Kd.

Embodiment 6: the generation of chimeric antibody and qualification

Use Qiagen RNeasy mini kit (products catalogue No.74104), then use Qiagen OneStep RT-PCR kit (products catalogue No.210210), from the hybridoma producing anti-CD98 monoclonal antibody, extract total serum IgE.Use and RT-PCR is carried out to mouse heavy chain and the special primer sets of sequence of light chain.To each RNA sample, set up 12 single heavy chains by using the degeneracy forward primer mixture of leader sequence covering mouse variable region and 11 light chain RT-PCR react.Reverse primer is arranged in the constant region of mouse heavy chain and light chain.The RT-PCR product reacted from the first round is taken turns in PCR second and increases further.Half nested primers using antagonist variable region special sets up vertical 12 single heavy chains and 11 light chain RT-PCR react.In the enterprising performing PCR reaction of sepharose, and cut heavy chain and light chain PCR primer from described gel, and be cloned in sequencing vector.To the cloning and sequencing of the 10-20 in each hybridoma to determine the variable region of anti-CD98 monoclonal antibody.Then, same for variable region of heavy chain frame (in-frame) is cloned in the carrier containing human IgG1's light chain constant region sequence, and same for variable region of light chain frame (in-frame) is cloned in the carrier containing human kappa light chain constant-region sequences.Produce chimeric antibody from the transient transfection of HEK293 Freestyle cell, and use the method purifying described in embodiment 3.

As described in Example 4, the binding analysis to mouse monoclonal anti-CD 98 antibody is carried out to the chimeric antibody of purifying.Fig. 3 A shows the competitive binding assay result of 8-34B, 18-2A2.1,18-2A2.2 and 18-2A7.1 chimeric antibody.As shown in Figure 2, and " homotype " is the contrast IgG2a antibody not being bonded to CD98 to reference antibody 1-4.Result shown in Fig. 3 A shows that chimeric antibody remains the epitope binding specificity of the murine antibody that they are originated.Colon carcinoma cell line DLD1 is used to determine the binding affinity of mouse and inosculating antibody CD98 monoclonal antibody by facs analysis as described in Example 5.Show Kd value (scope is at 0.9nM to 4.5nM) in figure 3b, show that all these recombinant antibodies remain the high affinity that be bonded to CD98 suitable with they parent murine antibody.As described in example 5 above, the clone of three kinds of AML primary tumor samples and expression cynomolgus monkey CD98 (cynCD98) is also used to carry out facs analysis to the chimeric mAb of purifying.Result shown in Fig. 3 C shows that all chimeric antibodies remain the combination to people CD98 on AML cell and cynCD98.

By the binding affinity (people such as Lipschultz, Methods 20:310-318, summarizes in 2000) using the combination/dissociation rate determined value of BIACORE system to determine mouse and inosculating antibody CD98 monoclonal antibody.As shown in table 2, the avidity of chimeric antibody is similar to the avidity of parent murine antibody.Also show the data (see embodiment 7) of humanized antibody 8-34B H2 L1.

Table 2

Embodiment 7: the preparation of humanized antibody

By the CDR of mouse heavy chain and light variable domains being transplanted to the humanization form having prepared mouse monoclonal antibody 8-34B in people acceptor framework as shown in Figure 4.Humanization 8-34B light variable domains L1 (SEQ ID NO:15) is constructed by the CDR of mouse light chain being transplanted to people's receptor sequence (GenBank accession number No.ACJ71709.1).By constructing 8-34B humanization light variable domains L2 (SEQ ID NO:16) with two residues (S63T and the D70E amino-acid substitution of being numbered by Kabat, see Fig. 4 A) in mouse monoclonal antibody corresponding residue substitutions people acceptor light chain FR3.8-34B humanized heavy chain H1 (SEQ ID NO:17) is constructed by the CDR of mouse heavy chain being transplanted to people's receptor sequence (GenBank accession number No.137782).By constructing 8-34B humanized heavy chain variable domains H2 (SEQ ID NO:18) with 1 residue in mouse monoclonal antibody corresponding residue substitutions people acceptor heavy chain FR2 and 2 residues in FR3 (thus result in the amino-acid substitution of I48L, V71K and the F78V according to Kabat numbering, see Fig. 4 B).8-34B humanized heavy chain variable domains H3 (SEQ ID NO:19) with the addition of two extra replacements to mouse residue in FR3, V67L and T73N, as shown in Figure 4 B.

Below show the H2 heavy chain of humanized antibody 8-34B and the nucleic acid of L1 variable region of light chain and aminoacid sequence:

Humanization 8-34B variable region of heavy chain H2

CAGGTGCAGCTGCAGGAGTCCGGCCCCGGCCTGGTGAAGCCCTCCGAGACCCTGTCCCTGACCTGCACCGTGTCCGGCTTCTCCCTGACCTCCTACGGCGTGCACTGGATCCGCCAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCCTGATCTGGGCCGGCGGCTCCATCAACTACAACTCCGCCCTGATGTCCCGCGTGACCATCTCCAAGGACACCTCCAAGAACCAGGTGTCCCTGAAGCTGTCCTCCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCAAGGGCCACATGTACTCCTACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ?ID?NO：25)

QVQLQESGPGLVKPSETLSLTCTVS gFSLTSYGVHwIRQPPGKGLEWLG lIWAGGSINYNSALMSrVTISKDTSKNQVSLKLSSVTAADTAVYYCAR kGHMYSYAMDYwGQGTLVTVSS (SEQ ID NO:18; CDR underscore represents)

Humanization 8-34B variable region of light chain L1

GACATCGTGATGACCCAGTCCCCCGACTCCCTGGCCGTGTCCCTGGGCGAGCGCGCCACCATCAACTGCAAGTCCTCCCAGTCCCTGCTGAACTCCGGCAACCAGAAGACCTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCGAGTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAACGACTACTCCTACCCCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG(SEQ?ID?NO:24)

DIVMTQSPDSLAVSLGERATINC kSSQSLLNSGNQKTYLtWYQQKPGQPPKLLIY wASTRESgVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC qN dYSYPPWTfGQGTKVEIK (SEQ ID NO:15; CDR underscore represents)

By being transplanted to from the mouse heavy chain of chimeric antibody 18-2A7.1 and the CDR of light variable domains the humanization form having prepared mouse monoclonal antibody 18-2A in people acceptor framework.Humanization 18-2A7.1 light variable domains L1 (SEQ ID NO:20) is constructed by the CDR of mouse light chain being transplanted to people's receptor sequence (GenBank accession number No.ACJ71709.1).By constructing 18-2A7.1 light variable domains L2 (SEQ ID NO:21) with some the people's Framework residues of corresponding residue substitutions from mouse monoclonal antibody.18-2A7.1 humanized heavy chain variable domains H1 (SEQ ID NO:22) is constructed by the CDR of mouse heavy chain being transplanted to people's receptor sequence.By constructing 18-2A7.1 humanized heavy chain variable domains H2 (SEQ ID NO:23) with some the people's Framework residues of corresponding residue substitutions from mouse monoclonal antibody.

Below show the heavy chain of humanized antibody 18-2A7.1 and the nucleic acid of variable region of light chain and aminoacid sequence:

Humanization 18-2A7.1 variable region of heavy chain H1

CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCAACGCCTTCACCAACTACCTGATCGAGTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCGTGATCAACCCCGGCTCCGGCATCACCAACTACAACGAGAAGTTCAAGGGCAAGGCCACCATCACCGCCGACAAGTCCACCTCCACCGCCTACATGGAGCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTACTGCTCCGGCTCCGCCAACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ?ID?NO：26)

QVQLVQSGAEVKKPGSSVKVSCKAS gNAFTNYLIEwVRQAPGQGLEWMG vINPGSGITNYNEKFKGkATITADKSTSTAYMELSSLRSEDTAVYYCSG sANWFAYwGQGTLVTVSS (SEQ ID NO:22; CDR underscore represents)

Humanization 18-2A7.1 variable region of light chain L1

GACATCGTGATGACCCAGTCCCCCGACTCCCTGGCCGTGTCCCTGGGCGAGCGCGCCACCATCAACTGCAAGTCCTCCCAGTCCCTGCTGTACTCCTCCAACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCGACTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCGCTACTACGGCTACCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG(SEQ?ID?NO：27)

DIVMTQSPDSLAVSLGERATINCKSS qSLLYSSNQKNYLAwYQQKPGQPPKLLIY wASTRDSgVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC qR yYGYPWTfGGGTKVEIK (SEQ ID NO:20; CDR underscore represents)

Humanization 18-2A7.1 variable region of heavy chain H2

CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCAACGCCTTCACCAACTACCTGATCGAGTGGATCCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGTGATCAACCCCGGCTCCGGCATCACCAACTACAACGAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCACCTCCACCGCCTACATGGAGCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTACTGCTCCGGCTCCGCCAACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ?ID?NO：28)

QVQLVQSGAEVKKPGSSVKVSCKAS gNAFTNYLIEwIRQAPGQGLEWIG vINPGSGITNYNEKFKGkATLTADKSTSTAYMELSSLRSEDTAVYYCSG sANWFAYwGQGTLVTVSS (SEQ ID NO:23; CDR underscore represents)

Humanization 18-2A7.1 variable region of light chain L2

GACATCGTGATGACCCAGTCCCCCGACTCCCTGGCCGTGTCCCTGGGCGAGCGCGCCACCATCAACTGCAAGTCCTCCCAGTCCCTGCTGTACTCCTCCAACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCGACTCCGGCGTGCCCGACCGCTTCACCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCGCTACTACGGCTACCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG(SEQ?ID?NO：29)

DIVMTQSPDSLAVSLGERATINCKSS qSLLYSSNQKNYLAwYQQKPGQPPKLLIY wASTRDSgVPDRFTGSGSGTDFTLTISSLQAEDVAVYYC qR yYGYPWTfGGGTKVEIK (SEQ ID NO:21; CDR underscore represents)

Embodiment 8: the in-vivo tumour of anti-CD-98 mediated monoclonal antibody suppresses

Such as, have studied the Antibody Efficacy that growth and metastasis of tumours is formed in or normotopia cancer xenograft models subcutaneous mouse.Described antibody can be uncombined, maybe can be bonded to therapeutical agent, as understood in the art.

As described in Example 1, create the monoclonal antibody of anti-CD-98, and carry out Purification and Characterization as mentioned above.Chimeric or humanized antibody as above can also be used.Prepare therapeutic monoclonal antibodies or comprised the mixed antibody of mixture of each monoclonal antibody, and use it for the treatment of the mouse accepting subcutaneous or the injection of normotopia tumor xenograft.

By at female SCID or nu ^±the right side of mouse is injected at PBS (not containing magnesium or calcium) and BDMatrigel (BD Biosciences) with 1 × 10 in the mixture of the ratio of 1:1 ⁷individual cancer cells produces Subcutaneous tumor.The cumulative volume of every injected in mice is 200ml, and wherein 50% is Matrigel (BD Biosciences).Once tumour reaches 65-200mm ³between size, then by mouse random packet.Administration of antibodies weekly, and measure once with twice body weight and tumour respectively weekly.As described in, calculate gross tumor volume people such as (, (2009) Neoplasia 11:355-364) van der Horst.As negative control, to mouse IgG or the PBS of injected in mice purifying; Or identify the monoclonal antibody of the purifying of the antigen except CD98.

Embodiment 9:CD-98 monoclonal antibody is on the impact of B cell lymphoma xenograft growth in mouse

Ramos (B cell lymphoma) clone derives from ATCC and cultivates according to the code of supplier.Animal derives from Charles River laboratory.

Under CB.17 background, at 1 × 107 viable cell of right side subcutaneous injection in the PBS (without magnesium or calcium) of 1:1 ratio and the mixture of BD Matrigel (BD Biosciences) of all large immunodeficiency type SCID female mices of 4-6.The cumulative volume of every injected in mice is 200 μ l, and wherein 50% is Matrigel (BD Biosciences).Once tumour reaches 65-200mm ³between size, then by mouse random packet.Administration of antibodies weekly, and measure once with twice body weight and tumour respectively weekly.As described in, calculate gross tumor volume people such as (, (2009) Neoplasia 11:355-364) van der Horst.All experiments are carried out in the group of each experimental point at least 7 animals.Experimentation on animals is carried out according to Igenica Inc. institutional review board-the care of animal and the code using committee member club to ratify.

Use Graphpad Prism software package and apply the significance,statistical between the two tail t inspection computing group of Xue Shengshi and control group.The p value being less than 0.05 is considered to significant.Calculate doubling time and evolution time analysis, as people such as Daniel, described in (2007) Blood 110:4037-4046.

By Rituximab (anti-CD20 antibodies) as positive treatment control antibody.Antibody HB121 is IgG2a negative control.Rituximab and anti-CD 98 antibody 8-93A and 18-3A are IgG1 antibody, and every other anti-CD 98 antibody is IgG2a antibody.

Show induction of strong Tumor growth inhibition in B cell lymphoma (Ramos) tumour set up with anti-CD 98 antibody process.It should be noted that anti-CD 98 antibody process is better than Rituximab (see, Fig. 5 A, Fig. 5 B) in induced tumor growth-inhibiting.

Anti-CD 98 antibody also shows the development time of the handled RAMOS tumour of significant prolongation.As mentioned above, calculate the tumour doubling time of the tumor regrowth long data from Fig. 5 A-C, and use it for and predict development time (TTP) further.Then, to every animal extrapolation TTP in treatment group, until will 2000mm be reached ³, and as Kaplan-Meier curve tracing, as shown in figures 6 a-c.As shown in Fig. 6 A-Fig. 6 C, multiple anti-CD 98 antibody extends in development time and is better than Rituximab in Ramos tumour.

In order to evaluate the possible maximum therapy effect of anti-CD 98 antibody, increase the starting tumor volume of set up Ramos tumour.Table 3 shows the treatment effect of anti-CD 98 antibody in the Ramos Xenograft Tumor Models increased in the initial size of gross tumor volume.Can find out, even if the gross tumor volume increased, anti-CD 98 antibody remains Tumor growth inhibition (TGI).

Table 3

Embodiment 10: the impact that anti-CD-98 monoclonal antibody suppresses tumor growth in vivo

Use the code described in embodiment 7, in several heteroplastic transplantation model, test the impact of anti-CD98 monoclonal antibody 8-34B and 18-2A.

DLD-1 (colorectal carcinoma), A549 (nonsmall-cell lung cancer), Ramos (B cell lymphoma) and OCI-AML-3 (acute myelocytic leukemia) clone derive from ATCC, and cultivate according to the code of supplier.Animal derives from Charles River laboratory.Under CB.17 background, the immunodeficiency type NOD.SCID female mice in all for 4-6 ages is used for sarcoma tumor model, under CB.17 background, the immunodeficiency type SCID female mice in all for 4-6 ages is used for Ramos and DLD-1 tumor model, and by the immunodeficiency type nu in 4-6 age in week ^±female mice is used for A549 and OCI-AML-3 tumor model.By Rituximab (IgG1 anti-CD20 antibodies) or Erbitux (IgG1 anti-egfr antibodies) as positive control antibodies, and the IgG2a antibody for incoherent antigen is used as negative control.DC101 is rat anti-mouse VEGFR2/KDR IgG1 mAb (ATCC No.HB-11534) and is used as positive control.Carry out as described in Example 9 injecting, antibody treatment and statistical computation.

As Figure 6-9, anti-CD98 monoclonal antibody 18-2A is effective inhibitor of tumor growth in colorectal carcinoma (DLD-1), nonsmall-cell lung cancer (A549), burkitt's lymphoma (Ramos) and AML (OCI-AML-3) xenotransplantation.The effect of 18-2A is advantageously compared with the effect of Erbitux (Fig. 9 with Figure 10) with Rituxan (Fig. 7).

Anti-CD98 monoclonal antibody 8-34B suppresses the growth of tumour in Ramos and AML (OCI-AML-3) xenotransplantation, as shown in Figure 7 and Figure 8.

Embodiment 11: there is the anti-CD98 monoclonal antibody in the mouse system of different immune deficiency background

Use different mouse pedigrees, in RAMOS heteroplastic transplantation model as described in Example 9, test three kinds of anti-CD98 monoclonal antibody 8-300B, 8-34B and 18-2A.IgG2a antibody is used as negative control.Three kinds of immune-deficient mice pedigrees are less to height immune impairment.SCID mouse lacks functional B and T cell, but remains natural killer (NK) cell function and some complement functions.NOD.SCID mouse lacks complement function and only has part NK function, and NSG (NOD/SCID/gamma) mouse lacks NK function.

As shown in figure 11, when evaluating in the mouse system that immunocompetence is stronger, in RAMOS heteroplastic transplantation model, the Tumor growth inhibition effect of anti-CD 98 antibody raises, and this shows that the anti-tumor in vivo activity of anti-CD 98 antibody is that the combination suppressed due to immunological effect subfunction and CD98 activity causes.The result lacked in the NSG mouse of whole ADCC and CDC function shows that anti-CD 98 antibody can also suppress the biological function of CD98 in RAMOS xenotransplantation, and CD98 for RAMOS tumour tumor growth and/or maintain may be crucial.

Embodiment 12: effect in the body of mouse and inosculating antibody CD98 monoclonal antibody

In RAMOS heteroplastic transplantation model, by comparing of effect in the body of mouse anti-CD 98 antibody 8-34B and 18-2A and chimeric antibody 8-34B-ch and 18-2A-ch7.1, as described in example 9 above.Antibody is tested at 0.5mg/kg dosage (about 30nm).Results verification shown in Figure 12 is fitted together to anti-CD 98 antibody with Tumor suppression growth in the effective gonosome being similar to their parent mouse counterparts.

Embodiment 13: the epitope mapping of Humanized monoclonal antibodies IGN523

materials and methods

Reagent.By FREESTYLE ^tMcHO-S cell (Invitrogen) maintains and is supplemented with GlutaMAX ^tMfREESTYLE ^tMcHO expresses in substratum (Invitrogen).Control antibodies 4F2 and MEM108 derives from Santa Cruz Biotechnology and Thermo Scientific respectively.Use suitable Zenon antibody labeling test kit (Invitrogen), with Alexa Fluor 647 traget antibody.

Plasmid construction.Build total length CD98, CD98 Point mutont and mouse and people CD98 mosaic by gene chemical synthesis (GeneWiz) and be cloned in pCDNA3.1 carrier (Invitrogen).Build CD98ECD, CD98ECD Point mutont and CD98ECD mosaic by gene chemical synthesis and be cloned in pDisplay carrier (Invitrogen).Whole construct is confirmed by DNA sequencing.

The fluorescence-activated cell sorting (FACS) being bonded to the IGN523 of CD98 is analyzed.By using electroporation transfection CD98 mosaic, CD98 point mutation construct and people's wild type control construct of Nucleofector 4D unit (Lonza).Mixed with Transfection solution by construct, then transient transfection is to FREESTYLE ^tMin CHO-S cell.The CHO-S cell of 24 hours results transfection after transfection.To cell quantification, then use IGN523, MEM108 or 4F2 antibody staining.Before dyeing, use suitable Zenon antibody labeling test kit (Invitrogen), with Alexa Fluor 647 traget antibody.Miltenyi MACSQuant analyser (Miltenyi Biotec) gathers flow-data, and uses FlowJo software 9.5.3 version (Tree Star, Inc.) to carry out data analysis.

determine the land of IGN523 on CD98

In order to determine the region participating in the CD98 that IGN523 combines, replace about 40 of people CD98 adjacent amino acid whose regions (SEQ ID NO:1) with the respective regions (SEQ ID NO:96) of mouse CD98, and use facs analysis to monitor these to replace the impact combined the entirety of IGN523.Figure 13 shows displacement and defines 13 mouse-people CD98 chimeric constructs to the mouse sequence region in described human sequence.Figure 14 shows the combination of IGN523 and control antibodies and mouse CD98 (Mu), people CD98 (Hu) and 13 mouse-human chimera.Result shows that displacement is that IGN523 is bonded to needed for CD98 to the region in chimeric constructs 10.On the contrary, can find out that the region that responsible control antibodies combines is present in displacement in the region in mosaic 11 and 12.

Figure 15 shows the sequence in the people CD98 district that wherein IGN523 combines, as use mouse-people CD98 chimeric constructs differentiated, and the position of this sequence in the three-dimensional arrangement of CD98.The region that mosaic 10 limits is made up of the amino-acid residue T358-N405 of people CD98.Amino acid T358-G368 (underscore) to be embedded in crystalline structure and can not to be the part of bonding interface.The non-conservation residue between people and mouse sequence is shown with overstriking.Compared with people, the displacement of D391 site N result in extra glycosylation site in mouse sequence.

there is in CD98 the Fine Mapping of the IGN523 of single or multiple sudden change.

In order to define IGN523 epi-position further, the change of single or multiple amino acid is incorporated in the region that mouse-human chimera 10 defines.Prepared 4 constructs, the non-homogeneous residue from a part of mouse CD98 sequence is incorporated in people CD98 sequence by each construct.Construct 4.1 is containing sudden change 1371L, D374Q, A375G and A376 disappearance.Construct 4.2 is containing sudden change M383A and E384.Construct 4.3 is containing sudden change D391N, F395I, P396F and D397H.Construct 4.4 is containing sudden change G400R, A401P and A404L.IGN523 is prevented to be bonded to CD98 (Figure 16) with contained sudden change in the facs analysis display construct 4.1 of the Chinese hamster ovary celI of each construct transfection.Sudden change contained in construct 4.4 also have impact on the combination (Figure 16) of IGN523 and CD98 to a certain extent.

In order to determine which hydrophobic residue take part in the bonding interface of IGN523 and people CD98, by replacing with highly charged amino acid (as aspartic acid or l-asparagine) the single mutated constructs that these hydrophobic residue create hydrophobic residue in target ring region.By the transfection of single mutated constructs difference in Chinese hamster ovary celI, and by facs analysis to determine the combination with IGN523.As shown in figure 17, hydrophobic residue A377 and L378 negatively have impact on IGN523 combination to the sudden change of Charged acids.In less degree, the sudden change of residue I398 and A401 shows IGN523 in conjunction with similar negative influence.

Other constructs prepared containing multiple sudden change are bonded to other important residues of CD98 with discriminating to IGN523.As shown in figure 18, the multiple sudden changes (D374Q, D397H, G400R and A401P) in construct M1 entirely prevented IGN523 and combine, and this demonstrate the meaning of these residues.Construct M2 (D374E and A375E) and M3 (D397S and I398T) also causes combining reduction.The existence using other experiment (not shown)s of the construct comprising A376 disappearance to show this residue seemingly epi-position ring region is correctly folding required.

Based on using mouse-human chimera, hydrophobic residue sudden change and the binding of multiple sudden change, determine that following amino acid is a part for IGN523 epi-position: D374, A377, L378, D397, I398, G400 and A401.

pepscan is analyzed

As these mutation researches supplement, use pepscan analysis (PepScan) analyze further IGN523 in conjunction with epi-position.

The synthesis of peptide: in order to rebuild the epi-position that target molecules is interrupted, uses chemical connection peptides (CLIPS) patented technology (Pepscan) on the skeleton of Pepscan to synthesize structure peptide library.Substantially the chemical bond carrying out peptide on skeleton as follows: cysteine side chain multiple in peptide is coupled to one or two CLIPS template.The solution of two for 0.5mM T2 CLIPS template 1,3-(brooethyl) benzene is dissolved in bicarbonate of ammonia (20mM, pH7.9)/acetonitrile (1:1 (v/v)).This solution is joined in peptide array, thus causes two cysteine side chain existing in the solid phase binding peptide of CLIPS template and peptide array (455 orifice plates have 3 μ l holes) to combine.When covering completely in the solution, vibrate peptide array 30-60 minute in the solution gently.Finally, with excessive H ₂o thoroughly cleans peptide array, and 70 DEG C of supersound process 30 minutes in the lysis buffer containing the solution of 1%SDS/0.1% beta-mercaptoethanol in PBS (pH7.2), then at H ²supersound process 45 minutes again in O.In addition see people such as Timmerman, (2007), J.Mol.Recognit.20:283-99; With people such as Slootstra, (1996), the method described in Molecular Diversity1:87-96.

ELISA screens: the combination testing antibody and often kind of synthetic peptide in based on the ELISA of PEPSCAN.Peptide array is cultivated together with first antibody solution (spending the night at 4 DEG C).After cleaning, cultivate 1 hour together 25 DEG C of antibody peroxidase binding substancess that peptide array and 1/1000 are diluted (SBA, catalog number No.2010-05).After cleaning, add the 3%H of peroxidase substrate 2,2'-azino-two-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μ l/ml ₂o ₂.After 1 hour, measure colour developing.With charge coupled device (CCD)-photographic camera and image processing system, colour developing is carried out quantitatively.

Data processing: the value deriving from CCD camera, in the scope of 0-3000mAU, is similar to standard 96 orifice plate ELISA microplate reader.To result quantitatively and be stored in Peplab database.Occasionally, bubble is contained in hole, lead to errors on the occasion of.Manual examination (check) card, any worth caused by bubble is divided into 0.

Synthesis quality control: in order to verify the quality of synthetic peptide, the positive that parallel projects is independent and negative control peptide group.Screen these (people such as Posthumus, J.Virology, 1990,64:3304-3309) with antibody 57.9.

The result of variable-length peptide screening is shown in Figure 19.The ELISA result of often kind of peptide shows as sea line.The starting point of described line and terminal represent which residue is included in described peptide, and the Y value of described line shows the ELISA result obtained this peptide.It is right that the ELISA result of peptide shows ³⁹⁵fPDIPGA ⁴⁰¹the main combination of (SEQ ID NO:42) and right ³⁷⁹pGQP ³⁸²the secondary combination of (SEQ ID NO:43).Global analysis's display of 29 single position alanine substitution groups is right ³⁹⁴sFDIPGAVASANMTV ⁴⁰⁷the combination of (SEQ ID NO:44) is the strongest.Figure 20 shows the combination of the single position alanine substitution peptide group of best combination, and wherein each residue of peptide SEQ ID NO:44 is by alanine substitution, if or Original Residue be L-Ala, then replaced by glycine.Analyze the display dependency the strongest to residue F395, P396, D397 and I398, it seems to define the core of this epi-position.

Then, by CLIPS conformation matrix structure (conformational matrix structures) for the peptide in differentiate in peptide screening two regions is measured the combination of antibody to conformational epitope to combining.Figure 21 shows the thermal map of the data that representative obtains from CLIPS conformation matrix structure two portions sequence of people CD98 merged.Result demonstrates the high dependency to secondary structure.To inciting somebody to action ³⁹⁵fPDIPGAVSAN ⁴⁰⁵(SEQ ID NO:70) and ³⁷²gLDAAALPGQP ³⁸²the peptide that (SEQ ID NO:50) combines observed best combination.These two kinds of peptides are used as the basis of screen mutation, as shown in figure 22.SEQ1 shows peptide sequence, and DIF1 shows the position suddenlyd change in peptide.Gray area shows the peptide with non-mutated sequence.Last row show the difference of ELISA value between wild-type and mutant peptide.Higher value display sudden change has strong side effect to combination.P379, G380, D397 and I398 differentiate as important in conjunction with residue by screen mutation.

Figure 23 show determine to Humanized monoclonal antibodies IGN523 in conjunction with the position of important amino-acid residue on the surface of people CD98.Figure 23 A shows the position of the residue differentiated by mosaic and mutation research, and Figure 23 B shows and analyzes by Pepscan the resi-dues determined.Figure 23 C shows two groups of basic overlapping residues, thus confirm high bright ring region be IGN523 in conjunction with epi-position.

Embodiment 14: the anti-tumor in vivo of the anti-CD98 monoclonal antibody IGN523 of humanization is active

Use the code described in embodiment 8, in several heteroplastic transplantation model, test the impact of anti-CD98 Humanized monoclonal antibodies IGN523.Carry out as described in Example 9 injecting, antibody treatment and statistical computation.In RAMOS (RA.1) and DAU Burkitt lymphoma model, the standard of IGN523 and care drug Rituximab is compared (Figure 24 and Figure 25).In the NSCLC heteroplastic transplantation model IGN-LNG-12 in patient source, IGN523 is compared (Figure 26) with its maximum tolerated dose and carboplatin.Also in AML heteroplastic transplantation model G-1, test IGN523 (Figure 27).The tumour used minimally in NOD/SCID mouse goes down to posterity, and cultivates without any intermediate cell, to keep the heterogeneity of primary tumor.

In all cases, IGN523 demonstrates significant Tumor growth inhibition.Enjoyably, the tumour in IGN-LNG-12 patient source causes NOD-SCID Mouse Weight to reduce, this relevant with tumor load (Figure 26 A and Figure 26 B).Although cause significant Tumor growth inhibition at its maximum tolerated dose carboplatin, it further shows the increase that body weight reduces.On the other hand, IGN523 treatment creates the antitumor action similar with carboplatin, but is reduction of the body weight that IGN-LNG-12 causes and reduces.These data show that IGN523 process is equally effective with carboplatin, and body weight can not be caused to reduce in this NOD-SOD model.

Data show in Burkitt lymphoma model RAMOS (RA.1) and DAU, patient source lung tumor xenografts IGN-LNG-12 in and in AML model G-1, demonstrate significant Tumor growth inhibition.In addition, the Tumor growth inhibition of IGN523 respectively with antitumor and anticancer agent carboplatin or Rituximab quite (table 4).

Table 1

Be investigated the dose-response relationship of IGN523 in IGN-LNG-12 tumour.Use " therapeutic dose scheme ", select IGN-LNG-12 to determine the dose response of antagonist.At the 12nd day and 19 days, between 1mg/kg to 30mg/kg, dosage used IGN523 (Figure 15).In IGN-LNG-12 lung tumor, relative to control group, the dosage of 10 to 30mg/kg creates the maximum tumor growth reduction of 50-66%.

Comprehensive, in body, efficacy data shows that IGN523 causes significant Tumor growth inhibition in multiple heteroplastic transplantation model, and it is at least worked as with standard clinical Reagent evaluation.

Embodiment 15: to the receptor binding specificities of the anti-CD98 monoclonal antibody IGN523 of humanization

Table 5 shows the percent sequence homology of the percent sequence homology of CD98 extracellular domain (BCD) between indicated species and the CD98 epi-position of IGN523 combination.As shown in table 5, the homology between the people that IGN523 combines and the CD98 epi-position of cynomolgus monkey is 96%.Use multiple method (as surface plasma resonance (SPR, Biacore), bio-layer interferometry (Octet) or receptor binding specificities ELISA) are determined IGN523 and are bonded to people and cynomolgus monkey CD98 with high affinity, but in conjunction with other species, mouse, rat, rabbit, dog and pig CD98 homologue (table 5) can not be comprised because homology reduces.Biacore and Octet data show that the KD of IGN523 for people CD98 is in the scope of 2 to 6nM, for the KD of cynomolgus monkey CD98 in the scope of 8 to 14nM.ELISA Binding number according to the show IGN523 is 9ng to the EC50 of people, is 39ng to the EC50 of cynomolgus monkey CD98.

Table 5

ND=does not determine.

In order to confirm for toxicologic study, cynomolgus monkey is relative species, with frozen tissue section dyeing (Figure 29) of IGN523 to the kidney of people and cynomolgus monkey, placenta and brain.In the freezing microtome section of people's kidney and placenta (positive control), observe film and the tenuigenin dyeing of medium extremely strong renal cells (kidney) and trophoderm epithelial cell (placenta), this similar with the staining power in corresponding cynomolgus monkey tissue slice (if not identical).With regard to people and cynomolgus monkey brain, observed the similar weak tenuigenin to medium nerve fibers net and cytoplasmic granule epithelial cell (cytoplasmic granule epithelium) and dye.Generally speaking, seem dyeing in cynomolgus monkey tissue and people organize in suitable.Based on avidity and dyeing data, cynomolgus monkey is considered to the species be applicable to evaluating IGN523 security.

Embodiment 16: single dose pharmacokinetic study in cynomolgus monkey

In male and female cynomolgus monkeys, carry out the exploration non-GLP single dose intravenous pharmacokinetic research of IGN523 with 1,3,10 and the dosage of 100mg/kg (N=2, often kind of sex/group).In monkey, after every kg body weight single 1h IV infusion 1,3,10 and 100mg IGN523, observed significant IGN523 dose dependent kinetics.Therefore, in studied whole dosage range, be not suitable for IGN523 in the basic assumption of the used relevant linear lag without containing in the application of analytical procedure, and show parameter and compare for being described property between dosage group.Although only have rated 2 animals to often kind of sex at each dosage level, in the male PK relative to jenny composes, there is no notable difference.In each dosage group, under the dosage in the scope of the every kg body weight of 1 to 100mgIGN523, mean concentration-time that is male and jenny composes similar (table 6).

The PK parameter of table 6:IGN523

Τ _1/2λ: non-transformation period eventually, CL: clearance rate; V _ss: volume of distribution

Generally speaking, in the dosage range of 1 to 100mg/kg, " apparent " plasma C L of IGN523 reduces 10 times.In the dosage range of 1 to 100mg/kg, average " apparent " MRT and average " apparent " T _1/2improve 12 to 13 times.1,3 or 10mg/kg dosage under, average " apparent " V _ssindifference, but in 100mg/kg dosage group, average " apparent " V _ssratio 1,3 or 10mg/kg group height about 30 to 40%.All these find to show that in monkey, the non-linear disposal feature of IGN523 may be because the disposition of drug (TMDD) of target mediation causes.Previously TMDD model was used for the non-linear disposal describing other monoclonal antibodies.TMDD is created when the cell surface target of antibody to dense distribution has specificity, described cell surface target great expression, thus the interaction of target-antibody to represent under low dosage important removing approach (Mager2001, Mager2003, Luu2012) quantitatively.

Embodiment 17: in cynomolgus monkey, dosage GLP studies repeatedly

GLP multiple doses IV in cynomolgus monkey uses in toxicologic study the toxicology, PK and the immunogenicity that have studied IGN523.This study provides pertinent clinical terminal, Toxicokinetics, immunogenicity (generation of anti-IGN523 antibody) and about organizing the histopathologic extensive data of (comprising injection site) widely, and use the representative its preparing materials used in clinical trial is carried out.In addition, also been evaluated selected safety pharmacology terminal (neurobehavioral, electrocardiogram(ECG, Respiratory behavior).

Dosage is weekly 1 intravenously, 60 minutes infusions, uses 8 weeks.The dosage used is the IGN523 of 10,30 and 100mg/kg, 1 time weekly (amounting to 9 dosage), is then 4 weeks decubations (table 5) without treatment.4 week decubation was considered to be enough to IGN523 is removed completely and the reversibility being enough to evaluate any genotoxic potential.Estimate that maximum dose level is about MTD, and additionally provide except desired in patients and repeatedly expose significantly.Immunogenicity (anti-IGN523 antibody) and Toxicokinetics are monitored.

Table 7: the treatment group of proposed cynomolgus monkey GLP toxicologic study

Group	Process (mg/kg)	Buck number	Jenny number	Treatment schedule
					1	0	5	5	1 time weekly, totally 9 dosage
2	10	5	5	1 time weekly, totally 9 dosage
					3	30	5	5	1 time weekly, totally 9 dosage
4	100	5	5	1 time weekly, totally 9 dosage

Table 8 comprises the detailed overview of the research and design for multiple doses cynomolgus monkey GLP toxicologic study.Selected safety pharmacology terminal (neurobehavioral, electrocardiogram(ECG, Respiratory behavior) is evaluated by the background of the dose study repeatedly of the GLP in cynomolgus monkey.

Table 8: the sheet format overview of cynomolgus monkey GLP toxicologic study

The haemolysis possibility of embodiment 18:IGN523 is evaluated

The haemolysis possibility of cynomolgus monkey whole blood in-vitro evaluation IGN523 will be used.The result of this test is differentiated may act on any of oxyphorase by being used for.Will, on collecting whole blood same day, non-human primates whole blood be used to study.Table 8 contains the summary of the GLP research and design that IGN523 haemolysis possibility is evaluated.

Table 2: proposed haemolysis possibility evaluates the general introduction of GLP research

Group	Process	Preparation	Sample number	Blood volume	Process volume
						1	Test article	1 × expection concentration	3	0.5ml	0.5ml
2	Test article	2 × expection concentration	3	0.5ml	0.5ml
						3	Test article	4 × expection concentration	3	0.5ml	0.5ml
4	Negative control	Salt solution	3	0.5ml	0.5ml
						5	Positive control	Distilled water	3	0.5ml	0.5ml

Embodiment 19: stimulate in Acute Venous and around blood vessel

Devise stimulate and local tissue tolerance research to evaluate the short term toxicity of compound in direct injection region under high density.As a part for this research, differentiate will to be subject to the effect of the tissue of initial exposure to expection by using around compound intravenously and blood vessel.Rabbit is the standard animal model of this evaluation.

The tissue cross reactivity research of embodiment 20:IGN523

As FDA guide, for what describe in detail in the production of the monoclonal antibody product of people's use and the consideration main points (Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use) (in February, 1997) in testing, use a complete set of people's tissue characterization tissue cross reactivity spectrum of IGN523.Include in table 9 to study organize table look-up.

Table 3: the general introduction of the GLP tissue cross reactivity research proposed

1. suprarenal gland	17. lymphoglandula
		2. bladder	18. ovaries
3. hemocyte	19. pancreas
		4. marrow	20. parathyroid glands
5. mammary gland	21. hypophysis
		6. cerebellum	22. placentas (if available)
7. pallium	23. prostate glands
		8. colon	24. skins
9. endothelium	25. spinal cords
		10.	26. spleens
11. uterine tubes	27. voluntary muscles
		12. gi tract	28. testis
13. hearts	29. thymus gland
		14. kidneys (renal glomerulus, tubule)	30. Tiroidina
15. livers	31. ureters

16. lungs

32. uterus (uterine cervix, uterine endometrium))

Embodiment 21: the security of IGN523 and the clinical I phase of pharmacokinetics in recurrent or refractory AML patient evaluated is studied

major objective

The major objective of this research is

● evaluate security and the tolerance of the IGN523 used according to the timetable using from every circumferential recurrent or refractory AML (AML) patient dose

● when weekly × 4 use, determine maximum tolerated dose (MTD) and the dose-limiting toxicity (DLT) of IGN523

● according to security, pharmacokinetics and Pharmacodynamic Data, differentiate the recommendation 2 phase dosage (RP2D) of IGN523

by-end

● evaluate the incidence that anti-IGN523 antibody is formed

● differentiate the pharmacokinetics of IGN523 in recurrent or intractable AML patient

● preliminary assessment is carried out to the anti-leukocythemia liveness of IGN523 in recurrent or intractable AML patient

● to predicting that the biomarker of IGN523 anti-leukocythemia liveness carries out preliminary assessment

method

With MTD or RP2D, use standard 3+3 to design additional amplification group, open dosage is carried out to about 6 dosage groups and increases research

experimenter's number

Dosage escalation: about 21-30

Dosage increases: 20

research medicine

IGN523 medicament production is provided as 20mL10mg/mL solution in the disposable vial of 25mL.The IGN523 medicament production of proper volume is diluted to 250mL and in 1 hour internal jugular vein infusion.In I phase dose escalation study (the code summary see in 10.2.1 joint), can weekly with 8 ascending-dose process patient groups up to 30mg/kg.Continuous process will provide lasting clinical benefit (namely lack disease progression and do not have unacceptable toxicity) more than 8 weeks for patient.Under given conditions (10.2.1 joint described in), the dosage escalation in patient can be allowed, thus under relevant dose, at utmost accumulate data and under possible sub-therapeutic dose minimum degree reduction process patient.

diagnosis and main selection criteria

There is no the recurrent of effective standard treatment or intractable AML

● measurable disease must be had

● age >18 year,

● eastern united states tumour cooperative groups (Eastern Cooperative Oncology Group, ECOG) shows state 0-2

● the predicted life at least 12 weeks

● platelet count >25000/mm3 (can be maintained by hematometachysis)

● the Upper Limit of Normal Value (ULN) of AST (SGOT)/ALT (SGPT) <2.5 × mechanism

● the ULN of total bilirubin <1.5 × mechanism

● the ULN of creatinine <2 × mechanism or the CrCl >50mL/min of calculating or measurement, for having the possible women of fertility and the male sex, agrees to use enough contraceptives

● (hormone or obstruct contraception; Ascetic), before research starts and in the whole process participating in research

● understand and be ready to sign the ability of written informed consent document

exclusion standard

● in circulation 1 before the 1st day, in 4 weeks, employ monoclonal antibody therapy or in 2 weeks, employ chemotherapy or radiotherapy (allow to reach 72 hours in circulation 1 before the 1st day and use hydroxyurea to control periphery protoblast counting)

● the unsolved acute toxicity grade >1 of NCI CTCAE v4.0 in previous anti-cancer therapies

● previous allogeneic stem cell is transplanted needs immunosuppressive therapy at present

● there are severe allergy or anaphylaxis history to monoclonal antibody therapy

● known leukemic pia arachnoid or CNS illness

● uncontrolled concomitant disorders, include, but is not limited to occurent or Active infection, symptomatic congestive heart failure, unstable angina pectoris, arrhythmia or will restriction to the mental disorder/social condition of the cooperation that research requires.

● before the 1st day of circulation 1, carried out recent major operation in 4 weeks

● conceived or nursing women

diagnosis plan

Based on following fundamental principle, after recruiting CD98 expression study, retrospective analysis is from the patient AML sample (and not analyze as patient screening part, and be used as the condition of research qualification) of peripheral blood (or marrow):

● compared with similar " classification " cell from normal bone marrow sample, CD98 difference process LAN in CD34+/CD33+ and the CD34+/CD33-subgroup of most of AML patient (about 94%).

● be not enough to the method for the clinical verification to Patient Sample A's detection and quantitative CD98 expression of patient's (or getting rid of patient's participation) that the I phase proposed by selection participation is studied.

● the I phase measures and the qualified design by the future clinical be used for for patient may be needed to select research of analytical results is given information.

test products, administration form, initial dose

IGN523

Intravenous infusion

Initial dose: be not more than viewed nothing in GLP multiple doses cynomolgus monkey toxicity research and observe 1/6 of people's dose,equivalent (HED) of side effect level (NOAEL)

group's initial sum treatment time length

Before any other patient in this group at least 1 day is used to the first patient dosage in each new dosage group, thus allow to observe and may affect successive patients and to recruit or dosage uses possible serious and/or great acute (such as, infusion is correlated with) toxicity of decision.

Patient will accept weekly the intravenous dosages of IGN523, accepts 8 weeks (two circulations in 28 days).For meeting the patient continuing clinical benefit (namely lacking disease progression) and acceptable safety standards, the dosage outside 8 weeks is used and is reached 1 year by allowing.

dosage escalation in patient

In order at utmost collect the information of relevant dose and minimally patient be exposed to possible suboptimal dosage, the dosage escalation in patient can be allowed under the following conditions:

● before any dosage escalation, patient must complete at least two circulations of 28 days under the dosage level that they are original distributed

● patient only can by their dosage escalation to by least one IGN523 of 28 days use 3-6 patient dose group circulate know confirmation maximum dose level level

● the dosage escalation in all patients determines to monitor based on researchist's integrative medicine feeling whether it is in the judgement of the maximum benefit of patient

the definition of DLT

DLT will be any following adverse events thought about the researchist of (and not owing to the obvious recognizable reason of another kind) with IGN523 occurred during the 1-28 days of circulation 1:

● 3 or 4 grades of non-blood toxicity, except

-occur in 24 hours during completing infusion or after completing infusion and reduce infusion rates, Supportive Care and/or use solve in reflunomide 24 hours reversibility 3 grades of nonallergic infusion toxicity (comprise symptom, as fever, feel cold/shiver with cold, Nausea and vomiting, itch, headache, rhinitis, fash, weakness and/or anoxic (when the sign/symptom breathing no more difficult)) outside

-of short duration if (namely continue <48 hour) and without clinical tumor Cell lysis syndrome symptom (i.e. creatinine >1.5 × ULN, arrhythmia, sudden death or epileptic seizures), then 3 or 4 grades of hyperuricemias, hyperphosphatemia or hypocalcemias, or 3 grades of hyperpotassemias

● 3 or 4 grades of thrombopenia (previously there is no thrombopenia and in the patient needing blood transfusion to support), it causes hemorrhage, or can not to be improved in 2 weeks when not carrying out platelet transfusion >=baseline value 80%

● 3 or 4 grades of neutrophilic leukocytes reduce (reduce previously not having neutrophilic leukocyte and in the patient needing somatomedin to support), it is relevant with fever (oral cavity or tympanic temperature are 100.4 °F/38 DEG C), or can not to be improved in 2 weeks when not carrying out somatomedin support >=baseline value 80%

dLT window

The 1-28 days of circulation 1

dosage escalation regimens

Dosage escalation only may occur after reaching the 28th day by each individuality in given group.Stand disease progression and exited from research before the 28th day but will be not valuable without the patient of DLT for DLT, and by replaced.According to following scheme, between group, carry out dosage escalation by with the increase (or less, if observe significant AE) up to 100%:

If ● given dose level 0/3 patient, there is DLT, then can recruit 3 patients at next dosage level

● if in given dose level, the patient of >2/3 stands DLT, then will stop dosage escalation, and this dosage will be claimed more than MTD.

If ● stand DLT given dose level 1/3 patient, then will recruit 3 patients at least again in same dose level.If 0 experienced by DLT in these 3 patients, then continue next dosage level (it can be the dosage increase of <100%).If 1 or multidigit experience DLT, then stop dosage escalation, and this dosage will be claimed more than MTD in this group.

● once exceed MTD, if the increase <30% of dosage escalation before, so then minimum 6 valuable patients can be recruited to be evaluated as MTD at a upper dosage level.If the increase >30% of dosage escalation before, then can evaluate at least one the middle dosage level between two maximum dose level levels.

● cause the maximum dose level level of generation DLT to be declared to be one that MTD in being less than in 1/3 of minimum 6 patients.

the dosage escalation council

The council will be increased progressively by internal dose to make decision: agree to proceed dosage escalation, amendment dosage escalation regimens or termination research, described internal dose increase progressively the council represented by medical monitoring personnel, drug safety and other especially research team members form, and when each research group completes and researchist consult.Before subsequent groups being made to dosage escalation and determining, this council will examine from the whole available data of current group and whole available security data of a upper group.All research experimenters will accept research therapy until the development of disease progression, unacceptable toxicity, disobedience or experimenter agree to exit or researchist determines to exit.

evaluate the standard of effect

Peripheral blood counts

Bone marrow aspirates and examination of living tissue

Security

Security final result is measured as follows:

The incidence of DLT and character,

Adverse events and incidence and seriousness

pK samples

PK evaluation will be carried out according to following timetable:

1st day dosage: predose, and the dosage after 30 minutes, 4 hours, 24 hours and 48 (or 72 hours)

8th, 15 and 21 days dosage: predose, and the dosage after 30 minutes, 4 hours and 48 (or 72 hours)

Subsequent dose: predose and the dosage after 30 minutes

amplification group

In order to obtain the preliminary proof of other securities, tolerance and pharmacokinetic data and clinical activity, at MTD or RP2D by nearly 20 extra patient enrolment with recurrent or intractable AML to the group that increases.Although MTD will be determined mainly through I phase DLT observation period viewed security (DLT) data, but other data of safety that RP2D also will consider outside DLT window, and the information that it gathers during can also being included in I phase dosage escalation, comprises PK and target takies data.

Claims (70)

1. specific binding is to the antibody of the separation of people CD98 or its functional fragment, and wherein, described antibody or functional fragment are bonded to and comprise the residue A 377 of people CD98, the epi-position of D397, I398, G400 and A401.

2. the antibody of separation according to claim 1 or functional fragment, wherein, described epi-position also comprises residue D374 and L378 of people CD98.

3. the antibody of separation according to claim 2 or functional fragment, wherein, described epi-position also comprises residue P379 and G380 of people CD98.

4. the antibody of separation according to claim 3 or functional fragment, wherein, described epi-position also comprises residue F395 and P396 of people CD98.

5. the antibody of separation according to claim 3 or functional fragment, wherein, described epi-position also comprises residue Q381, P382 and P399 of people CD98.

6. the antibody of separation according to claim 1 or functional fragment, wherein, described epi-position also comprises other residues any one or more in the group selecting D374, L378, P379, G380, Q381, P382, F395, P396 and P399 of freeman CD98 to form.

7. specific binding is to the antibody of the separation of people CD98 or its functional fragment, and wherein, described antibodies is to the epi-position of residue P379, G380, D397 and I398 comprising people CD98.

8. the antibody of separation according to claim 7 or functional fragment, wherein, described epi-position also comprises residue F395 and P396 of people CD98.

9. the antibody of separation according to claim 8 or functional fragment, wherein, described epi-position also comprises residue Q381, P382, P399, G400 and A401 of people CD98.

10. the antibody of separation according to claim 8 or functional fragment, wherein, described epi-position also comprises residue D374, A377 and L378 of people CD98.

The antibody of 11. separation according to claim 7 or functional fragment, wherein, described epi-position also comprises other residues any one or more in the group selecting D374, A377, L378, Q381, P382, F395, P396, P399, G400 and A401 of freeman CD98 to form.

The antibody of 12. separation according to claim 1 or functional fragment, wherein, described antibody or functional fragment are bonded to the epi-position of residue D374, A377, L378, P379, G380, Q381, P382, F395, P396, D397, I398, P399, G400 and the A401 comprising people CD98.

13. 1 kinds of specific bindings are to the antibody of the separation of people CD98 or its functional fragment, and wherein, described antibody or functional fragment are bonded to the epi-position be included in the amino-acid residue 369-405 of people CD98.

14. 1 kinds of specific bindings are to the antibody of the separation of people CD98 or its functional fragment, and wherein, described antibody or functional fragment are bonded to the epi-position be made up of the amino-acid residue 369-405 of people CD98.

15. antibody according to claim 1 or functional fragments, wherein, described antibody is monoclonal antibody.

16. antibody according to claim 15 or functional fragments, wherein, described monoclonal antibody is humanized antibody, people's antibody or chimeric antibody.

17. antibody according to claim 1 or functional fragments, wherein, described fragment is Fab, F (ab') 2, Fv or Sfv fragment.

18. 1 kinds of antibody or its functional fragment be separated, it comprises whole three the heavy chain complementary determining regions (CDR) from the heavy-chain variable domains with the aminoacid sequence being selected from SEQ ID NO:8 and SEQ ID NO:12, and/or from having whole three light chain CDR of light variable domains of the aminoacid sequence being selected from SEQ ID NO:10 and SEQ ID NO:14.

19. 1 kinds of antibody or its functional fragment be separated, it comprises whole three the heavy chain CDR from the heavy-chain variable domains with the aminoacid sequence being selected from SEQ ID NO:8 and SEQ ID NO:12, and from having whole three light chain CDR of light variable domains of the aminoacid sequence being selected from SEQ ID NO:10 and SEQ ID NO:14.

20. antibody according to claim 19, wherein, described antibody comprises the heavy-chain variable domains sequence being selected from SEQ ID NO:8 and SEQ ID NO:12.

21. antibody according to claim 19, wherein, described antibody comprises the light variable domains sequence be made up of SEQ ID NO:10 and SEQ ID NO:14.

22. antibody according to claim 20, wherein, described antibody also comprises the light variable domains sequence be made up of SEQ ID NO:10 and SEQ ID NO:14.

23. antibody according to claim 22, wherein, described antibody comprises the heavy-chain variable domains sequence of SEQ ID NO:8 and the light variable domains sequence of SEQ ID NO:10.

24. antibody according to claim 22, wherein, described antibody comprises the heavy-chain variable domains sequence of SEQ ID NO:12 and the light variable domains sequence of SEQ ID NO:14.

25. humanized antibodies according to claim 16, wherein, described antibody comprises the heavy-chain variable domains sequence being selected from SEQ ID NO:22 and SEQ ID NO:23.

26. humanized antibodies according to claim 16, wherein, described antibody comprises the light variable domains sequence being selected from SEQ ID NO:20 and SEQ ID NO:21.

27. humanized antibodies according to claim 25, also comprise the light variable domains sequence being selected from SEQ ID NO:20 and SEQ ID NO:21.

28. humanized antibodies according to claim 27, wherein, described antibody comprises the light variable domains sequence of SEQ ID NO:20 and the heavy-chain variable domains sequence of SEQ ID NO:22.

29. humanized antibodies according to claim 27, wherein, described antibody comprises the light variable domains sequence of SEQ ID NO:21 and the heavy-chain variable domains sequence of SEQ ID NO:23.

30. humanized antibodies according to claim 27, wherein, described antibody comprises the light variable domains sequence of SEQ ID NO:21 and the heavy-chain variable domains sequence of SEQ ID NO:22.

31. antibody being bonded to the epi-position substantially identical with antibody according to claim 30.

32. bonding agents being bonded to the epi-position substantially identical with the antibody according to any one of claim 1-31.

33. bonding agents according to claim 32, wherein, described bonding agent suppresses the growth of the tumour expressing CD98.

34. bonding agents according to claim 32, it is antibody or its functional fragment.

35. bonding agents according to claim 32, it is the Kunitz type inhibitor of anticalin, adnectin, affine body, DARPin, fynomer, affitin, affilin, Avimers, kink rhzomorph peptide or engineering design.

36. 1 kinds of bonding agents that can be bonded to CD98, wherein, the antibody according to any one of claim 1-31 replaces described bonding agent in competitive binding assay.

37. 1 kinds of bonding agents that can be bonded to CD98, wherein, the antibody according to any one of claim 1-31 replaced by described bonding agent in competitive binding assay.

38. bonding agents according to claim 36, wherein, described bonding agent is antibody or its functional fragment.

39. according to bonding agent according to claim 37, and wherein, described bonding agent is antibody or its functional fragment.

40. according to claim 1-31,34 and 38-39 according to any one of antibody or functional fragment, wherein, described antibody or fragment are bonded to cytotoxic reagent.

41. antibody according to claim 40 or functional fragments, wherein, described cytotoxic reagent is selected from chemotherapeutics, medicine, growth inhibitor, toxin or radio isotope.

42. according to claim 1-31,34 and 38-39 according to any one of antibody or functional fragment, wherein, described antibody or fragment are bonded to detectable mark.

43. antibody according to claim 40 or functional fragments, wherein, described detectable mark is selected from radio isotope, metal chelator, enzyme, fluorescent chemicals, bioluminescent compound and chemiluminescence compound.

The transgenic animal of 44. generation monoclonal antibody according to claim 15.

The hybridoma of 45. generation monoclonal antibody according to claim 15.

46. comprise coding according to claim 1-31,34 and 38-39 according to any one of antibody or the carrier of polynucleotide of its fragment.

47. comprise according to claim 1-31,34 and 38-43 according to any one of antibody or the pharmaceutical composition of functional fragment and pharmaceutically acceptable carrier.

48. 1 kinds are suppressed the method for growth of cancer cells expressing CD98, described method comprise described cell is exposed to according to claim 1-31,34 and 38-43 according to any one of antibody or functional fragment.

49. methods according to claim 48, wherein, described cancer cells comes from and is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or the cancer of these cancer metastasis cancers any.

50. methods according to claim 49, wherein, described cancer cells comes from acute myelocytic leukemia.

51. 1 kinds of methods being used for the treatment of cancer in experimenter, comprise and use pharmaceutical composition according to claim 47 to described experimenter.

52. methods according to claim 51, wherein, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

53. methods according to claim 52, wherein, described cancer is acute myelocytic leukemia.

54. methods according to claim 51, wherein, described experimenter is recurrent or refractory AML.

55. methods according to claim 51, wherein, described antibody or functional fragment and one or more chemotherapy compounds are combined and use to described experimenter, wherein said chemotherapy compound is selected from bendamustine hydrochloride, endoxan, ifosfamide, fludarabine, cytosine arabinoside, gemcitabine, prednisone, prednisolone, methylprednisolone, taxol, docetaxel, vinorelbine, vincristine(VCR), Etoposide, irinotecan, anthracycline, Dx, cis-platinum, carboplatin and Rituximab.

56. methods according to claim 51, wherein, described cancer increases relevant with the expression of CD98 on cell surface.

57. 1 kinds of methods detecting the existence of CD98 in biological sample, be included in allow under the condition that is combined with CD98 of described antibody by described biological sample and according to claim 1-31,34 and 38-43 according to any one of antibody contacts, and detect between described antibody and CD98 whether form mixture.

58. methods according to claim 57, wherein, described biological sample comes from the Mammals suffering from or suspect and suffer from cancer, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

59. 1 kinds of diagnosis express with CD98 the method increasing relevant cancer, comprise by test cell with according to claim 1-31,34 and 38-43 according to any one of antibody contacts; The expression level of CD98 is determined by the combination detecting described antibody and CD98; With by the CD98 expression level in described test cell compared with the CD98 expression level in compared with control cells, wherein compared with compared with control cells, the instruction of the expression level of CD98 higher in described test cell exists to express with CD98 increases relevant cancer.

60. methods according to claim 59, wherein, described test cell comes from the patient suspecting and suffer from cancer, described cancer is selected from bladder cancer, mammary cancer, colon and rectum carcinoma, cancer of the stomach, esophagus cancer, lung cancer, laryngocarcinoma, kidney, oral carcinoma, ovarian cancer or prostate cancer, or sarcoma, melanoma, neurospongioma, lymphoma or leukemia, or these cancer metastasis cancers any.

61. methods according to claim 60, wherein, described method comprises the expression level determining described test cell CD98 on the surface, with compared with the expression level of described test cell expression level and the compared with control cells CD98 on the surface of CD98 on the surface.

62. methods according to claim 61, wherein, described test cell is cancer cells, and described compared with control cells is the normal cell of homologue's type.

63. claim 1-31,34 and antibody according to any one of 38-43 or the purposes of functional fragment in medicine preparation, wherein, described medicine is used in the method for the growth suppressing the cancer cells of expressing CD98.

64. according to claim 1-31,34 and 38-43 according to any one of antibody or functional fragment, for suppressing the growth of cancer cells expressing CD98.

The purposes of 65. pharmaceutical compositions according to claim 47 in medicine preparation, wherein, described medicine is used for the treatment of in the method for the cancer in experimenter.

66. 1 kinds comprise according to claim 1-31,34 and 38-43 according to any one of antibody or the pharmaceutical composition of functional fragment and pharmaceutically acceptable carrier, be used for the treatment of the cancer in experimenter.

67. claim 1-31,34 and antibody according to any one of 38-43 or the purposes of functional fragment in medicine preparation, wherein, described medicine is for detecting in the method for the existence of CD98 in biological sample.

68. according to claim 1-31,34 and 38-43 according to any one of antibody or functional fragment, for detecting in the method for the existence of CD98 in biological sample.

69. claim 1-31,34 and antibody according to any one of 38-43 or the purposes of functional fragment in medicine preparation, wherein, described medicine is expressed in the method increasing relevant cancer with CD98 for diagnosing.

70. according to claim 1-31,34 and 38-43 according to any one of antibody or functional fragment, express in the method increasing relevant cancer with CD98 for diagnosing.