NZ626513B2 - 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-CD98 ANTIBODIES AND METHODS OF USE THEREOF FIELD OF THE INVENTION The present invention relates generally to anti—CD98 antibodies and to methods of using such antibodies.

BACKGROUND CD98 (also referred to as CD98 heavy chain; 42F heavy chain; ) is a type II embrane glycoprotein composed of 529 amino acid residues. The protein comprises a 75 amino acid N-terminal intracellular cytoplasmic domain, a single transmembrane domain, and a 426 amino acid C—terminal extracellular domain (Parmacek et al., Nucleic Acids Res. 17: 1915-1931, 1989). CD98 covalently links via a de bond to one of several light chains 5, 6, 7, 8, 10, or 1 1), which are L~type amino acid transporters. This interaction is required for the cell surface expression and amino acid transport function of the light chains. CD98 also associates with integrin [3 subunits, thereby regulating integrin signaling that controls cell proliferation, survival, ion, and epithelial adhesion/polarity (Cai et al., J. Cell Sci. 118: 889-899, 2005).

CD98 was originally identified as a cell surface antigen ated with lymphocyte activation (Haynes et al., J. Immunol. 126: 1409-1414, 1981). CD98 has since been identified in all cell types with the ion of platelets and is sed at the highest levels in the gastrointestinal (GI) tract and the tubules of the kidney (Verrey et al., Pflugers Arch. 440: 503—5 12, 2000). Upregulation of CD98 has been observed in intestinal mation. Recently, intestinal CD98 expression was shown to have a crucial role in lling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in intestinal epethilial cells has therefore been suggested as a promising therapeutic strategy for the treatment and prevention of inflammatory inal diseases, such as inflammatory bowel disease (IBD) and s-associated cancer (Nguyen et al., J. Clin. Invest. 121: 747, 2011). CD98 is also overexpressed on the cell surface of almost all tumor cells, regardless oftissue of origin (Itoh et al., Jpn.

J. Cancer Res. 92: 1313-1321, 2001).

Increased expression of one of the light chains that binds CD98, L-type amino acid transporter 1 (LATl; also known as SLC7A5) has also been observed in many types of human cancer cells, including breast cancer, colon cancer, oral cancer, ovarian cancer, esophageal cancer, glioma and leukemia (Fan et al., Biochem. Pharmacol. 80: 811—818, 2010). Increased amino acid supply may be required to support the high growth rate of cancer cells, both by providing the amino acid building blocks for protein sis, and by stimulating growth via mammalian target of rapamycin (mTOR) (Fan et al., supra; Imai et al., Anticancer Res. 30: 4819-4828, 2010). The expression of LATl and CD98 is significantly higher in metastatic sites of human cancers than in the primary sites, suggesting that overexpression of LATl/CD98 may be essential for ssion and metatstasis of human cancers. ln particular, LAT1/CD98 overexpression appears to be required for tumor metastasis in patients with colon cancer. (Kaira et al., Cancer Sci. 99: 2380-2386, 2008).

The expression pattern and functions of CD98 and LAT1 suggest these proteins as promising targets for treatment of a variety of human cancers. Inhibitors of LAT1 activity have demonstrated mor activity in a number of cancer types, including non-small cell lung cancers (Imai et al., supra), colon cancer cells (Oda et al., Cancer Sci. 101: 173-179, 2010), oral cancer cells (Kim et al., Biol. Pharm. Bull. 33: 1117-1121, 2010), and breast cancer cells (Shennan and Thomson, Oncol. Rep. 20: 885-889, 2008).

LAT1 has also been suggested as a target for treatment of n cancer (Fan et al, supra).

A murine monoclonal antibody to CD98, fied as HBJ 127, was found to inhibit lymphocyte proliferation (Yagita and Hashimoto, J. Immunol. 136: 2062-2068, 1986) and to inhibit the growth of bladder tumor and ma cells (Yagita et al., Cancer Res. 46: 489, 1986). The epitope for the HJ127 antibody was found to be residues 442AFS444 of human CD98 (Itoh et al., 2007). A ent murine monoclonal antibody to CD98 was shown to significantly inhibit tumor cell growth in vitro for glioma, prostate and colon cancer cells (Papetti and Herman, Am. J. Pathol. 159: 165-178, 2001). onal monoclonal antibodies to human CD98 have been disclosed in U.S. Publication No. 20100143367. These monoclonal antibodies bind to epitopes within amino acid regions 372-530 or 104-371 of CD98. Five of these antibodies were found to t amino acid uptake in a bladder cancer cell, and three of these antibodies were shown to suppress tumor growth in a mouse model.

As sed herein, analysis of fresh primary acute myelogenous leukemia (AML) tumor samples from patients using surface tagged antigen profiling (sTAg) of the cell e proteome identified the transmembrane protein CD98 as being present at high density on the surface of AML tumor cells. CD98 is therefore a target for the treatment of AML, for example, by using binding agents such as antibodies which specifically bind to CD98. Binding agents specific for CD98, such as D98 antibodies, were also shown in various in vivo xengraft models to have utility in ng not only AML but various cancers, such as sarcoma, lymphoma, non-small cell lung cancer (NSCLC) and colorectal cancer.

The invention relates to dies to CD98 that are useful in the diagnosis and treatment of various types of human cancers. [0008a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general dge in the field. [0008b] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

SUMMARY [0008c] According to a first aspect, the invention provides an isolated antibody or a functional fragment thereof comprising all three heavy chain CDRs from a heavy chain variable domain having an amino acid sequence set forth in SEQ ID NO: 12, and all three light chain CDRs from a light chain variable domain having an amino acid sequence set forth in SEQ ID NO: 14. [0008d] According to a second aspect, the invention provides a binding agent e of binding to CD98 which is an dy or a onal fragment thereof, wherein the antibody of the ion displaces the binding agent in a itive binding assay, or wherein the binding agent displaces the antibody of the invention in a competitive binding assay.

] According to a third aspect, the invention provides a vector comprising a polynucleotide encoding the antibody, fragment or binding agent of the invention.

] According to a fourth aspect, the invention provides a pharmaceutical composition that comprises the antibody, functional fragment or binding agent of the invention, and a pharmaceutically acceptable carrier. [0008g] According to a fifth aspect, the invention es use of the antibody, functional fragment or binding agent of the invention in the manufacture of a medicament for inhibiting growth of a cancer cell that expresses CD98. [0008h] According to a sixth , the invention provides use of the antibody, functional fragment or binding agent of the invention, or the pharmaceutical composition of the invention in the manufacture of a ment for treating a cancer in a subject. [0008i] According to a seventh aspect, the invention provides a method of detecting the presence of CD98 in a biological , comprising contacting the biological sample with the antibody, onal fragment or binding agent of the invention under conditions permissive for binding of the antibody, functional fragment or binding agent to CD98, and detecting whether a complex is formed between the dy, functional fragment or binding agent and CD98. [0008j] According to an eighth aspect, the invention provides a method of diagnosing a cancer ated with sed expression of CD98, comprising contacting an in vitro or ex vivo test cell with the antibody, functional fragment or binding agent of the invention; determining the level of expression of CD98 by detecting binding of the antibody, functional fragment to CD98; and comparing the level of expression of CD98 in the test cell with the level of expression of CD98 in a control cell, wherein a higher level of expression of CD98 in the test cell as compared to the control cell indicates the presence of a cancer associated with increased expression of CD98.

Using in-solution ng of intact AML tumor cell surfaces, followed by high-resolution, solution-based liquid chromatography coupled tandem mass spectrometry (LCMS /MS), CD98 was identified as being present at high density on the surface of a majority of AML cell subtypes as compared to normal cells including ping blood cells. Thus, the invention provides anti-CD98 antibodies and s of using the such antibodies in the treatment of AML and other cancers, ing but not limited to lymphoma, sarcoma, nonsmall cell lung cancer and colorectal cancer. 2012/066347 In an embodiment, the invention provides an isolated antibody or a functional fragment thereof that specifically binds to human CD98, wherein the antibody or functional fragment binds to an epitope comprising residues A377, D397, I398, G400 and A401 of human CD98. In some embodiments, the epitope further comprises residues D374 and L378 of human CD98. In some embodiments, the epitope further comprises residues P3 79 and G3 80 of human CD98. In some embodiments, the epitope r comprises residues F395 and P396 of human CD98. In some ments, the epitope r comprises residues Q381, P382 and P399 of human CD98. In some embodiments, the epitope further comprises any one or more additional residues selected from the group consisting of D3 74, L378, P379, G3 80, Q381, P382, F395, P396 and P399 of human CD98. [0011} In an ment, the invention provides an isolated dy or a functional fragment thereof that specifically binds to human CD98, wherein the antibody binds to an epitope comprising residues P379, G380, D397 and I398 of human CD98. In some embodiments, the epitope further comprises residues F395 and P396 of human CD98. In some ments, the epitope further comprises residues Q381, P382, P399, G400 and A401 of human CD98. In some embodiments, the epitope further ses residues D374, A377 and L378 of human CD98. In some embodiments, the epitope further comprises any one or more additional residues selected from the group consisting of D374, A377, L3 78, Q381, P3 82, F395, P396, P399, G400 and A401 of human CD98. [0012} In some embodiments, the invention provides an isolated antibody or a functional fragment thereof, wherein the antibody or functional nt binds to an epitope sing residues D374, A377, L378, P379, G380, Q381, P382, F395, P396, D397, I398, P399, G400 and A401 of human CD98.

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

In some embodiments, the onal antibody of the invention is a humanized, human or chimeric antibody. In some embodiments, the antibody onal nt of the invention is an Fab, F(ab')2, Fv or scFv fragment. [0015} n an ment, the invention provides an isolated antibody or a functional fragment thereof comprising all three heavy chain complementarity determining s (CDRs) from a heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 31, and SEQ ID NO: 35, and/or all three light chain CDRs from a light chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 33, and SEQ ID NO: 37.

In an ment, the invention provides an isolated antibody or a functional fragment thereof comprising all three heavy chain CDRs from a heavy chain variable domain having an amino acid 2012/066347 sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 31, and SEQ ID NO: 35, and all three light chain CDRs from a light chain variable domain having an amino acid sequence selected from the group ting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 33, and SEQ ID NO: 37. In some embodiments, the antibody or functional fragment thereof comprises all heavy and light chain complementarity ining regions (CDRs) from: (a) the dy designated 8—34B; (b) the antibody designated 18—2A 2.2; (c) the antibody designated 18— 2A 7.1; (d) the antibody designated 1—47C; or (e) the antibody designated 1—1 15A. In some ments, the antibody or functional fragment thereof comprises all heavy and light chain CDRs from the antibody designated 8—34B. In some embodiments, the antibody or functional fragment thereof comprises all all heavy and light chain CDRs from the antibody designated 18—2A 2.2. In some embodiments, the dy or functional fragment thereof comprises all all heavy and light chain CDRs from the dy designated 18—2A 7.1. In some embodiments, the antibody or functional fragment thereof comprises all all heavy and light chain CDRs from the antibody designated 1—47C. In some embodiments, the antibody or functional fragment thereof comprises all all heavy and light chain CDRs from the antibody designated 1-1 15A.

[0017] In some embodiments, the antibody comprises a heavy chain le domain sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 31, and SEQ ID NO: 35. In some embodiments, the antibody comprises a light chain variable domain sequence consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 33, and SEQ ID NO: 37. In some embodiments, the antibody comprises a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 31, and SEQ ID NO: 35, and further comprises a light chain le domain sequence consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 33, and SEQ ID NO: 37.

In an embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 4 and the light chain variable domain sequence of SEQ ID NO: 6. In an embodiment, the antibody ses the heavy chain variable domain sequence of SEQ ID NO: 8 and the light chain variable domain ce of SEQ ID NO: 10. In an embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 12 and the light chain variable domain sequence of SEQ ID NO: 14. In an embodiment, the antibody ses the heavy chain le domain sequence of SEQ ID NO: 31 and the light chain variable domain sequence of SEQ ID NO: 33. In an embodiment, 3O the dy comprises the heavy chain variable domain sequence of SEQ ID NO: 35 and the light chain variable domain sequence of SEQ ID NO: 37.

In an embodiment, the invention provides humanized antibodies. In some embodiments, the humanized antibody comprises a heavy chain variable domain sequence selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23. In some embodiments, the humanized antibody comprises a light chain variable domain sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO: 21. In some embodiments, the humanized antibody comprises a heavy chain le domain sequence selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23, and further comprises a light chain variable domain sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO: 21. In some embodiments, the humanized antibody comprises a light chain variable domain sequence selected from SEQ ID NO: 15 and SEQ ID NO: 16, and a heavy chain variable domain sequence ed from SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. In an embodiment, the humanized antibody comprises the light chain variable domain ce of SEQ ID NO: 15 and the heavy chain variable domain sequence of SEQ ID NO: 18.

In an embodiment, the humanized antibody comprises the light chain variable domain sequence of SEQ ID NO: 20 and the heavy chain variable domain sequence of SEQ ID NO: 22.

In an embodiment, the humanized antibody comprises the light chain variable domain sequence of SEQ ID NO: 2l and the heavy chain le domain sequence of SEQ ID NO: 23. In an embodiment, the zed antibody comprises the light chain variable domain ce of SEQ ID NO: 20 and the heavy chain le domain sequence of SEQ ID NO: 23. In an embodiment, the humanized antibody comprises the light chain le domain sequence of SEQ ID NO: 21 and the heavy chain variable domain sequence of SEQ ID NO: 22. In an embodiment, the ion provides an antibody that bind to the same epitope as a humanized antibody sing the light chain variable domain sequence of SEQ ID NO: 21 and the heavy chain variable domain sequence of SEQ ID NO: 22. In an alternative embodiment, the invention comprises a g agent that binds to essentially the same epitope as an antibody from bin 1 or bins 3—7 as shown in Fig. l.

In a further embodiment, the invention comprises a binding agent that binds to essentially the same epitope as any of the antibodies disclosed above. In some embodiments, the binding agent inhibits the growth ofa tumor expressing CD98. In some embodiments, the binding agent is an antibody or a functional fragment thereof. In other embodiments, the binding agent is an anticalin, an adnectin, an affibody, a DARPin, a r, an affitin, an affilin, an avimer, a cysteine—rich knottin peptide, or an ered Kunitz-type inhibitor.

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

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

In some embodiments, the invention provides an antibody that binds to CD98, wherein the antibody comprises a heavy chain variable domain having 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% ce identity to an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18; SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, and SEQ ID NO; 35. In some embodiments, the antibody comprises a light chain variable domain having 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% ce identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 33, and SEQ ID NO: 37. In some embodiment, the antibody comprises a heavy chain variable domain having 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% sequence ty to an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18; SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, and SEQ ID NO: 35, and the antibody further comprises a light chain variable domain having 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% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 33, and SEQ ID NO: 37.

In some embodiments, the invention es an antibody that is a variant of any of the above dies having one or more amino acid substitutions, deletions, insertions or modifications, and which retains a biological function of the antibody. In some embodiments, the invention provides an antibody that binds to CD98 expressed on the cell surface and inhibits the growth of the cell. In some embodiments, the anti—CD98 dy binds to CD98 expressed on the cell e and inhibits cell eration. In some embodiments, the anti—CD98 antibody binds to CD98 expressed on the cell surface and induces cell death. In some embodiments, the ion provides an antibody that is a variant of any one of the above antibodies having improvements in one or more of a property such as binding affinity, specificity, thermostability, expression level, or function, glycosylation, reduced immunogenicity, or solubility as compared to the unmodified antibody.

In some embodiments, the invention provides any one of the above antibodies or functional fragments, wherein theantibody or fragment is conjugated to a cytotoxic agent. In various embodiments, the cytotoxic agent is selected from a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, or a radioactive isotope. In some embodiments, the invention provides any one of the above antibodies or functional fragments, wherein theantibody or nt is conjugated to a detectable marker. In various embodiments, the detectable marker is selected from a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound and a chemiluminescent compound.

In an embodiment, the invention provides a hybridoma that produces a monoclonal antibody of the invention. In an embodiment, the ion es a transgenic animal that produces a monoclonal dy of the invention.

In some embodiments, a polynucleotide encoding any of the above antibodies is provided. In an embodiment, a vector comprising the polynucleotide is provided. In an embodiment, a host cell comprising the vector is provided. In an ment, the host cell is prokaryotic. In an embodiment, the host cell is an E. coli cell. In another embodiment, the host cell is otic. In an ment, the host cell is a Chinese r Ovary (CHO) cell. In an embodiment, a method of making an anti—CD98 antibody is provided, wherein the method comprises culturing the host cell under conditions suitable for expression of the polynucleotide encoding the antibody, and isolating the antibody.

In one embodiment, the invention provides a pharmaceutical composition comprising any of the above antibodies or functional fragments thereof, antibody conjugates, or binding agents of the invention. In a further embodiment, the invention provides a method of ting growth of cancer cells that express CD98, the method comprising exposing the cells to any one or more of the above antibodies or functional fragments thereof, antibody conjugates, or g agents of the invention.

In various embodiments, the cancer cells are from a cancer selected from r, , colon, , gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, ma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers.

In an embodiment, the invention provides a method for treating a cancer in a subject comprisingadministering to the subject a pharmaceutical composition comprising any of the above antibodies or functional fragments f, antibody conjugates, or binding agents of the invention. In various embodiments, the cancer is selected from bladder, breast, colon, rectal, gastric, esophageal, lung, l5 laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers. In some ments, the cancer is acute myeloid leukemia. In some embodiments, the subject has relapsed or refractory acute myeloid leukemia. In some embodiments, the cancer is associated with increased expression of CD98 on the e of a cell.

In some embodiments, the subject is administered one or more chemotherapeutic compound in combination with the antibody or functional fragment, wherein the chemotherapeutic nd is selected from bendamustine hydrochloride, cyclophosphamide, ifosfamide, fludurabine, cytarabine, gemcitabine, sone, prednisolone, methylprednisolone, paclitaxel, docetaxel, vinorelbine, vincristine, etoposide, irinotecan, anthracycline, adriamycin, cisplatin, carboplatin and rituximab.

[0031] In an embodiment, a method of detecting the presence of CD98 in a biological sample is provided, the method comprising ting the biological sample with any of the above antibodies under ions sive for binding of the antibody to CD98, and detecting whether a complex is formed between the antibody and CD98. In some ments, the biological sample is from a mammal having or suspected of having a cancer of cells or tissues including, but not limited to, bladder, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers.

In an embodiment, a method of diagnosing a cancer associated with increased expression of CD98 is provided, the method sing contacting a test cell with any of the above antibodies; determining the level of expression of CD98 by detecting binding of the antibody to CD98; and comparing the level of expression of CD98 by the test cell with the level of expression of CD98 by a control cell, n a higher level of expression of CD98 by the test cell as compared to the control cell indicates the presence of a cancer associated with increased expression of CD98. In some embodiments, the test cell is a cell from a patient suspected of having a cancer selected from r, breast, colon, rectal, esophageal, lung, laryx, , oral, ovarian, or prostate cancer, or a sarcoma, glioma, lymphoma or leukemia,, or a metatasis of any of these cancers. In an ment, the method comprises determining the level of expression of CD98 on the surface of the test cell and comparing the level of expression of CD98 on the surface of the test cell with the level of expression of CD98 on the surface of the control cell. In some embodiments, the test cell is a cancer cell and the l cell is a normal cell of the same tissue type.

In an embodiment, the ion provides a use of any of the above antibodies or functional fragments in the in the manufacture of a medicament, n the medicament is for use in a method of inhibiting growth of cancer cells that express CD98. In various embodiments, the cells are from a cancer is selected from bladder, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers.

In an embodiment, the ion provides any of the above antibodies or functional fragments for use in inhibiting the growth of cancer cells that s CD98. In various embodiments, the IS cells are from a cancer is selected from bladder, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers.

In an embodiment, the invention provides a use of a pharmaceutical ition comprising any of the above antibodies or functional fragments in the manufacture of a medicament, wherein the medicament is for use in a method of treating cancer in a subject. In various ments, the cancer is selected from bladder, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, ma or leukemia, or a metatasis of any of these cancers. In some embodiments, the cancer is acute myeloid leukemia. In some embodiments, the subject has ed or refractory acute myeloid leukemia. In some embodiments, the cancer is associated with increased expression of CD98 on the surface of a cell. In some embodiments, the t is administered one or more herapeutic compound in combination with the antibody or functional fragment, wherein the chemotherapeutic compound is selected from bendamustine hydrochloride, cyclophosphamide, ifosfamide, fludurabine, cytarabine, gemcitabine, prednisone, solone, methylprednisolone, paclitaxel, docetaxel, vinorelbine, vincristine, etoposide, irinotecan, anthracycline, ycin, cisplatin, carboplatin and rituximab.

In an embodiment, the invention provides a pharmaceutical composition comprising any of the above dies or functional fragments and a pharmaceutically acceptable carrier, for use in treating cancer in a subject. In s embodiments, the cancer is selected from bladder, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers. In some embodiments, the cancer is acute myeloid leukemia. In some embodiments, the subject has relapsed or refractory acute d leukemia. In some embodiments, the cancer is ated with sed expression of CD98 on the surface of a cell. In some embodiments, the subject is administered one or more chemotherapeutic compound in combination with the antibody or functional fragment, wherein the chemotherapeutic compound is selected from bendamustine hydrochloride, cyclophosphamide, ifosfamide, fludurabine, cytarabine, gemcitabine, prednisone, prednisolone, methylprednisolone, paclitaxel, docetaxel, vinorelbine, vincristine, etoposide, irinotecan, anthracycline, adriamycin, tin, carboplatin and U: rituximab.

In an embodiment, the invention provides the use of any of the above antibodies or functional fragments in the manufacture of a medicament, wherein the medicament is for use in a method for detecting the presence of of CD98 in a biological sample. In some embodiments, the method comprisescontacting the biological sample with any of the above antibodies under conditions permissive for binding of the antibody to CD98, and detecting whether a complex is formed between the antibody and CD98. In some embodiments, the biological sample is from a mammal having or suspected of having a cancer of cells or tissues including, but not limited to, bladder, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate , or a sarcoma, melanoma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers.

In an embodiment, the invention provides any of the above antibodies or functional fragments for use in a method of detecting the presence of CD98 in a ical sample. In some embodiments, the method comprisescontacting the biological sample with any of the above antibodies under conditions sive for binding of the antibody to CD98, and detecting whether a complex is formed between the antibody and CD98. In some embodiments, the ical sample is from a mammal having or ted of having a cancer of cells or tissues including, but not limited to, r, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, lymphoma or ia, or a sis of any of these cancers.

In an embodiment, the invention provides the use of any of the above antibodies or functional fragments in the manufacture of a medicament, wherein the medicament is for use in a method of diagnosing a cancer associated with increased expression of CD98. In some embodiments, the method comprises contacting a test cell with any of the above dies; determining the level of expression of CD98 by detecting binding of the antibody to CD98; and ing the level of expression of CD98 by the test cell with the level of sion of CD98 by a control cell, wherein a higher level of expression of CD98 by the test cell as ed to the control cell indicates the presence of a cancer associated with increased expression of CD98. In some embodiments, the test cell is a cell from a t suspected of having a cancer selected from bladder, breast, colon, rectal, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, glioma, lymphoma or leukemia,, or a metatasis of any of these cancers.

In an ment, the method ses determining the level of sion of CD98 on the surface of the test cell and comparing the level of expression of CD98 on the surface of the test cell with the level of expression of CD98 on the surface of the control cell. In some embodiments, the test cell is a cancer cell and the control cell is a normal cell of the same tissue type.

In an embodiment, the ion provides any of the above antibodies or functional fragments for use in a method of diagnosing a cancer associated with increased expression of CD98. In some embodiments, the method comprises contacting a test cell with any of the above antibodies; determining the level of expression of CD98 by detecting g of the antibody to CD98; and ing the level of expression of CD98 by the test cell with the level of expression of CD98 by a control cell, wherein a higher level of expression of CD98 by the test cell as compared to the control cell indicates the presence of a cancer associated with increased expression of CD98. In some embodiments, the test cell is a cell from a patient suspected of having a cancer selected from bladder, breast, colon, rectal, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a a, glioma, lymphoma or Ieukemia,, or a metatasis of any of these cancers. In an embodiment, the method comprises determining the level of expression of CD98 on the surface of the test cell and comparing the level of expression of CD98 on the surface of the test cell with the level of expression of CD98 on the surface of the control cell. In some ments, the test cell is a cancer cell and the control cell is a normal cell of the same tissue type.

In another embodiment of the invention, an article of manufacture, or “kit”, containing materials useful for the ent of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container.

Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds an antibody or an antibody—drug conjugate (ADC) composition which is effective for treating the condition, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibdy or ADC. The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer. Alternatively, or additionally, the article of cture may further comprise a second (or third) container comprising a pharmaceutically—acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered , 's solution and se solution. It may further include other materials ble from a cial and user oint, ing other buffers, diluents, s, needles, and es.

BRIEF DESCRIPTION OF THE FIGURES Fig. I shows the protein expression level of CD98 that was fied and quantified by sTAg analysis in the AML, CLL, CRC specimens and relevant normal controls. Lines indicate the mean of% normalized spectral abundance factor (NSAF) in positive samples.

Fig. 2 is a graph showing the results of epitope binning for 39 anti—CD98 antibodies.

Fig. 3 shows the binding properties of chimeric anti-CD98 monoclonal antibodies 8— 34B, l8—2A 2.l, l8-2A 2.2, and l8-2A 2.7. Fig. 3A is a graph showing the results of epitope g for chimeric anti—CD98 monoclonal antibodies. The four reference antibodies are as in Fig. 1. “lsotype” is a control antibody of the same isotype that does not bind CD98. Fig. 3B shows the Kd of ic anti— CD98 monoclonal antibodies as determined by FACS analysis with colon cancer cell line DLDl. Fig. 3C shows the s of FACS analysis of three AML y tumor samples and a cell line expressing cynomolgus monkey CD98 (cynCD98), stained with chimeric anti—CD98 monoclonal dies.

Fig. 4 shows the construction of the humanized 8—3413 antibodies. Fig. 4A shows the of the sequences of the murine 8—34B light chain variable domain (IGN 34) aligned to the sequence human acceptor sequence (AC) and the humanized light chains L1 and L2. The CDRs according to Kabat numbering are shown in red, and the substitutions in L2 as compared to L1 are ined. Fig. 4A discloses SEQ ID NOS 6, 38, 15-16 and 38, respectively, in order of appearance. Fig. 4B shows the of the sequences of the murine 8—34B heavy chain variable domain (IGN 34) aligned to the sequence human acceptor sequence (AC) and the humanized heavy chains H1, H2 and H3. The CDRs according to Kabat ing are shown in red, and the substitutions in H2 and H3 as ed to H3 are underlined. Fig. 4B discloses SEQ ID NOS 4, 39, 17-19 and 40, respectively, in order of ance.

Fig. 5 shows that D98 antibody treatment induces strong tumor growth inhibition in established Ramos tumors. Tumor volumes at which treatment was initiated increased from (A) ~75mm3, (B) ~150mm3 to (C) 3. Dosing ofthe antibodies was stopped at day 29 (A) or day 22 (B) and tumor regrowth was measured for the duration of the study. Rituximab (anti—CD20 antibody) was used as a positive therapeutic control dy, and antibody HB121 (ATCC) was used as an IgGZa isotype negative control.

Fig. 6 shows that anti—CD98 antibodies prolong significantly time to progression of treated RAMOS tumors. Tumor doubling time of previous tumor regrowth data (Figs. 4A—C) was 2O calculated and used for further prediction of time to progression (TTP). TTP was then extrapolated for each animal within the treatment groups, until 2000mm3 would have been d and graphed as a Kaplan—Meier curve.

Fig. 7 shows the inhibition ofin vivo tumor growth in a lymphoma xenograft by the D98 onal antibody 18-2A as compared to rituxan and a negative control lgG2a. Arrows indicate administration of antibody treatment.

Fig. 8 shows the inhibition of in vivo tumor growth in an acute myeloid leukemia xenograft by the anti—CD98 monoclonal dies 18—2A and 8—34B as compared to a negative control lgG2a. Arrows indicate administration of antibody treatment.

Fig. 9 shows the inhibition of in vivo tumor growth in a colorectal cancer xenograft by the anti—CD98 monoclonal antibody 18—2A as compared to erbitux and a negative control lgG2a (first study) and to DCl 01 + CTX (cyclophosphamide) and a negative control IgGZa (second study). DC101 is a rat anti-mouse VEGFRZ/KDR IgGi mAb (ATCC No. HB-11534) and serves as a positive control.

Arrows te administration of antibody treatment.

Fig. 10 shows the inhibition ofin vivo tumor growth in a non-small cell lung carcinoma xenograft by the D98 monoclonal antibody 18—2A as compared to Erbitux (anti-EGFR) and a negative control IgG2a. Arrows indicate administration of antibody treatment.

Fig. l 1 shows the effect of anti—CD98 monoclonal antibodies on in vivo tumor growth of a lymphoma xenograft in mouse strains with ent immunodeficient backgrounds: (A) NSG mice; (B) NODSCID mice, and (C) SCID mice.

Fig. 12 shows a comparison of the effect of ic anti—CD98 monoclonal antibodies (18-2A—ch7.1 and 8—34B-ch) as compared to their parent murine monoclonal antibodies (18— 2A and 8-34B) on in vivo tumor growth of a lymphoma xenograft.

Fig. 13 illustrates the regions ofthe mouse CD98 sequence (SEQ ID NO: 96) that were substituted into the human CD98 sequence (SEQ ID NO: 1) to form the 13 mouse-human CD98 chimera constructs used to map the epitope on human CD98 bound by humanized monoclonal antibody IGN523.

Fig. 14 shows the binding of humanized monoclonal antibody IGN523 and a control antibody to each of the 13 mouse-human CD98 chimera ucts as determined by FACS analysis.

Fig. 15 shows the sequence of the region of human CD98 within which IGN 523 binds, as identified using the mouse—human CD98 chimera constructs, and the location of this sequence within the three-dimensional structure of CD98. Amino acids T358-G368 (underlined) are buried in the crystal structure and are unlikely to be part of the binding interface. nserved residues between the human and mouse sequences are shown in bold.

Fig. 16 shows the binding of IGN523 to four constructs made by introducing nonhomologous residues from mouse CD98 into the targeted loop region of the human sequence.

Construct 4.1 consists of mutations: l37lL, D374Q, A3756 and on of A376. Construct 4.2 consists of mutations: M383A, and E384K. Construct 4.3 consists of mutations: D391N, F3951, P396F and D397H. Construct 4.4 consists of mutations: G400R, A401 P and A404L. Binding was detected by FACS analysis of CHO cells transfected with the respective constructs.

Fig. 17 shows the binding of IGN523 to single mutation constructs of hydrophobic residues in the targeted loop region. Each indicated hydrophobic residue was tuted with a highly charged amino acid as shown. Binding was detected by FACS is of CHO cells transfected with the respective constructs.

Fig. 18 shows the binding of IGN523 to constructs containing multiple mutations of residues in the ed loop region, as detected by FACS analysis of CHO cells transfected with the tive constructs. M1 containts mutations D374Q, D397H, G400R and A401P. M2 ns mutations D374E and A375 E. M3 contains mutations D397S and 1398T.

Fig. 19 shows the results of a variable—length peptide screen for epitope mapping of humanized monoclonal antibody . ELISA results for each peptide are shown as a horizontal line.

Start and end points of the lines indicate which residues are included in the e. The Y—value of the line shows the ELISA result ed for that peptide. The results indicated dominant binding for 395FPDIPGA401 and secondary binding for 379PGQP382 (shaded s).

Fig. 20 shows the results of a best—binding single—positions alanine-replacement peptide set. Each residue was ed by A (or G if the orginal amino acid was A). The height at which the replacement letter is d in the graph is the obtained ELISA value for that mutated peptide. The central line and shaded interval indicate the reference ELISA value.

Fig. 21 shows heat maps representing the data obtained from CLIPS conformational matrix structures that combined two partial sequences of human CD98 (SEQ ID NOS 45—59 shown on the X axis, and SEQ ID NOS 60—74 shown on the Y axis).

Fig. 22 shows the results of a mutagenesis screen of strongly-binding es from the matrix analyses shown in Fig. 21. SEQI shows the sequence of the peptide and DIF l indicates where the on is located in the peptide. Grey flelds indicate peptides having non—mutated ces. The last column shows the ence in ELISA value between wild-type and mutated peptide. High values l0 indicate that the mutation has a strong negative effect on binding.

Fig. 23 shows the location in the sequence and on the surface of human CD98 of the amino acid residues determined to be important for binding of humanized monoclonal antibody IGN523.

Fig. 23A shows the location in the sequence of residues determined by the chimera and nesis studies (bold), by Pepscan analysis (gray) or both (shaded). Fig. 238 shows the location of the es determined by the chimera and mutagenesis studies (dark gray). Fig. 23C shows the location of residues determined by Pepscan analysis (light gray). Fig. 23D shows the overlap of both sets of residues (black).

Fig. 24 shows the tion of in vivo tumor growth in a RAMOS (RA. 1) Burkitt lymphoma xenografi by the humanized monoclonal antibody IGN523 as compared to Rituximab and a negative control IgG. Antibodies were dosed interperitoneally at 10 mg/kg on days 1 l, 17 and 25. 2O Arrows te administration of antibody treatment.

Fig. 25 shows the inhibition of in vivo tumor growth in a DAU Burkitt lymphoma xenograft by the humanized monoclonal antibody IGN523 as compared to rituxan and a negative control IgG. Antibodies were dosed interperitoneally at 10 mg/kg on days 20 and 26. Arrows indicate stration of antibody treatment.

[0067] Fig. 26A shows the inhibition ofin vivo tumor growth in a IGN-LNG-l 2 lung tumor xenograft by the humanized monoclonal antibody IGN523 as compared to carboplatin and a negative control IgG. IGN523 and carboplatin were dosed interperitoneally on days 17, 24 and 31 at 10 mg/kg or 75 mg/kg, respectively. Arrows indicate administration of treatment. Fig. 268 shows body weight measurements corresponding to mice in Fig. 26A treated with the indicated reagents. latin was dosed at its maximum tolerated dose, which induced body weight loss in ID mice.

Fig. 27 shows the inhibition of in vivo tumor growth in a KG—l acute myeloid leukemia xenograft by the humanized monoclonal antibody IGN523 as compared to n and a negative control IgG. Antibodies were dosed interperitoneally at 15 rug/kg on days 21, 28 and 34.

Arrows indicate administration of antibody treatment.

Fig. 28 shows dose dependent inhibition ofin vivo tumor growth in a lung tumor xenograft by the humanized onal antibody 1GN523. The antibody was dosed intraperitoneally at the indicated doses on days 12 and 19.Arrows indicate stration of antibody treatment.

Fig. 29 shows staining of human and cynomolgus monkey frozen tissue sections by humanized monoclonal dy IGN523. Cryosections of human and cynomolgus monkey kidney, cerebrum, and placenta were d with 10 ug/mL of 1GN523. Modifications of the methods of Tuson, Fung, and Hierck for immunohistochemistry were used to eliminate the requirement for ng of 1GN523 and to preclude cific reactivity between the secondary labeled anti—human IgG and IgG endogenous to the tissues to be examined (Fung 1992, Hierck 1994, Tuson 1990). Sections were cut at approximately 5 um. All slides were lly assessed for the adequacy of tissue elements and staining, then evaluated and subjectively graded by the Study Pathologist for intensity of staining. Representative images are shown at 40x magnification with the exception of human um (20X).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION General Techniques The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies bed in Sambrook et al., lar Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in lar Biology (F. M. Ausubel, et al. eds., (2003)); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Z. An, ed, Wiley, Hoboken NJ. (2009); Monoclonal Antibodies: Methods and Protocols, M. Albitar, ed., Humana Press, Totawa, NJ. (2010); and Antibody Engineering, 2nd Ed, Vols 1 and 2, Kontermann and Dubel, eds., Springer-Verlag, Heidelberg, 2010.

DEFINITIONS AND ABBREVIATIONS Definitiglfi For purposes of reting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the tion set forth below shall control.

The term , as used herein, refers to any native CD98 from any vertebrate , including mammals such as primates (e.g. humans, cynomolgus monkey (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. The amino acid and encoding c acid sequences of human CD98 are provided below as SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0074] MSQDTEVDMKEVELNELEPEKQPMNAASGAAMSLAGAEKNGLVKIKVAE DEAEAAA AAKFTGLSKEELLKVAGS PGWVRTRWALLLLFWLGWLGMLAGAVVI IVRAPRCRELPAQKWWHTGALYRI GDLQAFQGHGAGNLAGLKGRLDYLSSLKVKGLVLGPIHKNQKDDVAQTDLLQI DPNFGSKEDFDSLLQSA KKKS IRVILDLTPNYRGENSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDIENLKDAS SFLAEWQNI T KGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGSTGEHTKSLVTQYLNATGNRWCSWSLSQ FLPAQLLRLYQLMLFTLPGTPVFSYGDEIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANM TVKGQSEDPGSLLSLFRRLSDQRSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAG LQASDLPASASLPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA(SEQIDIQOJ) w07fl ATGAGCCAGGACACCGAGGTGGATATGAAGGAGGTGGAGCTGAATGAGTTAGAGCC CGAGAAGCAGCCGATGAACGCGGCGTCTGGGGCGGCCATGTCCCTGGCGGGAGCCGAGAAGAATGGTCTG GTGAAGATCAAGGTGGCGGAAGACGAGGCGGAGGCGGCAGCCGCGGCTAAGTTCACGGGCCTGTCCAAGG TGCTGAAGGTGGCAGGCAGCCCCGGCTGGGTACGCACCCGCTGGGCACTGCTGCTGCTCTTCTG GCTCGGCTGGCTCGGCATGCTTGCTGGTGCCGTGGTCATAATCGTGCGAGCGCCGCGTTGTCGCGAGCTA CCGGCGCAGAAGTGGTGGCACACGGGCGCCCTCTACCGCATCGGCGACCTTCAGGCCTTCCAGGGCCACG GCGCGGGCAACCTGGCGGGTCTGAAGGGGCGTCTCGATTACCTGAGCTCTCTGAAGGTGAAGGGCCTTGT GCTGGGTCCAATTCACAAGAACCAGAAGGATGATGTCGCTCAGACTGACTTGCTGCAGATCGACCCCAAT TTTGGCTCCAAGGAAGATTTTGACAGTCTCTTGCAATCGGCTAAAAAAAAGAGCATCCGTGTCATTCTGG ACCTTACTCCCAACTACCGGGGTGAGAACTCGTGGTTCTCCACTCAGGTTGACACTGTGGCCACCAAGGT GAAGGATGCTCTGGAGTTTTGGCTGCAAGCTGGCGTGGATGGGTTCCAGGTTCGGGACATAGAGAATCTG AAGGATGCATCCTCATTCTTGGCTGAGTGGCAAAATATCACCAAGGGCTTCAGTGAAGACAGGCTCTTGA TTGCGGGGACTAACTCCTCCGACCTTCAGCAGATCCTGAGCCTACTCGAATCCAACAAAGACTTGCTGTT GACTAGCTCATACCTGTCTGATTCTGGTTCTACTGGGGAGCATACAAAATCCCTAGTCACACAGTATTTG AATGCCACTGGCAATCGCTGGTGCAGCDGGAGTTTGTCTCAGGCAAGGCTCCTGACTTCCTTCTTGCCGG CTCAACTTCTCCGACTCTACCAGCTGATGCTCTTCACCCTGCCAGGGACCCCTGTTTTCAGCTACGGGGA TGAGATTGGCCTGGATGCAGCTGCCCTTCCTGGACAGCCTATGGAGGCTCCAGTCATGCTGTGGGATGAG TCCAGCTTCCCTGACATCCCAGGGGCTGTAAGTGCCAACATGACTGTGAAGGGCCAGAGTGAAGACCCTG GCTCCCTCCTTTCCTTGTTCCGGCGGCTGAGTGACCAGCGGAGTAAGGAGCGCTCCCTACTGCATGGGGA CTTCCACGCGTTCTCCGCTGGGCCTGGACTCTTCTCCTATATCCGCCACTGGGACCAGAATGAGCGTTTT GTGCTTAACTTTGGGGATGTGGGCCTCTCGGCTGGACTGCAGGCCTCCGACCTGCCTGCCAGCG CCAGCCTGCCAGCCAAGGCTGACCTCCTGCTCAGCACCCAGCCAGGCCGTGAGGAGGGCTCCCCTCTTGA GCTGGAACGCCTGAAACTGGAGCCTCACGAAGGGCTGCTGCTCCGCTTCCCCTACGCGGCCTGA(SEQID N02) The term “CD98” encompasses “full—length,” unprocessed CD98 as well as any form of CD98 that results from processing in the cell. The term also encompasses naturally occurring variants or mutations of CD98, e.g., splice variants, allelic variants, SNP ts and isoforms. The CD98 polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. A "native sequence CD98 ptide" comprises a polypeptide having the same amino acid ce as the ponding CD98 polypeptide derived from nature. Such native sequence CD98 polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence CD98 polypeptide” specifically encompasses naturally—occurring truncated or secreted forms of the specific CD98 polypeptide (cg, an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide.

The term "antibody” is used in the broadest sense and specifically covers, for example, single anti-CD98 monoclonal antibodies (including agonist, nist, neutralizing antibodies, full length or intact monoclonal antibodies), anti—CD98 antibody itions with polyepitopic city, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain anti—CD98 antibodies, and fragments of anti-CD98 antibodies, as defined below.

The term “immunoglobulin” (lg) is used interchangeable with antibody herein. An antibody can be human, humanized and/or affinity matured. [0078} An “antigen” is a predetermined antigen to which an dy can selectively bind.

The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide. [0079} An antibody “which binds” an antigen of interest is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross—react with other proteins. In such embodiments, the extent of binding of the antibody to a “non—target” protein will be less than about 10% of the g of the antibody to its ular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an dy to a target molecule, the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a ecific interaction. Specific binding can be ed, for example, by determining binding ofa molecule compared to g ofa control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non—labeled target. In this case, c binding is indicated ifthe g of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10'4 M, alternatively at least about 10‘5 M, atively at least about 10'6 M, alternatively at least about 10‘7 M, atively at least about 10'8 M, alternatively at least about 10'9 M, atively at least about 10"0 M, alternatively at least about 10'11 M, alternatively at least about 10’12 M, or greater. In one embodiment, the term “specific binding” refers to g where a molecule binds to a particular polypeptide or e on a particular polypeptide t substantially binding to any other polypeptide or polypeptide epitope. [0080} The term “anti—CD98 antibody” or “an antibody that binds to CD98” refers to an antibody that is capable of binding CD98 with sufficient affinity such that the antibody is useful as a diagnostic and/or eutic agent in targeting CD98. Preferably, the extent of g of an anti-CD98 antibody to an unrelated, non-CD98 protein is less than about 10% of the binding of the antibody to CD98 as measured, e.g., by fluorescence activated cell sorting (FACS) analysis or a radioimmunoassay (RIA). An antibody that “specifically binds to” or is “specific for” CD98 is defined as above. In n embodiments, an antibody that binds to CD98 has a dissociation nt (Kd) of S 1 MA, 5 100 nM, S nM, S 1 nM, or 5 0.1 nM. In certain embodiments, anti-CD98 antibody binds to an epitope of CD98 that is conserved among CD98 from different species.

An "isolated antibody" is one that has been identified and separated and/or recovered from a component of its natural nment. Contaminant components of its natural environment include, but are not limited to, materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method (Lowry et al., J. Bio. Chem. 193: 265-275, 1951) and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup ator, or (3) to homogeneity by SDS—PAGE under ng I5 or ucing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. [0082} The basic 4-chain dy unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case ofIgGs, the n unit is generally about 0 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more de bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the 0t and y chains and four CH domains for u and 8 es. Each L chain has at the N—terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first nt domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the ure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

The “variable region” or “variable ” or “V domain” of an dy refers to the terminal s ofthe heavy or light chain ofthe antibody. The variable domain ofthe heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” The term "variable" refers to the fact that n segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its ular antigen. However, the variability is not evenly distributed across the l 10—amino acid span of the variable domains. Instead, the V regions consist of WO 78377 2012/066347 relatively invariant stretches called framework regions (FRs) of 1530 amino acids separated by r regions of extreme variability called “hypervariable regions” that are each 9—12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a B—sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the B—sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunolooical Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991)). The constant domains are not ed directly in binding an dy to an antigen, but exhibit various or functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). [0084} An “intact” antibody is one comprising an antigen—binding site as well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e. g. human native sequence constant domains) or amino acid sequence variant f.

Preferably, the intact antibody has one or more effector functions.

"Antibody nts" se a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include, without tion, Fab, Fab”, F(ab')2, and Fv fragments; diabodies and di—diabodies (see, e.g. Holliger, P. et a1 (1993) Proc. Natl. Acad. Sci. 90:6444-8; Lu, D. et al. (2005) J. Biol. Chem. 280: 19665—72; Hudson et al., Nat. Med. 9:129-134 (2003); WO 93/11161; and U.S. Patent Nos. 5,837,242 and 6,492,123); single—chain antibody molecules (see, e.g. US. Patent Nos. 4,946,778; 5,260,203; 5,482,858 and ,476,786); dual variable domain antibodies (see, e.g. US. Patent No. 7,612,181); single le domain antibodies (SdAbs) (see, e.g. n et al., Immunogenetics 50: 98-101, 1999; Streltsov et al., Proc Natl Acad Sci USA. 444—12449, 2004); and multispecific antibodies formed from antibody fragments. [0086} A “functional fragment” of a therapeutic antibody will t at least one if not some or all of the ical functions attributed to the intact antibody, the function comprising at least specific binding to the target antigen.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier "monoclonal" is not to be construed as ing production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first bed by Kohler et al., , 2562495 (1975), or may be made using recombinant DNA s in bacterial, eukaryotic animal or plant cells (see, e.g., US.

Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody ies using the ques described in Clackson et al., Nature, 352:624—628 (1991) and Marks et al., J. Mol.

Biol., 222:581—597 (1991), for example. [0088} The monoclonal antibodies herein include ”chimeric" dies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies d from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another dy class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see US. Patent No. 567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851—6855 (1984)). [0089} "Humanized" forms of non—human (e.g., rodent) antibodies are chimeric antibodies that contain l sequence d from the non—human antibody. For the most part, humanized antibodies are human immunoglobulins ient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non—human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, corresponding non-human es e framework region (FR) residues of the human immunoglobulin. Furthermore, humanized antibodies may comprise es that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will se substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non—human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized dy optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that ofa human immunoglobulin. For further details, see Jones et al., Nature 3212522—525 (1986); Riechmann et al., Nature 332:323—329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review es and patents and references cited therein: Vaswani and Hamilton, Ann.

Allergy, Asthma and Immunol, 1:105-1 15 (1998); Harris, Biochem. Soc. Transactions, 23: 103 5—1038 (1995); Almagro and Fransson, Front. Biosci. 13: 1619—1633 (2008); US. Patent Nos. 5,585,089; ,693,762; 6,1803 70; and 6,054,297). [0090} A “human antibody” is one which ses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody sing man antigen~binding residues. Human antibodies can be produced using various ques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991) and yeast display libraries (Chao et al., Nature Protocols 1: 755—768 (2006)). Also available for the preparation of human monoclonal dies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al, J. Immunol, 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such dies in response to nic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, A., Curr. Opin. Biotechnol. 1995, 6(5):561-6; emann and Taussing, Curr. Opin. Biotechnol. 1997, 8(4):455-8; and US. Pat. Nos. 6,075,181 and 584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3 557-3562 (2006) regarding human antibodies generated via a human B—cell hybridoma technology.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an dy variable domain that are hypervariable in sequence and/or form urally defined loops. Generally, antibodies comprise six hypervariable s; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on ce variability and are the most commonly used (Kabat et al., Sequences eins ofImmunological Interest, 5th Ed. Public Health Service, al Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 1962901—917 ).

The end of the Chothia CDR—Hl loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; ifneither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable s represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” hypervariable regions are based on an analysis of the ble complex crystal structures. The residues from each of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact Ll L24-L34 L24-L34 L26—L32 L30—L36 L2 L50-L56 L50—L56 L50—L52 L46-L55 L3 L89-L97 L89-L97 L91—L96 6 H1 H31—H35B H26—H3SB H26—H32 H30—H35B (Kabat Numbering) H1 5 H26-H35 H26-H32 H30—H35 ia Numbering) H2 H50-H65 1150-1158 H53-H55 H47—H58 H3 H95-H102 H95-H102 H96-H101 H93—H1 01 [0092} Hypervariable regions may comprise “extended hypervariable regions” as follows: 24—36 or 24-34 (L1), 46-56 or 50—56 (L2) and 89-97 or 89—96 (L3) in the VL and 26—35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93—102, 94—102, or 95—102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions. As used , the terms “HVR” and “CDR” are used interchangeably. [0093} “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues herein defined.

The term “variable domain residue numbering as in Kabat" or “amino acid position numbering as in Kabat”, and variations thereof, refers to the ing system used for heavy chain variable domains or light chain variable domains of the compilation of dies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health e, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR ofthe variable domain. For e, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after e 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of gy of the sequence of the antibody with a “standar ” Kabat numbered sequence.

The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1—107 of the light chain and residues 1-1 13 of the heavy chain) (e.g, Kabat et (1]., Sequences ofImmunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an globulin heavy chain nt region (e.g, the EU index reported in Kabat et (1]., supra). The “EU index as in Kabat” refers to the residue ing of the human IgG1 EU antibody. Unless stated otherwise , nces to residue numbers in the variable domain of antibodies means residue ing by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system.

An “affinity d” antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the y of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For review, see Hudson and Souriau, Nature Medicine 9 :129— 134 (2003); Hoogenboom, Nature Biotechnol. 23 : 1105-1 1 16 (2005); Quiroz and Sinclair, Revista lngeneria Biomedia 4 : 39-51 (2010).

A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of st.

“Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site ofa molecule (e.g, an antibody) and its binding partner (e.g, an n). Unless ted otherwise, as used herein, “binding affinity” refers to intrinsic g affinity which reflects a 1:] interaction between members of a binding pair (e.g,, antibody and antigen).

The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

Low—affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high— affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of ing binding affinity are known in the art, any of which can be used for es of the t invention. Specific illustrative embodiments are described in the following. [0100} “Or better” when used herein to refer to binding affinity refers to a stronger binding between a molecule (e.g. antibody) and its binding partner, and is represented by a r numerical Kd value. For example, an antibody which has an affinity for an antigen of “.6 nM or better”, the dy’s affinity for the antigen is <.6 nM, i.e. .59 nM, .58 nM, .57 nM etc. or any value less than .6 nM. [0101} In one embodiment, the “Kd” or “Kd value” according to this invention is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay that measures solution g y of Fabs for antigen by equilibrating Fab with a l concentration of(1251)-labeled n in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti—Fab antibody—coated plate (Chen, et al., (1999).]. Mol Biol 293:865-881). According to another embodiment the Kd or Kd value is measured by using surface plasmon resonance assays using, for example, a BIAcoreTM—2000 or a BlAcoreTM-3 000 (BlAcore, lnc., Piscataway, NJ). [0102} An “on-rate” or “rate of association” or “association rate” or “k0 according to this ion can also be determined with the same surface plasmon resonance technique described above using, for example, a BIAcoreTM—2000 or a BIAcoreTM-3000 (BIAcore, Inc., away, NJ).

The phrase antially similar,” or “substantially the same”, as used herein, denotes a sufficiently high degree of similarity between two numeric values ally one associated with an antibody of the invention and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic ed by said values (e.g., Kd values). The difference between the two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% as a function of the value for the reference antibody. [0104} The phrase “substantially reduced,” or “substantially different”, as used herein, 3O denotes a sufficiently high degree of difference between two c values (generally one associated with an antibody of the invention and the other associated with a reference antibody) such that one of skill in the art would consider the ence between the two values to be of statistical significance within the t of the biological characteristic measured by said values (e.g., Kd values, HAMA se). The difference between said two values is preferably greater than about 10%, preferably greater than about 20%, preferably r than about 30%, preferably greater than about 40%, preferably greater than about 50% as a function of the value for the reference antibody.

An antibody that “inhibits the growth of tumor cells sing a CD98 polypeptide” or a “growth inhibitory” antibody is one which results in measurable growth inhibition of cancer cells expressing or pressing the appropriate CD98 polypeptide. The CD98 polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferred growth inhibitory anti-CD98 antibodies inhibit growth of CD98-expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by r than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibody being tested. In one embodiment, growth inhibition can be measured at an antibody concentration of about 0.1 to 30 ug/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the dy. Growth inhibition of tumor cells in viva can be ined in various ways such as is described below. The antibody is growth inhibitory in vivo if administration of the anti-CD98 antibody at about 1 ug/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

An antibody that "induces apoptosis" is one that s programmed cell death as determined by binding of annexin V, ntation of DNA, cell shrinkage, dilation of endoplasmic lum, cell fragmentation, and/or formation of membrane vesicles (called tic bodies). The cell is usually one that overexpresses a CD98 polypeptide. Preferably the cell is a tumor cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA ntation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. ably, the antibody which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding ve to untreated cell in an annexin binding assay. [0107} An antibody that “induces cell death” is one that causes a viable cell to become nonviable. The cell is of a cell type that specifically expresses or overexpresses a CD98 polypeptide.

The cell may be cancerous or a normal cell of the particular cell type. The CD98 ptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is ed and ed by a cancer cell. Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by dy-dependent cell— mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be performed using heat inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether the antibody is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue (see Moore et al.

Cytotechnology 1721-1 1 (1995)) or 7AAD can be assessed relative to untreated cells. Preferred cell death-inducing antibodies are those which induce PI uptake in the PI uptake assay in BT474 cells.

WO 78377 Antibody “effector functions” refer to those ical activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of dy effector functions e: Clq binding and complement dependent cytotoxicity; Fc or binding; antibody—dependent cell—mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell or); and B cell activation.

The term “Fc region” herein is used to define a C-terminal region of an globulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C—terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise dy populations with all K447 residues removed, dy populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as disclosed, for example, in definitions herein A e sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native ce human Fc regions include a native ce human IgG] Fc region (non—A and A allotypes); native sequence human IgG2 Fc region; native sequence human lgG3 Fc region; and native sequence human lgG4 Fc region as well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid ce which s from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution 3O compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and ably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native ce Fc region and/or with an Fc region of a parent ptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

Antibody—dependent cell—mediated xicity” or “ADCC” refers to a form of cytotoxicity in which secreted I g bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen—bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are tely required for such g. The primary cells for mediating ADCC, NK cells, express Fclell only, whereas monocytes express Fcle, Fclel and Fclell. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and U) Kinet, Annu. Rev. lmmunol. 91457—92 (1991). To assess ADCC activity ofa molecule ofinterest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed.

Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. (USA) 952652—656 (1998).

[0114] “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an dy. The red FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an lgG dy (a gamma receptor) and includes receptors of the Fcle, Fclel and Fclell subclasses, including allelic variants and alternatively spliced forms of these receptors. Fclel ors include FclelA (an "activating receptor”) and FcyRIlB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic s thereof (see review M. in Daeron, Annu. Rev. lmmunol. 15:203—234 (1997)). FcRs are ed in Ravetch and Kinet, Annu. Rev. l. 9:457-492 (1991); Cape] et al., lmmunomethods 4:25-34 (1994); and de Haas et al., J. Lab.

Clin. Med. 0—41 (1995). Other FcRs, including those to be identified in the future, are assed by the term "FcR" herein. The term also es the neonatal receptor, FcRn, which is sible for the transfer of maternal lgGs to the fetus (Guyer et al., J. Immunol. 1 17:5 87 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Antibody variants with ed or diminished binding to FcRs are described, for example, in , and US. Patent Nos. 7,183,387; 7,332,581; and 7.335,742. See also, e.g., Shields et al. J Biol. Chem. 9(2):6591—6604 (2001).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate n. To assess complement tion, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. lmmunol. Methods 2021163 (1996), may be performed. ptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc ) and increased or decreased C1q binding capability are described, e.g., in US Patent No. 6,194,551 Bl and WO 1999/51642. See also, e.g., ldusogie et a]. J. Immunol. 164: 4178—4184 (2000).

The CD98 polypeptide “extracellular domain” or “ECD” refers to a form of the CD98 polypeptide that is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a CD98 polypeptide ECD will have less than 1% of such transmembrane and/or cytoplasmic s and preferably, will have less than 0.5% of such domains. The transmembrane domain of CD98 comprises amino acid residues 76—103 (Parmacek et al., Nucleic Acids Res. 17: 1915-1931, 1989). The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified. Optionally, therefore, an extracellular domain of a CD98 polypeptide may se amino acids from about 98-108 to 529 of the sequence of CD98 as disclosed in Parmacek et al, supra.

“Percent (%) amino acid ce identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate ce that are with the amino acid residues in the reference polypeptide ce, after aligning the sequences and ucing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. ent for purposes of determining percent amino acid sequence identity can be achieved in s ways that are within the skill in the art, for instance, using publicly ble computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning the full length of the sequences, including any thms needed to achieve maximal alignment over sequences being compared.

A “modification” of an amino acid residue/position, as used herein, refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/positions. For example, typical ations include substitution of the e with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (generally fewer than 5 or 3) amino acids nt to said residue/position, and deletion of said residue/position.

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

An antibody binds "essentially the same e" as a reference antibody, when the two antibodies recognize cal or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody. Usually, the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.

“Epitope mapping” is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes may be linear epitopes or conformational epitopes.

Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three—dimensional ure.

“Epitope binning”, as defined herein, is the process of grouping antibodies based on the epitopes they ize. More particularly, epitope binning comprises methods and systems for discriminating the epitope recognition properties of different antibodies, combined with computational processes for clustering antibodies based on their epitope recognition properties and identifying antibodies having distinct g specificities.

A "disorder" is any condition or disease that would benefit from treatment with an substance/molecule or method of the invention. This includes chronic and acute disorders including those pathological conditions which predispose the mammal to the disorder in question. Non—limiting examples of disorders to be treated herein include cancerous ions such as r, breast, colon, rectal, gastric, esophageal, lung, laryx, , oral, n, or prostate cancer, or a sarcoma, melanoma, glioma, lymphoma or ia, as well as ases of these cancers.

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

[0125] “Tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and ous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative er” and “tumor” are not mutually exclusive as referred to herein.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or id malignancies.

More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung , adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, astoma, cervical cancer, ovarian cancer, oral cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland oma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic oma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B—cell lymphoma, brain cancer, as well as head and neck cancer, and associated metastases.

A “CD98-expressing cell” is a cell that expresses nous or transfected CD98 on the cell surface. A “CD98—expressing cancer” is a cancer comprising cells that have CD98 protein present on the cell surface. A “CD98-expressing cancer” produces sufficient levels of CD98 on the surface of cells thereof, such that an anti-CD98 antibody can bind thereto and have a eutic effect with respect to the cancer. A cancer that “overexpresses” CD98 is one that has significantly higher levels of CD98 at the cell surface thereof, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. CD98 overexpression may be determined in a diagnostic or prognostic assay by ting increased levels of the CD98 protein present on the surface of a cell (e.g. via an immunohistochemistry assay; FACS analysis). Alternatively, or additionally, one may e levels of CD98-encoding nucleic acid or mRNA in the cell, e.g. via fluorescent in situ hybridization; (FISH; see WO98/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR R). Aside from the above assays, s in vivo assays are available to the skilled tioner. For example, one may expose cells within the body of the t to an antibody which is optionally labeled with a detectable label, e.g. a radioactive e, and binding of the antibody to cells in the patient can be evaluated, e.g. by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody. A CD98—expressing cancer includes, but is not limited to, bladder, , colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate , or a sarcoma, melanoma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers.

As used herein, “treatment” (and variations such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course ofthe individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment e ting occurrence or recurrence of e, alleviation of symptoms, diminishment of any direct or indirect pathological uences of the disease, preventing metastasis in the case of cancer, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development ofa disease or disorder or to slow the progression ofa disease or disorder.

The above parameters for ing successful treatment and improvement in the e are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or by determining the response rate (RR). Other nts for measuring efficacy include, for example, overall survival (OS), disease-free survival (DFS) and recurrence-free (or relapse-free) survival (RFS). Metastasis can be determined by staging tests and by bone scan and tests for m level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the e include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).

An “individual” is a vertebrate. In certain embodiments, the rate is a mammal.

Mammals e, but are not d to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, a mammal is a human.

An "effective " refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic or prophylactic result. A “therapeutically effective amount” of a substance/molecule of the invention may vary according to factors such as the disease state, age, sex, and weight ofthe individual, and the ability ofthe substance/molecule, to elicit a desired response in the individual. A eutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of e, the prophylactically ive amount would be less than the eutically effective amount. In the case of cancer, the therapeutically 2012/066347 effective amount of the drug may, for example, reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (tie, slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See preceding definition of “treating”. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or xic.

"Chronic" administration refers to administration of the agent(s) in a uous mode as d to an acute mode, so as to maintain the initial therapeutic effect (activity) for an ed period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and utive administration in any order.

"Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the logically acceptable carrier is an aqueous pH ed solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) ptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, gine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, hylene glycol (PEG), and PLURONICSTM.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the ation would be administered.

Such formulation may be sterile.

A “sterile” formulation is aseptic of free from all living microorganisms and their spores. An tive amount” of an antibody as disclosed herein is an amount sufficient to carry out a ically stated purpose. An “effective amount” may be determined cally and in a routine manner, in relation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of an dy or other drug effective to ” a e or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of “treating”. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. A “prophylactically effective amount” refers to an amount effective, at s and for periods of time necessary, to achieve the desired prophylactic result. lly but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically ive amount.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes ction of cells. The term is intended to include radioactive isotopes (e.g., At“, 1131, 1125, Y90, Rem, Rexgg, Sm153, Bim, P32 and radioactive isotopes of Lu), herapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and nts thereof such as nucleolytic s, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, , plant or animal origin, including fragments and/or variants thereof, and the various mor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells. {0139] A “toxin” is any substance capable of having a ental effect on the growth or proliferation of a cell.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy. Examples of chemotherapeutic agents e, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and amelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); deltatetrahydrocannabinol (dronabinol, MARINOL®); beta~ one; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-ll (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9—aminocamptothecin); bryostatin; callystatin; CC-lO65 (including its adozelesin, carzelesin and bizelesin synthetic ues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the tic analogues, KW-2189 and l); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; en mustards such as mbucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, omycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5—oxo-L—norleucine, ADRIAMYClN®, doxorubicin (including morpholino-doxorubicin, orpholino—doxorubicin, 2— pyrrolino—doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, ycin, potflromycin, puromycin, ycin, rodorubicin, streptoni grin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti—metabolites such as methotrexate and 5—fluorouracil (5-FU); folic acid analogues such as denopterin, rexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6—mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, dine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti—adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; xate; defofamine; demecolcine; uone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; bicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide x (JHS Natural Products, , Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; quone; 2,2,2"— trichlorotriethylamine; trichothecenes (especially T—2 toxin, verracurin A, n A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; s, e.g., TAXOL® paclitaxel ol— Myers Squibb Oncology, Princeton, NJ), ABRAXANETM Cremophor-free, albumin—engineered nanoparticle formulation of axel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, ); chloranbucil; gemcitabine (GEMZAR®); 6—thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; Vinblastine (VELBAN®); platinum; etoposide (VP-l6); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; onate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); ids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as ations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide; doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5—FU and leucovovin. Additional chemotherapeutic agents e cytotoxic agents useful as antibody drug conjugates, such as maytansinoids (DM1 and DM4, for example) and auristatins (MMAE and MMAF, for example).

Also included in the definition of "chemotherapeutic agent" are: (i) anti-hormonal agents that act to regulate or inhibit e action on tumors such as anti-estrogens and selective estrogen receptor tors (SERMs), including, for example, tamoxifen ding NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and ON® ifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)- imidazoles, aminoglutethimide, MEGASE® (megestrol e), AROMASIN® (exemestane; Pfizer), formestanie, ole, RIVISOR® (vorozole), FEMARA® (letrozole; is), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti—androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3—dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, pha, Raf and H—Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) mes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HERZ expression tors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rlL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti—angiogenic agents such as bevacizumab (AVASTlN®, Genentech); and pharmaceutically acceptable salts, acids and tives of any of the above.

[0142] The term "prodrug" as used in this application refers to a precursor or derivative form of a compound of the ion that may be less xic to cells compared to the parent compound or drug and is capable of being enzymatically or hydrolytically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 7, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate—containing prodrugs, thiophosphate-containing prodrugs, e—containing prodrugs, peptide-containing gs, D-amino acid-modified prodrugs, glycosylated prodrugs, am—containing gs, optionally substituted phenoxyacetamide-containing prodrugs, optionally substituted phenylacetamide-containing prodrugs, 5— fluorocytosine and other 5~fluorouridine prodrugs which can be ted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, compounds of the invention and herapeutic agents such as described above.

A “small molecule” is defined herein to have a molecular weight below about 500 Daltons.

An "isolated nucleic acid" is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native ce. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized ues or analogues biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule.

"Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The tides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide U! may comprise modified nucleotides, such as methylated nucleotides and their analogs.

“Oligonucleotide,” as used herein, generally refers to short, lly single-stranded, generally tic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms nucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0146] The cell that produces an anti—CD98 antibody of the invention will include the parent oma cell e.g., the omas that are deposited with the ATCC, as well as bacteria] and eukaryotic host cells into which nucleic acid encoding the antibodies have been introduced. Suitable host cells are sed below.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

COMPOSITIONS AND METHODS OF MAKING THE SAME

[0148] Antibodies that bind to CD98 are provided. Immunoconjugates comprising anti-CD98 antibodies are provided. dies and immunoconjugates of the invention are useful, e.g., for the diagnosis or treatment of disorders associated with altered expression, e.g., increased expression, of CD98. In certain embodiments, antibodies or immunoconjugates of the invention are useful for the diagnosis or treatment of a cell proliferative disorder, such as cancer.

Anti—CD98 Antibodies In one ment, the t invention provides anti—CD98 antibodies that may find use herein as therapeutic agents. Exemplary antibodies e polyclonal, onal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having ed affinity or other properties. 1. onal Antibodies The antibodies of the invention may comprise polyclonal antibodies. s of preparing polyclonal antibodies are known to the skilled artisan. onal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.

Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple aneous or intraperitoneal injections. The immunizing agent may include the CD98 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be genic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to e limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of nts which may be employed include Freund's complete adjuvant and MPL- TDM nt (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art Without undue experimentation. The mammal can then U! be bled, and the serum assayed for CD98 antibody titer. If desired, the mammal can be boosted until the dy titer increases or plateaus. 2. Monoclonal Antibodies The antibodies of the invention may alternatively be monoclonal antibodies. Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., US. Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, cytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59—lO3 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the al myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT—deficient cells. red fusion r myeloma cells are those that fuse efficiently, support stable evel production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that s t the unfused parental cells. Preferred myeloma cell lines are murine a lines, such as those derived from MOPC—2l and MPC—l 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 and derivatives e.g., X63—Ag8- 653 cells available from the American Type e Collection, Manassas, Virginia, USA. Human myeloma and mouse-human myeloma cell lines also have been described for the production of human onal antibodies r, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51—63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of onal antibodies directed against the antigen. ably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme—linked sorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 1071220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are fied, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59—103 (Academic Press, 1986)). le culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.

In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g,, by i.p. injection of the cells into mice.

The monoclonal antibodies ed by the subclones are suitably separated from the culture medium, s fluid, or serum by tional antibody purification ures such as, for example, affinity chromatography (e. g., using n A or protein G-Sepharose) or ion—exchange chromatography, hydroxylapatite chromatography, gel ophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and ced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The oma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the sis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 52256-262 (1993) and Plfickthun, Immunol. Revs. 130:151-188 (1992).

[0159] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage ies generated using the techniques bed in, e.g, Antibody Phage Display: Methods and Protocols, PM. O’Brien and R. Aitken, eds, Humana Press, Totawa N.J., 2002. In principle, synthetic antibody clones are selected by screening phage ies containing phage that display various fragments of dy variable region (Fv) fused to phage coat protein. Such phage libraries are screened for against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the nding clones in the library. The g clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.

Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non—covalently, as described in Winter et al., Ann. Rev. l., 12: 433-455 (1994).

Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage ies, which can then be searched for antigen- binding clones as described in Winter et al., supra. Libraries from immunized sources provide high— affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human dies to a wide range of non- self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725- 734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381—388 (1992).

Screening of the libraries can be accomplished by various techniques known in the art.

For example, CD98 can be used to coat the wells of adsorption plates, sed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin—coated beads, or used in any other method for panning display libraries. The selection of antibodies with slow iation cs (and good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309—3 14 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992).

Any of the D98 antibodies of the invention can be obtained by ing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-CD98 antibody clone using the Fv ces from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91—3242, da MD , vols. 1—3. 3. Antibody Fragments The present invention encompasses antibody fragments. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. For a review of certain dy fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of dy fragments. ionally, these fragments were derived via proteolytic digestion of intact dies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107—1 17 (1992); and Brennan et al., Science, 229281 ). However, these nts can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody nts can be isolated from the antibody phage libraries sed above. atively, FabKSH fragments can be directly recovered from E. coli and ally coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10:163—167 (1992)). According to another approach, F(ab')2 fragments can be ed directly from recombinant host cell culture. Fab and F(ab’)2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv). See W0 93/ 16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion ns may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment Such linear may also be a “linear antibody”, e.g., as described for example, in the references cited before. antibodies may be monospecific or multi-specific, such as bispeciflc.

The smallest antibody-derived binding ures are the separate variable domains (V domains) also termed single variable domain antibodies (SdAbs). Certain types of organisms, the camelids and cartilaginous fish, possess high affinity single V-like domains mounted on an ’Fc equivalent domain ure as part of their immune system. (Woolven et al., Immunogenetics 50: 98—101, 1999; Streltsov et al., Proc Natl Acad Sci USA. 101:12444-12449, 2004). The V-like domains (called VhH in camelids and V—NAR in sharks) typically display long e loops, which allow penetration of cavities of target antigens. They also ize isolated VH domains by masking hydrophobic surface patches; These VhH and V—NAR domains have been used to engineer sdAbs. Human V domain variants have been designed using selection from phage libraries and other approaches that have resulted in stable, high g VL- and VH—derived domains. 4. Humanized Antibodies The invention encompasses humanized antibodies. Various methods for humanizing non-human dies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non—human. These man amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et a]. (1986) Nature 321 :522~525; Riechmann et a]. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239: 1 534~1 536), by substituting hypervariable region ces for the corresponding sequences of a human antibody. {0168] In some cases, the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six complementarity determining regions (CDRs) of the parent rodent antibody are grafted onto a human antibody framework. Padlan et al. (FASEB J. 9: 9, 1995) determined that only about one third of the es in the CDRs actually contact the antigen, and termed these the “specificity ining residues,” or SDRs. In the technique of SDR ng, only the SDR residues are grafted onto the human antibody framework iri et al., Methods 36: 25-34, 2005).

The choice of human variable domains, both light and heavy, to be used in making the zed antibodies can be important to reduce antigenicity. According to the so—called "best-fit” method, the ce of the variable domain of a rodent antibody is screened against the entire library of known human le—domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. M01. Biol. 1. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993)J. Immunol., 1512623. In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, VLK up I (VLKI) and VH subgroup III (VHIII). In another method, human germline genes are used at the source of the framework regions.

In an alternative paradigm based on ison of CDRs, called umanization, FR homology is irrelevant. The method consists of ison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical ures with the non—human antibody, those with highest homology within the CDRs are chosen as FR . Finally, the non—human CDRS are grafted onto these FRs. (Tan et al., J. Immunol. 169: I 1 19- 1 125, 2002).

[0171] It is further generally desirable that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three—dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three— dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for e, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 5, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714- 2713, 1997). Inspection e displays permits analysis ofthe likely role of the residues in the functioning ofthe candidate globulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. in this way, FR residues can be selected and combined from the recipient and import sequences so that the desired dy characteristic, such as sed affinity for the target n(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0172] Another method for antibody humanization is based on a metric of dy humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple e humanized variants. (Lazar et al., Mol. Immunol. 44: 1986-1998, 2007).

[0173] In contrast to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods are based upon the generation of large libraries of zed variants and selection of the best clones using enrichment technologies or high throughput screening techniques. Antibody variants may be isolated from phage, ribosome and yeast display libraries as well as by bacterial colony ing. nboom, Nat. Biotechnol. 23: 1105-1 1 I6, 2005', Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21: 163—70, 2003; Schlapschy et al., Protein Eng. Des. Se]. 17: 847-60, 2004).

In the FR library approach, a tion of residue variants are introduced at specific positions in the FR followed by selection of the library to select the FR that best supports the grafted WO 78377 CDR. The residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (Foote and Winter, J. Mol. Biol. 224: 487-499, 1992), or from the more limited set oftarget es identied by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

In FR shuffling, whole FRs are combined with the non-human CDRs d of creating combinatorial libraries of selected residue variants. (Dall’Acqua et al., Methods 36: 43-60, 2005). The libraries may be screened for binding in a two-step selection process, first humanizing VL, followed by VH. Alternatively, a one—step FR shuffling process may be used. Such a process has been shown to be more efficient than the two—step ing, as the resulting antibodies exhibited improved biochemical and physico-chemical properties including enhanced sion, increased y and thermal stability (Damschroder et al, Mol. Immunol. 44: 3049—60, 2007) The eering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non—human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR—3 of non—human VH and VL chains and progressively replaces other regions of the non—human antibody into the human FRs, including the CDR—1 and CDR—2 of both VH and VL. This methodology lly results in epitope retention and identification of dies from multiple sub—classes with distinct human V- segment CDRs. Humaneering allows for isolation of antibodies that are 91—96 % homologous to human germline gene antibodies. (Alfenito, Cambridge Healthtech lnstitute‘s Third Annual PEGS, The Protein ering Summit, 2007). 5. Human Antibodies Human anti-CD98 antibodies of the invention can be constructed by combining Fv clone variable domain sequence(s) selected from human—derived phage display libraries with known human nt domain sequences(s) as described above. Alternatively, human monoclonal anti-CD98 antibodies of the invention can be made by the hybridoma method. Human a and mouse—human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. lmmunol., 133: 3001 (1,984); Brodeur et al., Monoclonal dy tion Techniques and Applications, pp. 51—63 (Marcel Dekker, Inc, New York, 1987); and Boerner et al., J.

Immunol, 147: 86 .

It is also possible to produce transgenic s (e.g. mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of nous immunoglobulin production. enic mice that express human antibody repertoires have been used to generate high—affinity human sequence monoclonal dies against a wide variety of potential drug targets. See, e.g., Jakobovits, A., Curr. Opin. Biotechnol. 1995, 6(5):561—6; Bruggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8(4):455—8; US. Pat. Nos. 6,075,181 and 6,150,584; and Lonberg et al., Nature Biotechnol. 23: 1117—1125, 2005).

Alternatively, the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro). See, e.g, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1):86—95 (1991); and US Pat No. 5,750,373.

Gene shuffling can also be used to derive human dies from non~human, e.g. rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non~ human antibody. According to this , which is also called “epitope imprinting” or “guided selection”, either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is ed with a repertoire of human V domain genes, creating a population of non—human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site yed upon removal of the corresponding non-human chain in the primary phage display clone, i.e. the epitope guides (imprints) the choice of the human chain partner.

When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93/06213; and Osbourn et al, s, 36, 61-68, 2005). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of man origin. Examples of guided selection to humanize mouse antibodies towards cell surface antigens e the —binding n present on ovarian cancer cells i et al., Cancer Res, 58, 991-996, 1998) and CD147, which is highly expressed on hepatocellular carcinoma (Bao et al., Cancer Biol. Ther., 4, 1374—1380, 2005).

A ial disadvantage of the guided selection approach is that shuffling of one antibody chain while keeping the other constant could result in epitope drift. In order to maintain the epitope recognized by the non-human dy, CDR retention can be applied (Klimka et al., Br. J. ., 83, 252—260, 2000; Beiboer et al., J. Mol. Biol., 296, 833-49, 2000) In this method, the non— human CDR-H3 is commonly retained, as this CDR is at the center of the antigen—binding site and has proven to be the most important region of the antibody for antigen recognition. In some instances, however, CDR—H3 and CDR—L3, as well as CDR—H3, CDR—L3 and CDR-L2 of the non-human antibody may be retained. 6. Bispecific Antibodies Bispecific dies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the g icities is for CD98 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CD98.

Bispeciflc antibodies may also be used to localize cytotoxic agents to cells that express CD98. These antibodies s a CD98—binding arm and an arm which binds a cytotoxic agent, such as, e.g., saporin, anti-interferon-d, Vinca alkaloid, ricin A chain, rexate or radioactive isotope hapten. Bispeciflc antibodies can be prepared as full length antibodies or antibody fragments (e. g. F(ab’)2 bispecific antibodies).

Methods for making bispeciflc antibodies are known in the art, such as, for e, by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities ein and Cuello, Nature, 305: 537 ). For r details of generating bispecific antibodies see, for example, Bispecific Antibodies, Kontermann, ed., Springer-Verlag, Hiedelberg . 7. alent Antibodies A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain ses (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. In certain embodiments, a multivalent dy comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, a multivalent antibody comprises (or consists of) four antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (for example, two polypeptide ), wherein the polypeptide chain(s) comprise two or more variable domains. For ce, the ptide chain(s) may comprise 1)n -VD2-(X2)n ~Fc, wherein VDl is a first variable domain, VD2 is a second variable domain, Fc is one ptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CHl—flexible linker—VH-CHl—Fc region chain; or VH-CHl-VI—I-CH1~Fc region chain. The multivalent antibody herein may further comprise at least two (for example, four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here se a light chain variable domain and, optionally, further se a CL domain. 8. Effector Function Engineering It may be desirable to modify the antibody of the ion with t to effector function, e.g., so as to enhance antigen-dependent cell—mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the dy. See, e.g. Lazar et al., Proc. Natl. Acad. Sci. USA 2006, 103(11):4005-4010; Presta, L.G., Curr. Opin. Immunol. 2008, 20(4):460—70; and US. Patent Nos. 7,183,387; 7,332,581; and 7.335,742.

Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond ion in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody—dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 9l- 1195 (I992) and Shopes, B. J. Immunol. 148:2918—2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross—linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the serum half life of the antibody, one may orate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in US. Patent 5,739,277, for e. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an lgG molecule (e.g., lgG], IgGg, lgGg, or lgG4) that is responsible for sing the in vivo serum half—life of the lgG molecule. 9. Alternative g Agents The invention encompasses non—immunoglobulin binding agents that cally bind to the same epitope as an anti—CD98 dy disclosed herein. In some embodiments, a binding agent is identified an agent that displaces or is displaced by an anti—CD98 antibody of the invention in a competive binding assay. These alternative binding agents may include, for example, any of the engineered protein scaffolds known in the art. Such scaffolds e, for example, anticalins, which are based upon the lin scaffold, a n structure characterized by a rigid beta-barrel that supports four ariable loops which form the ligand binding site. Novel binding specificities are engineered by targeted random mutagenesis in the loop regions, in combination with functional display and guided selection (Skerra (2008) FEBS J. 275: 2677—2683). Other suitable scaffolds may include, for example, adnectins, or monobodies, based on the tenth extracellular domain of human fibronectin llI (Koide and Koide (2007) Methods Mol. Biol. 352: 95-109); affibodies, based on the Z domain of staphylococcal protein A (Nygren et al. (2008) FEBS J. 275: 2668-2676)); DARPins, based on ankyrin repeat proteins p et al. (2008) Drug. Discov. Today 13: 695-701); fynomers, based on the SH3 domain of the human Fyn protein kinase Grabulovski et al. (2007) J. Biol. Chem. 282: 3196—3204); affitins, based on Sac7d from Sulfolobus acidolarius (Krehenbrink et al. (2008) J. Mol. Biol. 383: 105 8—1068); affilins, based on human y-B-crystallin (Ebersbach et al. (2007) J. Mol. Biol. 372: 172—185); s, based on the A domains of membrane receptor proteins (Silverman et al. (2005) Biotechnol. 23: 1556-1561); cysteine-rich knottin peptides (Kolmar (2008) FEBS J. 275: 2684—2690); and engineered -type inhibitors (Nixon and Wood (2006) Curr. Opin. Drug. Discov. Dev. 9: 261—268) For a , see Gebauer and Skerra (2009) Curr. Opin. Chem. Biol. 13: 245—255.

Antibody Variants

[0188] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be ble to improve the binding affinity and/or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity or solubility. In addition to the anti—CD98 antibodies described herein, it is contemplated that anti-CD98 dy variants can be prepared. Anti-CD98 antibody variants can be ed by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art will iate that amino acid changes may alter post-translational ses of the anti-CD98 antibody, such as changing the number or position of ylation sites or altering the membrane anchoring characteristics.

Variations may be a substitution, deletion or insertion of one or more eodons encoding the antibody or polypeptide that s in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the ing variants for ty exhibited by the full—length or mature native sequence.

Amino acid sequence insertions include amino- and/or carboxyl—terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an inal methionyl residue. Other insertional variants of the antibody molecule e the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for antibody— directed enzyme prodrug y) or a polypeptide which increases the serum half—life of the antibody. ntial modifications in the ical properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids may be d according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, 2nd Ed, pp. 73—75, Worth Publishers, New York ): (I) non—polar: Ala (A), Val (V), Leu (L), He (1), Pro (P), Phe (F), Trp (W), Met (M) (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn OJ), Gln (Q) (3) acidic: Asp (D), Glu (E) (4) basic: Lys (K), Arg (R), His(H) Alternatively, naturally occurring residues may be d into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lle; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0193] Non—conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted es also may be introduced into the conservative substitution sites or, into the remaining (non—conserved) sites.

The variations can be made using methods known in the art such as oligonucleotide— mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site—directed mutagenesis r et al., Nucl. Acids Res, 13:4331 (1986); Zoller et al., Nucl. Acids Res, 1016487 ), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos.

Trans. R. Soc. London SerA, 3172415 (1986)) or other known techniques can be performed on the cloned DNA to produce the anti—CD98 antibody variant DNA.

Any cysteine residue not involved in maintaining the proper conformation of the anti- CD98 dy also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ) may be added to the anti» CD98 antibody to improve its stability (particularly where the dy is an antibody fragment such as an Fv fragment). Cysteine—engineered antibodies, which can be used to generate antibody—drug conjugates, are described, for example, in .

In an embodiment, an anti—CD98 antibody molecule of the invention is a "de~ immunized" antibody. A "de~immunized” anti-CD98 antibody is an antibody derived from a humanized or chimeric anti—CD98 antibody, that has one or more tions in its amino acid sequence resulting in a reduction of immunogenicity of the antibody, compared to the respective original —immunized antibody. One of the ures for generating such antibody mutants involves the identification and removal of T-cell epitopes of the antibody le. In a first step, the immunogenicity of the antibody le can be determined by several methods, e. g. by in vitro determination of T—cell epitopes or in silico prediction of such epitopes, as known in the art. Once the critical es for T—cell epitope on have been fied, mutations can be made to remove immunogenicity and retain antibody activity. For review, see, e.g., Jones et al., Methods in Molecular Biology 525: 405-423, 2009.

In vitro y maturation In an embodiment, antibody variants having an ed property such as affinity, stability, or expression level as compared to a parent antibody is in vitro affinity maturation. Like the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection.

Libraries of antibodies are displayed as Fab, scFv or V domain fragments either on the surface of an organism (e.g., phage, bacteria or yeast) or in association (covalently or non-covalently) with their encoding mRNA or DNA. y selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the dies. Two or three rounds of mutation and selection using display methods such as phage y usually results in antibody fragments with affinities in the low nanomolar range. Preferred affinity matured antibodies will have lar or even picomolar affinities for the target antigen.

Phage display is the most widepread method for display and selection of antibodies.

The antibodies are displayed on the surface of Pd or M13 bacteriophages as fusions to the iophage coat protein. Selection involves exposure to antigen to allow phage-displayed antibodies to bind their targets, a process referred to as “panning.” Phage bound to antigen are recovered and infected in bacteria to produce phage for further rounds of selection. For review, see Hoogenboom, Methods. Mol. Biol. 178: 1-37, 2002; Bradbury and Marks, J. Immuno. Methods 290: 29-49, 2004).

In the yeast display system (Boder et al., Nat. Biotech. 15: 553—57, 1997; Chao et al., Nat. Protocols 1:755-768, 2006), the antibody is displayed as single«chain variable fusions (scFv) in which the heavy and light chains are ted by a flexible linker. The scFv is fused to the adhesion t of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall through disulfide bonds to Agal p. y of a protein via Aga2p projects the protein away from the cell surface, minimizing ial interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen the library to select for dies with improved affinity or stability.

Binding to a soluble antigen of interest is determined by labeling of yeast with biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Variations in e expression of the antibody can be measured through fluorescence ng of either the hemagglutinin or c- Myc epitope tag flanking the scFV. Expression has been shown to correlate with the stability of the displayed protein, and thus antibodies can be selected for ed stability as well as affinity (Shusta et al., J. Mol. Biol. 292: 949-956, 1999). An additional advantage of yeast y is that displayed proteins are folded in the endoplasmic reticulum of the eukaryotic yeast cells, taking advantage of endoplasmic reticulum ones and quality-control machinery. Once tion is complete, antibody affinity can be conveniently ‘titrated’ while displayed on the surface of the yeast, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is the potentially smaller onal library size than that of other display methods; however, a recent approach uses the yeast cells’ mating system to create atorial diversity estimated to be 1014 in size (US Patent Publication 2003/0186,374; Blaise et al., Gene 342: 21 1—218, 2004).

In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system. The DNA library coding for a particular library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to surface-bound ligand, allowing simultaneous isolation of the antibody and its encoding mRNA h affinity capture with the ligand. The ribosome-bound mRNA is then reversed transcribed back into cDNA, which can then undergo mutagenesis and be used in the next round of selection. (Fukuda et al., Nucleic Acids Res. 34, e127, 2006). In mRNA display, a covalent bond between antibody and mRNA is established using cin as an adaptor molecule n et al., Proc.

Natl. Acad. Sci. USA 98, 3750-3755, 2001).

As these methods are performed entirely in vitro, they provide two main advantages over other selection logies. First, the diversity of the library is not limited by the transformation efficiency of ial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, for example, by non—proofreading polymerases, as no library must be transformed after any diversification step.

Diversity may be introduced into the CDRs or the whole V genes of the antibody libraries in a targeted manner or via random introduction. The former approach includes sequentially targeting all the CDRs of an antibody via a high or low level of mutagenesis or targeting isolated hot spots of somatic hypermutations (Ho, et al., J. Biol. Chem. 280: 607—617, 2005) or residues suspected affecting affinity on experimental basis or ural reasons. Random mutations can be introduced throughout the whole V gene using E. coli mutator strains, error—prone replication with DNA polymerases (Hawkins et al., J. Mol. Biol. 226: 889—896, 1992) or RNA replicases. Diversity may also be introduced by replacement of regions that are naturally diverse via DNA shuffling or similar techniques ((Lu et al., J. Biol. Chem 278: 43496-43507, 2003; US Pat. No. 5,565,332; US Pat. No. 250). Alternative techniques target hypervariable loops extending into framework-region residues (Bond et al., J. Mol. Biol. 348: 699-709, 2005) employ loop deletions and insertions in CDRs or use hybridization—based diversification (US Patent Publication No. 2004/0005709). Additional methods of generating diversity in CDRs are disclosed in US Pat. No. 7,985,840.

Screening of the libraries can be accomplished by s techniques known in the art.

For example, CD98 can be immobilized onto solid supports, columns, pins or cellulose/poly(vinylidene fiuoride) membranes/ other , expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with avidin—coated beads, or used in any other method for panning display libraries.

For review of in vitro affinity maturation s, see Hoogenboom, Nature Biotechnology 23: 1105—1116, 2005 and Quiroz and Sinclair, Revista Ingeneria Biomedia 4: 395 l, 2010 and references therein.

Modifications of Anti-CD98 Antibodies Covalent modifications of anti-CD98 antibodies are included within the scope of this invention. Covalent modifications include reacting targeted amino acid residues of an anti-CD98 antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N— or C- terminal residues of the anti-CD98 dy. Other ations include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of e and , phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a—amino groups of lysine, arginine, and histidine side chains (TE. Creighton, Proteins: Structure and lar ties, WH. Freeman & Co., San sco, pp. 7986 (1983)), acetylation of the N—terminal amine, and amidation of any C—terminal carboxyl group.

[0206] Other types of covalent modification of the anti—CD98 dy included within the of the dy or polypeptide scope of this invention include altering the native glycosylation pattern (Beck et al., Curr. Pharm. Biotechnol. 9: 482-501, 2008; Walsh, Drug . Today 15: 0, 2010), and linking the antibody to one of a variety of teinaceous rs, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in US Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The anti-CD98 antibody of the present invention may also be modified to form chimeric molecules sing an anti—CD98 antibody fused to another, heterologous polypeptide or amino acid sequence, e.g., an epitope tag (Terpe, Appl. Microbiol. Biotechnol. 60: 523—533, 2003) or the Fc region of an IgG molecule (Aruffo, “lmmunoglobulin fusion proteins” in Antibody Fusion Proteins, SM. Chamow and A. Ashkenazi, eds., Wiley—Liss, New York, 1999, pp. 221-242).

Preparation of Anti-CD98 Antibodies

[0208] Anti—CD98 antibodies may be produced by culturing cells transformed or transfected with a vector containing anti-CD98 antibody-encoding nucleic acid. Polynucleotide sequences ng polypeptide components of the antibody of the ion can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using tide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells.

Many vectors that are available and known in the art can be used for the e of the present invention.

Selection of an appropriate vector will depend mainly on the size of the c acids to be inserted into the vector and the particular host cell to be transformed with the . Host cells le for expressing antibodies of the invention include prokaryotes such as Archaebacteria and Eubacteria, including Gram-negative or Gram—positive organisms, eukaryotic microbes such as filamentous fungi or yeast, invertebrate cells such as insect or plant cells, and vertebrate cells such as mammalian host cell lines. Host cells are transformed with the above-described expression s and cultured in conventional nutrient media modified as appropriate for ng promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Antibodies ed by the host cells are purified using standard protein purification methods as known in the art.

Methods for dy production including vector construction, expression and purification are further bed in Pltickthun et al., (1996) in Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation (McCafferty, J ., Hoogenboom, H. R., and Chiswell, D. J., eds), 1 Ed., pp. 203—252, IRL Press, Oxford; Kwong, K. & Rader, C. E. coli expression and purification of Fab antibody fragments. Current protocols in protein e editorial board John E Coligan et al., Chapter 6, Unit 6.10 (2009); ana and Takekoshi, “Production of Antibody Fab Fragments in Escherischia coli,” in Antibody Expression and tion, M. Al-Rubeai, Ed., er, New York, 20] 1; Therapeutic Monoclonal dies: From Bench to Clinic (ed Z. An), John Wiley & Sons, Inc., Hoboken, NJ, USA.

It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare D98 antibodies. For instance, the riate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid—Phase Peptide sis, WH. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 852149—2154 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation. Various portions of the anti—CD98 antibody may be chemically synthesized separately and ed using chemical or enzymatic methods to produce the desired anti-CD98 U\ antibody. Alternatively, antibodies may be purified from cells or bodily fluids, such as milk, of a transgenic animal ered to express the antibody, as disclosed, for example, in US Pat. No. ,545,807 and US Pat. No. 5,827,690. lmmunoconjugates

[0211] The invention also provides immunoconjugates (interchangably referred to as ”antibody drug conjugates," or "ADCs") comprising any one of the anti-CD98 antibodies of the invention covalently bound by a synthetic linker to one or more cytotoxic agents. ADCs combine the high specificity of monoclonal antibodies with the pharmacological potency of xic molecules, allowing specific targeting of xic agents to tumor cells and avoiding the cific toxicity of most anti~ cancer drugs. For review, see, e.g. Carter and ,_Cancer J. 14: 9 (2008); Ducry and Stump, Bioconjugate Chem. 21 :5—1 3 (2010); Beck et al., Discov. Med. 10: 329—339 (2010).

Cytotoxic agents for use in the immunoconjugates of the invention may include chemotherapeutic agents, drugs or growth inhibitory agents as described above, toxins (e. g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or radioisotopes. In some embodiments, the immunoconjugate comprises a DNA binder (e.g., eamycin) or a tubulin depolymerization agent (e.g., a maytansinoid or an atin). The t invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g, a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

Enzymatically active toxins and nts thereof that can be used in the immunoconjugates ofthe invention include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP—S), ica charantia inhibitor, , crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. See, e.g., WO 93/21232.

[0214] A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include Atm, 1'3], 1125, Ygo, Rem, Rel“, Smm, Bim, P32, Pb”2 and ctive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintigraphic s, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine—123 again, —131, indium-111, fluorine—19, carbon—l3, nitrogen—15, oxygen~l 7, gadolinium, ese or iron. The radioisotopes may be incorporated in the conjugate in known ways as described, e.g., in Reilly, “The radiochemistry of monoclonal antibodies and es,” in Monoclonal Antibody and Peptide—Targeted Radiotherapy of Cancer, R.M. Reilly, ed., Wiley, Hoboken N.J., 2010. 2012/066347 The linker may be a “cleavable linker” facilitating e of the cytotoxic drug in the cell, but non—cleavable linkers are also contemplated herein. Linkers for use in the immunoconjugates of the invention include without limitation acid labile linkers (e.g., hydrazone linkers), disulfide—containing linkers, peptidase-sensitive linkers (e.g, peptide linkers such as citrulline-valine or phenylalanine—lysine), abile linkers, yl linkers (Chari et al., Cancer Research 52:127-131 (1992); US. Patent No. ,208,020), thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance (Kovtun et al., Cancer Res. 70: 2528-2537, 2010).

Conjugates of the dy and cytotoxic agent may be made using a y of bifunctional n coupling agents such as BMPS, EMCS, GMBS, HBVS, LC~SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, MBS, sulfo— SIAB, SMCC, and sulfo-SMPB, and SVSB nimidyl—(4—vinylsulfone)benzoate)). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1 098 (1987).

The invention further contemplates that conjugates of antibodies and cytotoxic agents may be prepared using any suitable methods as disclosed in the art, e.g., in Bioconjugate Technigues, 2nd Ed, G.T.

Hermanson, ed., Elsevier, San Francisco, 2008.

Conventional dy—drug conjugation strategies have been based on random conjugation chemistries involving the s—amino group of Lys residues or the thiol group of Cys residues, which results in heterogenous conjugates. Recently developed techniques allow site-specific conjugation to antibodies, resulting in homogeneous drug loading and avoiding ADC subpopulations with altered antigen-binding or pharmacokinetics. These include engineering of “thiomabs” comprising cysteine tutions at positions on the heavy and light chains that e reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter n binding (.lunutula et al., J. Immunol. Meth. 332: 41-52 (2008); Junutula et al., Nat. Biotechnol. 26: 925—932, 2008). In another , selenocysteine is cotranslationally inserted into an antibody sequence by recoding the stop codon UGA from ation to selenocysteine insertion, allowing site specific nt conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (Hofer et al., Proc. Natl. Acad. Sci. USA 105: 12451-12456 (2008); Hofer et a1, mistry 48(50): 12047—12057, 2009) Pharmaceutical ations

[0218] The antibodies or antibody-drug conjugates (ADC) of the invention may be administered by any route appropriate to the condition to be treated. The antibody or ADC will typically be administered parenterally, i.e., infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

For treating cancers, in one embodiment, the antibody or antibody-drug conjugate is administered via intravenous infusion. The dosage administered Via infusion is in the range of about 1 [Lg/m2 to about 10,000 [Lg/m2 per dose, generally one dose per week for a total of one, two, three or four doses. Alternatively, the dosage range is of about 1 [Lg/m2 to about 1000 pg/mz, about 1 [Lg/m2 to about 800 pg/mz, about 1 [Lg/m2 to about 600 pg/mz, about 1 [Lg/m2 to about 400 pg/mz, about 10 [Lg/m2 to about 500 ug/mz, about 10 ttg/m2 to about 300 ug/mz, about 10 pg/m2 to about 200 ug/m2, and about 1 ug/m2 to about 200 ug/mz. The dose may be administered once per day, once per week, multiple times times per week, but less than once per day, le times per month but less than once per day, multiple relieve or alleviate symptoms per month but less than once per week, once per month or intermittently to of the e. Administration may continue at any of the disclosed intervals until remission of the tumor or symptoms of the cancer being treated. Administration may continue after remission or relief of symptoms is achieved where such ion or relief is prolonged by such continued administration.

In one aspect, the invention further provides pharmaceutical formulations comprising at least one anti—CD98 antibody of the ion and/or at least one immunoconjugate thereof and/or at least one anti-CD98 antibody-drug ate of the invention. In some embodiments, a pharmaceutical formulation comprises 1) an anti-CD98 antibody and/or an anti—CD98 antibody-drug conjugate and/or an immunoconjugate thereof, and 2) a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical formulation comprises 1) an anti-CD98 dy and/or an immunoconjugate thereof, and optionally, 2) at least one additional therapeutic agent.

[0221] ceutical formulations comprising an antibody or immunoconjugate of the invention or the dyvdrug conjugate of the invention are prepared for storage by mixing the antibody or antibody-drug conjugate having the desired degree of purity with optional physiologically acceptable carriers, ents or stabilizers (Remington's ceutical Sciences 16th n, Osol, A. Ed. (1980)) in the form of aqueous solutions or lyophilized or other dried formulations. The formulations herein may also contain more than one active compound as necessary for the ular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an D98 antibody, it may be desirable to include in the one formulation, an additional antibody, e.g., a second anti—CD98 antibody which binds a different epitope on the CD98 ptide, or an antibody to some other target such as a growth factor that affects the growth of the particular cancer. Alternatively, or additionally, the composition may further se a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti—hormonal agent, and/or cardioprotectant. Such molecules are ly present in combination in amounts that are effective for the purpose intended.

The antibodies or immunoconjugates of the invention may be formulated in any 3O suitable form for delivery to a target cell/tissue, e.g, as microcapsules or macroemulsions (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980); Park eta1., Molecules 10: 146-161 (2005); Malik et al., Curr. Drug. Deliv. 4: 1 (2007)); as ned release formulations (Putney and Burke, Nature Biotechnol. 16: 153—157, (1998)) or in liposomes (Maclean et al., Int. J. Oncol. 11: 235—332 (1997); Kontermann, Curr. Opin. Mol. Ther. 8: 39—45 (2006)).

Therapeutic s An antibody or immunoconjugate of the invention may be used in, for example, in vitro, ex vivo, and in vivo therapeutic methods. In one aspect, the invention provides methods for 2012/066347 inhibiting cell growth or proliferation, either in vivo or in vitro, the method comprising exposing a cell to an anti—CD98 antibody or immunoconjugate thereof under conditions permissive for binding of the immunoconjugate to CD98. “Inhibiting cell growth or proliferation” means decreasing a cell’s growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death. In certain embodiments, the cell is a tumor cell. In certain embodiments, the cell is a bladder, breast, colon, rectal, gastric, geal, lung, laryx, , oral, ovarian, or prostate tumor cell, or a sarcoma, melanoma, glioma, lymphoma or leukemia cell.

In one aspect, an antibody or immunoconjugate of the ion is used to treat or prevent a cell proliferative er, such as cancer. In certain embodiments, the cell erative er is associated with increased expression and/or activity of CD98. For example, in certain embodiments, the cell proliferative disorder is associated with increased expression of CD98 on the surface of a cancer cell. Examples of cell proliferative disorders to be treated by the dies or immunoconjugates of the invention include, but are not limited to, bladder, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancers, or sarcomas, melanomas, gliomas, lymphomas or ias, or metatases of any of these cancers.

In one aspect, the invention provides methods for treating a cell proliferative disorder comprising administering to an dual an effective amount of an anti—CD98 antibody or immunoconjugate thereof. In certain embodiments, a method for treating a cell proliferative disorder comprises administering to an individual an effective amount of a pharmaceutical formulation 2O comprising an anti-CD98 antibody or D98 immunoconjugate and, optionally, at least one additional therapeutic agent, such as those provided below. In one embodiment, an anti-CD98 dy or immunoconjugate can be used for targeting CD98 on cancer cells by ting the antibody or immunoconjugate with CD98 to form an dy or immunoconjugate-antigen complex such that a conjugated cytotoxic agent of the immunoconjugate accesses the interior of the cell. In one embodiment, the bound antibody or immunoconj ugate is internalized into the cancer cell expressing CD98.

An anti-CD98 antibody or conjugate can be administered to a human for therapeutic purposes. Moreover, an anti~CD98 antibody or immunoconj ugate can be administered to a non-human mammal expressing CD98 with which the antibody cross—reacts (e.g., a primate, pig, rat, or mouse) for nary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic y of antibodies or immunoconjugates of the invention (e.g., testing of s and time s of administration).

Antibodies or immunoconjugates of the invention can be used either alone or in combination with other compositions in a therapy. For ce, an antibody or immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent and/or adjuvant. In certain embodiments, an additional therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, or a growth inhibitory agent. In some embodiments, a chemotherapeutic agent is an agent or a combination of agents such as an alkylating agent (for example, bendamustine hydrochloride, cyclophosphamide or ifosfamide) a nucleoside analog (for e, fludurabine, cytarabine or gemcitabine) a corticosteroid 2012/066347 (for example, prednisone, prednisolone or methylprednisolone), an anti—mitotic agent (for example, paclitaxel, docetaxel or vinorelbine), a vinca alkaloid (for example, vincristine or etoposide), a topoisomerase inhibitor (for example, irinotecan), an antibiotic (for example, anthracycline or adriamycin), a platinum analog (for example, cisplatin or carboplatin), a eutic antibody (for example, mab) or a combination of agents (for example CHOP or CVP) wherein the combination therapy is useful in the treatment of cancers. In some embodiments, the additional compound is a therapeutic antibody other than an anti-CD98 antibody (for example, rituximab).

Such combination therapies noted above encompass combined administration (where two or more eutic agents are included in the same or separate ations), and separate administration, in which case, administration of the antibody or immunoconjugate of the invention can and/or occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent adjuvant. Antibodies or immunoconjugates of the ion can also be used in combination with additional therapeutic regimens including without limitation radiation y and/or bone marrow and peripheral blood transplants.

[0229] An antibody or immunoconjugate of the invention (and any additional therapeutic agent or adjuvant) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and asal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody or immunoconjugate is suitably administered by 2O pulse infusion, particularly with declining doses of the antibody or immunoconjugate. Dosing can be by subcutaneous injections, depending in part any suitable route, e.g. by injections, such as intravenous or on whether the administration is brief or c.

Antibodies or immunoconjugates of the ion would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular er being treated, the particular mammal being d, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody or immunoconjugate need not be, but is optionally ated with one or more agents tly used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of dy or immunoconjugate present in the formulation, the type of disorder or treatment, and other s discussed above. These are generally used in the same dosages and with administration routes as bed herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirical nically determined to be appropriate.

For the prevention or treatment of e, the appropriate dosage of an antibody or immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents, such as chemotherapeutic agents) will depend on the type of disease to be treated, the type of antibody or conjugate, the severity and course of the disease, whether the antibody or immunoconjugate is administered for preventive or therapeutic purposes, previous therapy, the patients clinical history and response to the antibody or immunoconjugate, and the discretion of the attending physician. The antibody or immunoconjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the e, about I ug/kg to 100 mg/kg (e.g.

O. lmg/kg—20mg/kg) of antibody or conjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about I ug/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, ing on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms . One exemplary dosage of the dy or immunoconjugate would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) of dy or immunoconjugate may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody or immunoconjugate). An initial higher loading dose, followed by one or more lower doses I5 initial g dose of may be administered. An exemplary dosing n comprises administering an about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the antibody. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and .

Diagnostic methods and methods of detection In one aspect, anti-CD98 antibodies and immunoconjugates of the invention are useful for detecting the presence of CD98 in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue. In n embodiments, such tissues include normal and/or cancerous tissues that express CD98 at higher levels relative to other tissues, for e, bladder, breast, colon, rectal, gastric, esophageal, lung, laryx, kidney, oral, ovarian, or te cancer, or a a, melanoma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers.

In one aspect, the invention provides a method of detecting the presence of CD98 in a ical sample. In certain embodiments, the method comprises contacting the biological sample with an anti- CD98 antibody under conditions permissive for binding of the anti— CD98 dy to CD98, and ing whether a complex is formed between the anti— CD98 antibody and CD98.

In one aspect, the invention provides a method of diagnosing a disorder associated with increased expression of CD98. In certain embodiments, the method comprises contacting a test cell with an anti-CD98 antibody; ining the level of expression (either quantitatively 0r qualitatively) of CD98 by the test cell by detecting binding of the D98 antibody to CD98; and comparing the level of expression of CD98 by the test cell with the level of sion of CD98 by a control cell (e.g., a normal cell of the same tissue origin as the test cell or a cell that expresses CD98 at levels comparable to such a normal cell), wherein a higher level of expression of CD98 by the test cell as compared to the control cell indicates the presence of a disorder associated with increased expression of CD98. In certain embodiments, the increased expression corresponds to higher density of CD98 expression on the surface of a tumor cell as compared toa normal cell. In certain embodiments, the test cell is ed from an individual suspected of having a disorder associated with increased expression of CD98. In certain ments, the disorder is a cell proliferative disorder, such as a cancer or a tumor. Exemplary cell proliferative disorders that may be diagnosed using an antibody of the invention include bladder, breast, colon, , gastric, esophageal, lung, laryx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, ma, glioma, lymphoma or leukemia, or a metatasis of any of these cancers.

In certain embodiments, a method of diagnosis or detection, such as those described above, ses detecting g of an anti—CD98 antibody to CD98 expressed on the surface of a cell or in a membrane preparation obtained from a cell expressing CD98 on its surface. In certain ments, the method comprises contacting a cell with an D98 antibody under ions permissive for binding of the anti-CD98 antibody to CD98, and detecting whether a complex is formed between the anti—CD98 antibody and CD98 on the cell surface, An exemplary assay for detecting I5 binding of an anti—CD98 dy to CD98 expressed CD98 on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-CD98 dies to CD98.

Such methods include, but are not limited to, antigen-binding assays that are well known in the art, such as n blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, scent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In n embodiments, anti—CD98 dies are d. Labels e, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, on— dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., h an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes 321’, MC, 1251, 3H, and ml, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, ferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3— dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, B«galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose—6— phosphate dehydrogenase, heterocyclic es such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

In certain embodiments, anti—CD98 antibodies are immobilized on an insoluble matrix.

Immobilization entails separating the anti-CD98 antibody from any CD98 that remains free in solution.

This conventionally is accomplished by either bilizing the anti—CD98 antibody before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al.., US. 3,720,760), or by covalent coupling (for e, using glutaraldehyde cross-linking), or by insolubilizing the anti-CD98 antibody after formation of a complex between the anti-CD98 antibody and CD98, e.g., by immunoprecipitation.

Any of the above ments of diagnosis or detection may be carried out using an immunoconjugate of the invention in place of or in addition to an anti—CD98 antibody.

Assays Anti-CD98 antibodies and immunoconjugates of the invention may be characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

Activifl assays

[0241] In one aspect, assays are provided for identifying anti-CD98 antibodies or immunoconjugates thereof having ical activity. Biological activity may include, e.g., the ability to inhibit cell growth or proliferation (e.g., “cell killing” activity), or the ability to induce cell death, including programmed cell death (apoptosis). Antibodies or immunoconjugates having such biological activity in vivo and/or in vitro are also provided.

I5 [0242] In certain embodiments, an anti—CD98 antibody or immunoconjugate f is tested for its ability to inhibit cell growth or proliferation in vitro. Assays for inhibition of cell growth or proliferation are well known in the art. Certain assays for cell proliferation, ified by the “cell killing” assays described herein, measure cell viability. One such assay is the CellTiter—GloTM Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). That assay determines the number of viable cells in culture based on quantitation of ATP present, which is an indication of metabolically active cells. See Crouch et al (1993) J. Immunol. Meth. 160:81s88, US Pat.

No. 6602677. The assay may be ted in 96- or 384-well format, making it amenable to automated high—throughput screening (HTS). See Cree et al (1995) AntiCancer Drugs 6:398-404.

Another assay for cell proliferation is the “MTT” assay, a colorimetric assay that es the oxidation of 3—(4,5-dimethylthiazolyl)—2,5—diphenyltetrazolium bromide to an by mitochondrial ase. Like the CellTiter-GloTM assay, this assay indicates the number of metabolically active cells present in a cell culture. See, e.g., Mosmann (1983) J. l. Meth. 65:55— 63, and Zhang et al. (2005) Cancer Res. 65:3877—3882.

In one aspect, an anti—CD98 antibody is tested for its ability to induce cell death in vitro. Assays for induction of cell death are well known in the art. In some ments, such assays measure, e.g., loss of membrane integrity as indicated by uptake of propidium iodide (PI), trypan blue (see Moore et al. (1995) Cytotechnology, 1721-11), or 7AAD. In an exemplary PI uptake assay, cells are cultured in co's Modified Eagle Medium (D—MEM): Ham's F—12 (50:50) supplemented with 10% nactivated FBS (Hyclone) and 2 mM L—glutamine. Thus, the assay is performed in the absence of complement and immune effector cells. Cells are seeded at a y of3 x 106 per dish in 100 x 20 mm dishes and d to attach overnight. The medium is removed and ed with fresh medium alone or medium containing various concentrations of the antibody or immunoconjugate. The cells are incubated for a 3-day time period. Following treatment, monolayers are washed with PBS and detached by trypsinization. Cells are then fuged at 1200 rpm for 5 minutes at 4 °C, the pellet resuspended in 3 ml cold Ca2+ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaClz) and aliquoted into 35 mm strainer—capped 12 x 75 mm tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 . Samples are analyzed using a FACSCANTM flow cytometer and FACSCONVERTTM CellQuest software (Becton Dickinson). Antibodies or immunoconjugates that induce statistically significant levels of cell death as determined by PI uptake are thus identified.

In one aspect, an anti—CD98 antibody or conjugate is tested for its ability to induce sis (programmed cell death) in vitro. An exemplary assay for antibodies or immunconjugates that induce apoptosis is an annexin binding assay, for example, as in Zhang et al.

IO (BioTechniques 23: 525—53 1 Another exemplary assay for antibodies or immunconjugates that , 1997). induce apoptosis is a histone DNA ELISA colorimetric assay for ing internucleosomal degradation of genomic DNA. Such an assay can be performed using, e.g., the Cell Death Detection ELISA kit (Roche, Palo Alto, CA).

Cells for use in any of the above in vitro assays e cells or cell lines that lly s CD98 or that have been engineered to express CD98. Such cells include tumor cells that overexpress CD98 relative to normal cells of the same tissue origin. Such cells also include cell lines (including tumor cell lines) that s CD98 and cell lines that do not normally express CD98 but have been transfected with nucleic acid encoding CD98.

In one aspect, an anti-CD98 antibody or immunoconjugate thereof is tested for its ability to inhibit cell growth or proliferation in vivo. In certain embodiments, an anti-CD98 antibody or immunoconjugate thereof is tested for its ability to inhibit tumor growth in vivo. In vivo model systems, such as xenograft models, can be used for such testing. In an exemplary xenograft system, human tumor cells are uced into a suitably compromised non-human animal, e.g., a SCID mouse. An dy or immunoconj ugate of the invention is administered to the animal. The ability of the dy or immunoconjugate to inhibit or decrease tumor growth is measured. In certain ments of the above xenograft system, the human tumor cells are tumor cells from a human patient. Such cells useful for preparing xenograft models include without limitation cells sing exogenous CD98, and cells naturally expressing CD98. In certain embodiments, the human tumor cells are introduced into a suitably immunocompromised nomhuman animal by subcutaneous injection or by transplantation into a suitable site, such as a mammary fat pad.

Binding assays and other assays In one aspect, an anti-CD98 antibody is tested for its antigen binding activity. For example, in certain embodiments, an anti-CD98 antibody is tested for its ability to bind to exogenous or endogenous CD98 expressed on the surface of a cell. A FACS assay may be used for such testing.

[0249] A panel of onal antibodies raised against CD98 may be grouped based upon the epitiopes they recognize, a s known as epitope binning. Epitope binning is typically carried out using competition assays, which evaluate an antibody's ability to bind to an antigen in the presence of another antibody. In an exemplary competition assay, immobilized CD98 is incubated in a solution comprising a first labeled antibody that binds to CD98 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to CD98. The second antibody may be present in a hybridoma supernatant. As a control, immobilized CD98 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to CD98, excess d antibody is removed, and the amount of label associated with immobilized CD98 is ed. If the amount of label associated with immobilized CD98 is ntially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to CD98. In n embodiments, immobilized CD98 is present on the surface of a cell or in a membrane preparation obtained from a cell expressing CD98 on its surface.

High-throughput methods of epitope binning are also known in the art. See, for example, Jia et al., J. Immunol. Methods 2004, 288(1—2):91-98, describing a method of multiplexed competitive antibody binning for the characterization of onal antibodies; and Miller et al., J.

Immunol. Methods 2011, 2):118-25, bing epitope binning of murine monoclonal dies by a multiplexed pairing assay.

Epitope mapping Epitope mapping is the process of identifying the binding sites, or epitopes, of an antibody on its target protein antigen. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein.

Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought er upon folding of the protein into its three—dimensional structure.

A variety of methods are known in the art for mapping dy es on target protein antigens. These include nesis methods, peptide scanning methods, display methods, methods involving and mass spectroscopy, and structural determination.

[0254] The site directed mutagenesis method involves targeted site-directed mutagenesis where critical amino acids are identified by atically introducing substitutions along the protein sequence and then determining the effects of each substitution on antibody binding. This may be done by "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science 244: 1081—1085, or some other form of point mutagenesis of amino acid residues in human CD98. Mutagenesis s, however, may also reveal amino acid residues that are crucial to the overall three—dimensional structure of CD98 but that are not directly involved in antibody-antigen contacts, and thus other methods may be ary to confirm a functional epitope determined using this method.

Shotgun mutagenesis mapping utilizes a hensive plasmid—mutation y for the target gene, with each clone in the y bearing a unique amino acid mutation and the entire library covering every amino acid in the target protein. The clones that constitute the mutation y are individually arranged in microplates, expressed within living mammalian cells, and tested for immunoreactivity with dies of interest. Amino acids critical for dy epitopes are identified by a loss of reactivity and are then mapped onto a protein structure to visualize epitopes. By automating the analysis, new e maps can be d within days to weeks. Because it uses the native structure of proteins within mammalian cells, the technique allows both linear and conformational epitope structures to be mapped on complex ns. (Paes et al., J. Am. Chem. Soc. 131(20): 6952—6954 (2009); Banik and Doranz, Genetic ering and Biotechnology News 3(2): 25—28 (2010)).

The epitope bound by an anti-CD98 antibody may also be determined using peptide ng methods. In peptide scanning, libraries of short peptide sequences from overlapping segments of the target n, CD98 are tested for their ability to bind antibodies of interest. The peptides are synthesized and screened for binding, e.g. using ELISA or BIACORE, or on a chip, by any of the multiple methods for solid-phase screening (Reineke et al, Curr. Opin. Biotechnol. 12: 59-64, 2001) as in the “pepscan” methodology (WO 84/03 564; WO 93/09872). Such peptide screening methods may not be e of detecting some discontinuous functional epitopes, i.e. functional es that involve amino acid residues that are not uous along the primary sequence of the CD98 polypeptide chain.

A recently developed technology termed CLIPS (chemical linkage of es onto scaffolds) may be used to map conformational epitopes. The loose ends of the peptides are affixed onto synthetic scaffolds, so that the scaffolded peptide may be able to adopt the same spatial structure as the corresponding ce in the intact protein. CLIPS technology is used to fix linear peptides into cyclic structures (‘single-loop’ format), and to bring together different parts of a protein binding site (‘double— loop’, ‘triple-loop’, etc. format), so as to create conformational epitopes that may be assayed for antibody binding. (US Pat. No. 7,972,993).

[0258] The epitopes bound by antibodies of the invention may also be mapped using display techniques, including, for example, phage display, microbial display, and ribosome/mRNA display as described above. In these methods, libraries of peptide fragments are yed on the surface of the phage or cell. Epitopes are then mapped by screening mAbs against these fragments using selective binding assays. A number of computational tools have been developed which allow the prediction of conformational epitopes based upon linear affinity-selected peptides obtained using phage y. se et al., ormatics 23: 3244—3246 Methods are also available for the detection of , 2007). conformational epitopes by phage display. Microbial display systems may also be used to s properly folded nic fragments on the cell surface for identification of conformational epitopes (Cochran et al., J. Immunol. Meth. 287: 147—158, 2004; Rockberg et al., Nature s 5: 1039—1045, 2008).

Methods involving proteolysis and mass spectroscopy may also be used to determine dy epitopes (Baerga—Ortiz et al., Protein Sci. 2002 June; 1 1(6): 1300—1308). In limited proteolysis, the antigen is cleaved by different proteases, in the presence and in the absence of the antibody, and the fragments are identified by mass spectrometry. The epitope is the region of the antigen that becomes protected from proteolysis upon binding of the antibody (Suckau et al., Proc. Natl. Acad. Sci. USA 87: 9852, 1990). Additional proteolysis based methods include, for example, selective chemical modification (Fiedler et al., Bioconjugate Chemistry 1998, 9(2): 236—234, 1998), epitope excision (Van de Water et al., Clin. Immunol. Immunopathol. 1997, 85(3): 229—235, 1997), and the recently developed method of hydrogen-deuterium (H/D) exchange (Flanagan, N., Genetic Engineering and Biotechnology News 3(2): 25-28, 2010).

The epitope bound by antibodies ofthe present invention may also be determined by structural methods, such as X-ray crystal structure determination (e.g., ), molecular modeling and nuclear ic resonance (NMR) spectroscopy, including NMR determination of the H— D ge rates of labile amide hydrogens in lL—23R when free and when bound in a complex with an antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31:11335-11347; Zinn~Justin et al. (1993) Biochemistry 32:6884-6891).

Additional antibodies g to the same epitope as an antibody of the present invention may be obtained, for example, by screening of antibodies raised against CD98 for g to the epitope, by immunization of an animal with a peptide comprising a fragment of human CD98 comprising the epitope sequence, or by selection of antibodies using phage display for binding to the epitope sequence. Antibodies that bind to the same onal epitope might be expected to exhibit similar biological ties, such as blocking a biological activity of CD98, and such activities can be confirmed by functional assays of the dies.

Additional Activity Assays In one embodiment, an anti-CD98 antibody of the invention is an antagonist antibody that inhibits a biological activity of CD98. The anti-CD98 antibodies of the invention may be assayed to determine ifthey inhibit a biological activity of CD98, for example, binding to light chains. in order to determine whether CD98 antibodies of the invention inhibit binding to light chains, the y of the CD98 antibodies to inhibit amino acid uptake in cancer cell lines is conducted in accordance with the method described in Kim et al., Biochim. Biophys. Acta 1565: 2, 2002.

In one aspect, purified anti-CD98 antibodies can be further characterized by a series of assays including, but not limited to, inal cing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

In one ment, the invention contemplates an altered antibody that possesses some but not all effector functions, which make it a desirable candidate for many applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In certain ments, the Fc activities of the dy are measured to ensure that only the desired ties are maintained. In vitro and/or in vivo cytotoxicity assays can be ted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. An example of an in vitro assay to assess ADCC activity of a molecule of interest is described in US. Patent No. 5,500,362 or ,821,337. Useful effector cells for such assays e eral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or onally, ADCC activity of the molecule of interest may be ed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652— 656 (1998). Clq g assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. To assess ment activation, a CDC assay, e.g. as described in Gazzano—Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art.

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

EXAMPLES The following are examples of methods and compositions of the invention. It is understood that various other ments may be practiced, given the general description provided above.

EXAMPLE 1: IDENTIFICATION OF CD98 ON THE SURFACE OF ACUTE MYELOGENOUS LEUKEMIA (AML) TUMOR CELLS A total of 16 primary AML samples were obtained from Fred Hutchinson Cancer Research Center (FHCRC). Eleven samples from y donors were analyzed. To monitor the quality of individual AML s, hematoxylin and eosin staining of AML blasts were med. Only samples containing at least 75% tumor cells were analyzed. Additionally, analysis was performed on 23 primary chronic lymphocytic leukemia (CLL) samples ed from Billings Clinic or the University of Florida, and 27 primary colorectal omas (CRC) with 22 normal adjacent colon l samples obtained from The Cooperative Human Tissue Network (CHTN) or the National Disease Research Interchange (NDRI). Sample handling was optimized so as to maximally maintain cell viability during sample isolation. Optimal labeling times for AML, CLL and CRC samples were determined to allow for efficient labeling without compromise of cellular integrity.

Surface tagged antigen ng (sTAg) was used to identify and quantitatively profile the repertoire of surface proteins on cells in sixteen core AML samples, five bone marrow mononuclear cell (BMMC) control and six peripheral blood mononuclear cell (PBMC) control samples, core CLL s, 27 CRC samples, and 22 normal adjacent colon samples. The extracellular 3O domains of proteins associated with the AML tumor cell membranes of intact primary tumor cells were chemically tagged and then chromatographically enriched for tagged proteins using a solid-phase affinity resin. Eluted proteins were stored at -80°C prior to mass spectrometry analysis as described below.

Proteins enriched by the sTAg method were identified and quantified using high- resolution, shotgun liquid chromatography tandem mass spectrometry (MS). A hybrid ThermoFisher LTQ-Orbitrap Velos mass spectrometer, which combines the sensitivity of a linear ion trap with the high- resolution and mass cy afforded by the tionary orbitrap mass analyzer (Olsen et al., Mol.

Cell Proteomics -2769, 2009) d to a nanoflow liquid chromatography apparatus was employed for shotgun—based, bottoms—up proteomics to determine the identities and quantitative abundance measurements of proteins in the AML cell surface ment fractions (Yates et al., Annu.

Rev. Biomed. Eng. 11: 49-79, 2009). Tryptic digests from enriched surface proteins were separated by hydrophobicity via , nanoflow liquid chromatography as peptide masses and fragmentation patterns were recorded dynamically by the mass spectrometer. To determine peptide and n identities, the raw MS data was processed using the SEQUEST algorithm executed on a fast-processing Sorcerer 2 rm (Lundgren et al., Curr. Protoc. Bioinformatics, Chapter 13: Unit 13.3, 2009), to determine best- fit matches between experimental fragmentation patterns and those determined in-silico from the human proteome. Resulting matches were tically validated using the PeptideProphet (Keller et al., Anal.

Chem. 74: 5383—5392, 2002) and ProteinProphet (Nesvizhskii et al., Anal. Chem. 75: 4646-4658, 2003) software tools to ensure the lowest le false discovery rates (FDR) and thus ion of only robustly identified proteins in the candidate pool.

The relative quantitative levels of identified proteins in the sTAg samples were ined using the spectral counting method (Neilson et a1., Proteomics 1 1: 535-55 3, 2011). Spectral counting is based on the cal demonstration that the number of assigned (positively identified) spectra associated with peptides from each protein correlates strongly with that protein’s relative abundance in the original mixture (Liu et al.,Anal. Chem. 76:4193—4201, 2004). Spectral counts of identified peptides were obtained from proteomics analytical software platforms including Scaffold (Proteome Software) and ProteoIQ (NuSep) that display, sort and filter the results of SEQUEST~searched mass spectrometry data. Raw spectral counts were ormed to percent ized Spectral Abundance Factor (%NASF) values (Zybailov et al., J. Proteome Res. 5: 2339—2347, 2006) to account for differences in protein length and variability in sample size. Selected monoclonal antibodies were used to te the proteomic measurements using quantitative FACS as an ndent, al confirmatory measure of the sTAg mass ometry-based proteomic profiling of the primary tumor cell surface expression.

[0271] Using sTAg, the heterodimeric type II transmembrane glycoprotein CD98 having the amino acid sequence of SEQ ID NO: 1 was identified as being present at high density on the surface of AML, CLL and CRC tumor cells. As shown in Fig. 1, using sTAg CD98 was identified in 7 of 16 primary AML samples with a mean %NSAF of 0.11 and in 20 of 20 primary CLL samples with a mean %NSAF of 0.15. CD98 was identified in 5 out of 5 bone marrow mononuclear cells (BMMC) and 5 out of6 peripheral blood mononuclear cells (PBMC) samples with mean %NSAFs of 0.05 and 0.06 respectively. Furthermore, CD98 was identified in 11 of 27 primary CRC samples with a mean %NSAF of 0.10 and in only one normal adjacent colon sample with a mean %NSAF less than 0.01. Based on this analysis, CD98 is substantially enriched on a significant n of patient-derived AML, CLL, and CRC primary tumor specimens relative to relevant normal controls.

EXAMPLE 2: IDENTIFICATION OF CD98 IN TUMORS Antibody titration experiments were conducted with anti-CD98 onal antibody 8—34B (see Example 3) and the isotype control antibody HB-121 to establish dilutions that would result in minimal background and maximal detection of signal. Serial dilutions were performed using steam- based antigen retrieval (pH 6.0 citrate buffer) at 1:50, 1:100, 1:200, and 1:400 on formalin-fixed, U! paraffin-embedded (FFPE) tissues or on fresh frozen tissues. Frozen l cell lines (F244 and F244—P) and formalin fixed control cell lines (F244, RM, and F244-P) were provided by Igenica and prepared by LifeSpan. The dilutions of 8-34B of 1 :20 and 1:50 were selected for the study on formalin—fixed, paraffin-embedded tissues, s 8-34B at a dilution of 1:400 was selected for the study on fresh frozen tissues.

[0273] The principal detection system consisted of a Vector anti—mouse secondary antibody (BA—2000) and a Vector ABC—AP kit (AK-5000) with a Vector Red substrate kit (SK~5100), which was used to produce a fuchsia-colored deposit. Tissues were also stained with positive control antibodies (to CD31 and in) to ensure that the tissue antigens were preserved and accessible for immunohistochemical analysis. Only tissues that were positive for CD31 and vimentin staining were selected for the remainder of the study.

Antibody 8—34B, at dilutions of 1:20 and 1:50, showed positive staining within ten out of 15 malignant melanomas and four out of 18 lung carcinomas on formalin—fixed, paraffin» embedded tissues. In on, at a dilution of 1:400, antibody 8-34B showed positive staining of six of the six frozen lung carcinoma samples and also of two of the two frozen melanoma s.

[0275] Table 1: Frequency of positive CD98 staining in lung carcinomas and melanomas Carcinoma Frequency Non-small cell lung 4/18 (FFPE); 6/6 (Frozen) Melanoma 10/15 (FFPE); 2/2 (Frozen) EXAMPLE 3: PREPARATION OF ONAL ANTIBODIES TO CD98 Monoclonal antibodies were prepared in accordance with a general method as described in “Antibodies A Laboratory Manual” w and Lane 1988 CSH Press). Male Sva mice purchased from c Farms were used for immunization. Mice were immunized via subcutaneous injection in the flank with 106 human CD98 (huCD98) expressing tumor cells. On day 39 post zation, mice were boosted intraperitoneally with 5 million huCD98 expressing tumor cells. s were harvested on day 42. Individual splenocytes were prepared and fused with CRL—2016 myeloma cells (ATCC) using a PEG based method as generally described in odies A Laboratory Manual” w and Lane 1988 CSH Press) to establish hybridomas.

Hybridomas were grown in 384 well tissue culture plates and supernatants from individual wells were screened by ELISA for production of antibodies recognizing huCD98. Positive wells were then transferred to 48 well , expanded, and supernatants were collected for huCD98 binding ation by ELISA. Individual hybridomas producing anti-huCD98 antibodies were established as confirmed unique clones producing monoclonal anti—huCD98 antibodies by g single hybridoma cells in wells of 96 well . These cells were grown into colonies and the supernatant from these individual colonies was screened by ELISA to confirm monoclonal dy binding to huCD98.

Clonal hybridomas were ed into pristane treated Balb/C mice to produce ascites. Ascites was ted and purified using Gammabind sepharose (GE Healthcare product code 17-0885—01), Protein A IgG g buffer (Thermo Scientific part number 21001), and IgG elution buffer (Thermo Scientific part number 21004) following the l antibody purification protocol published by Thermo Scientific (Product Instructions #21001).

The nucleic acid and amino acid sequences for the heavy chain and light chain variable regions of the antibody 8-34B are shown below: 8-34B heavy chain variable region CAGGTGCAGCTGAAGGAGTCCGGCCCCGGCCTGGTGGCCCCCTCCCAGTCCCT GTCCATCACCTGCACCGTGTCCGGCTTCTCCCTGACCTCCTACGGCGTGCACTGGATCCGCCAG CCCCCCGGCAAGGGCCTGGAGTGGCTGGGCCTGATCTGGGCCGGCGGCTCCATCAACTACAACT CCGCCCTGATGTCCCGCCTGTCCATCTCCAAGGACAACTCCAAGTCCCAGGTGTTCCTGAAGAT GAACTCCCTGGAGACCGAGGACACCGCCATGTACTACTGCGCCCGCAAGGGCCACATGTACTCC TACGCCATGGACTACTGGGGCCAGGGCACCTCCGTGACCGTGTCCTCC (SEQ ID NO:3) HH8H QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWIRQPPGKGLEWLGEEEA GGSINYNSALMSRLSISKDNSKSQVFLKMNSLETEDTAMYYCARKGHMYSYAMDYWGQGTSVTV SS (SEQ ID NO:4; CDRs are ined) 8~34B light chain variable region “H83 GACATCGTGATGACCCAGTCCCCCTCCTCCCTGACCGTGACCGCCGGCGAGAA GGTGACCATGTCCTGCAAGTCCTCCCAGTCCCTGCTGAACTCCGGCAACCAGAAGACCTACCTG ACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCG AGTCCGGCGTGCCCGACCGCTTCACCGGCTCCGGCTCCGGCACCGAGTTCACCCTGACCATCTC CTCCGTGCAGGCCGAGGACCTGGCCGTGTACTACTGCCAGAACGACTACTCCTACCCCCCCTGG ACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO:5) unsq DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKTYLTWYQQKPGQPPKLL IY‘WEESTRESGVPDRFTGSGSGTEFTLTI SSVQAEDLAVYYCQNDYSYPPWTFGGGTKLEIK (SEQ ID NO:6; CDRs are underlined) The nucleic acid and amino acid sequences for the heavy chain and light chain variable regions of the antibody 18-2A 2.2 are shown below: 18—2A 2.2 heavy chain variable region CAGGTGCAGCTGCAGCAGTCCGGCGCCGAGCTGGTGAAGCCCGGCGCCTCCGT GAAGCTGTCCTGCAAGGCCTCCGGCTACACCTTCACCTCCTACTACATGTACTGGGTGAAGCAG CGCCCCGGCCAGGGCCTGGAGTGGATCGGCGTGATCAACCCCGGCTCCGGCATCACCAACTACA WO 78377 ACGAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCTCCAACACCGCCTACATGCA GCTGTCCTCCCTGTCCTCCGACGACTCCGCCGTGTACTTCTGCTCCGGCTCCGCCAACTGGTTC GCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCGCC (SEQ ID NO:7) “ESQ QVQLQQSGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGYEEE GSGITNYNEKFKGKATLTADKSSNTAYMQLSSLSSDDSAVYFCSGSANWFAYWGQGTLVTVSA (SEQ ID NO:8; CDRs are underlined) 18-2A 2.2 light chain variable region GACATCGTGATGTCCCAGTCCCCCTCCTCCCTGGCCGTGTCCGTGGGCGAGAA GGTGACCATGTCCTGCAAGTCCTCCCAGTCCCTGCTGTACTCCTCCAACCAGAAGAACTACCTG GCCTGGTACCAGCAGAAGCCCGGCCAGTCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCG ACTCCGGCGTGCCCGACCGCTTCACCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTC CTCCGTGAAGGCCGAGGACCTGGCCGTGTACTACTGCCAGCGCTACTACGGCTACCCCTGGACC GGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO:9) DIVMSQS PSSLAVSVGEKVTMSCKSSQSLLYS SNQKNYLAWYQQKPGQSPKLL IYWASTRDSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQRYYGYPWTFGGGTKLEIK (SEQ ID NO:lO; CDRs are underlined) The nucleic acid and amino acid sequences for the heavy chain and light chain variable regions of the antibody 18-2A 7.1 are shown below: 18—2A 7.] heavy chain variable region

[0294] CAGGTGCAGCTGCAGCAGTCCGGCGCCGAGCTGGTGCGCCCCGGCACCTCCGT GAAGGTGTCCTGCAAGGCCTCCGGCAACGCCTTCACCAACTACCTGATCGAGTGGATCAAGCAG CGCCCCGGCCAGGGCCTGGAGTGGATCGGCGTGATCAACCCCGGCTCCGGCATCACCAACTACA ACGAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCTCCAACACCGCCTACATGCA GCTGTCCTCCCTGTCCTCCGACGACTCCGCCGTGTACTTCTGCTCCGGCTCCGCCAACTGGTTC GCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCGCC (SEQ ID NO: ll) QVQLQQSGAELVRPGT SVKVSCKASGNAFTNYL IEWI KQRPGQGLEWI GV:N P GSGITNYNEKFKGKATLTADKSSNTAYMQLSSLSSDDSAVYFCSGSANWFAYWGQGTLVTVSA (SEQ ID NO:12; CDRs are underlined) 18—2A 7.1 light chain variable region NZ?“ GTGATGTCCCAGTCCCCCTCCTCCCTGGCCGTGTCCGTGGGCGAGAA GGTGACCATGTCCTGCAAGTCCTCCCAGTCCCTGCTGTACTCCTCCAACCAGAAGAACTACCTG TACCAGCAGAAGCCCGGCCAGTCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCG ACTCCGGCGTGCCCGACCGCTTCACCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTC CTCCGTGAAGGCCGAGGACCTGGCCGTGTACTACTGCCAGCGCTACTACGGCTACCCCTGGACC TTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO:l3) Hfl9fi SPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLL IYWASTRDSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQRYYGYPWTFGGGTKLEIK (SEQ ID NO:I4; CDRs are underlined) The c acid and amino acid sequences for the heavy chain and light chain le regions of the antibody 1-47C are shown below: l—47C heavy chain variable region HBOH CAGGTGCAGTTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCT GTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCACCTATGGTGTACACTGGGTTCGCCAG CCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATGTGGACTAATGGAATCACAAATTATAATT CGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAAT GAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGGAGGACACTACGGTAGT ACCTCCTATGCTATGGACTTCTGGAGTCAAGGA (SEQ ID NO: 30) [03B] QVQLKESGPGLVAPSQSLSITCTVSGFSLTTYGVHWVRQPPGKGLEWLGYMEE NGITNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARGGHYGSTSYAMDFWSQG (SEQ ID NO:3l; CDRs are underlined) l—47C light chain variable region “B0“ GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAAC TGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAACATGGTATCAGCAGAAA CAGGGAAAATCTCCTCAGCTCCTGGTCTATACTGCAAAAACCTTAGCAGATGGTGTGCCATCAA GGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGA TTTTGGGAGTTATTACTGTCAACATTTTTGGAATACTCCTTACACGTTCGGAGGGGGGACCAAG CTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATC (SEQ ID NO: 32) “HOS DIQMTQSPASLSASVGETVTITCRASGNIHNYLTWYQQKQGKSPQLLVYEAEE SRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWNTPYTFGGGTKLEIK (SEQ ID! NO: 33; CDRs are underlined) HBOQ ThenuddcaddandmnmoaddsammmesbrmehawychflnandhgnchQHVMMbk regions of the antibody 1-1 15A are shown below: 1-1 15A heavy chain variable region “Bum CAGGTGCAGCTGGAGGAGTCAGGACCTGGCCTGGTGGCGACCTCACAGAGCCT GTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAACTGTGGTGTACACTGGGTTCGCCAG CCTCAAGGAAAGGGTCTAGAGTGGCTGGGAGTGATATGGCCTAATGGAATCACAATTTATAATT CGGGTCTCATGTCCAGACTGAGTATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAAA GAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGGAGGACATTACGGTAGT AGCTCCTATGCTATGGACTACTGGAGTCAAGGA (SEQ ID NO: 34) WO 78377 2012/066347 QVQLEESGPGLVATSQSLSITCTVSGFSLTNCGVHWVRQPQGKGLEWLGyI—IIE NGITIYNSGLMSRLSISKDNSKSQVFLKKNSLQTDDTAMYYCARGGHYGSSSYAMDYWSQG (SEQ ID NO:35; CDRs are underlined) 1-115A light chain variable region GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAAC TGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAACATGGTATCAGCAGAAA CCGGGAAAATCTCCTCAACTCCTGGTCTATACTGCAAAAACCTTAGCAGATGGTGTGCCATCAA GGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGA TTTTGGGAGTTATTACTGTCAACATTTTTGGAATACTCCTTACACATTCGGAGGGGGGACCAAG CTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAAGC (SEQ ID NO: 36) DIQMTQSPASLSASVGETVTITCRASGNIHNYLTWYQQKPGKSPQLLVYTAKT LADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWNTPYT FGGGTKLEIK (SEQ ID NO:37; CDRS are underlined) EXAMPLE 4: ISOTYPING AND BINNING OF MONOCLONAL ANTIBODIES Individual hybridoma supematants containing antibodies that recognize huCD98 were assessed for isotype by detection on ELISA with isotype specific ary dies purchased from n Immunologicals (Goal x IgG] HRP — Product# 115206, Goat x IgGZa HRP — Product# 115-035~206, Goat x IgGZb HRP ~ Product# 115207, Goat x IgG3 HRP ~ Product# 115209).

A competition ELISA was performed to ish competitive binding bins. Individual anti-huCD98 isotyped antibody containing hybridoma supematants were allowed to bind to huCD98 in individual wells of an ELISA plate. After 1 hour, the wells were washed and fixed using 4% paraformaldehyde. Then individual anti—huCD98 isotyped antibody (of a different isotype) containing hybridoma supernatants were allowed to bind to huCD98 in individual wells of an ELISA plate for an hour. After washing, the wells were incubated with a specific secondary antibody (Jackson Immunologicals Goatx IgGZa HRP - Product# 115206) and detected with Supersignal ELISA Pico Chemiluminescent substrate (Thermo Scientific — t# 37069). dual IgG2a e antibodies that were able to bind in the presence of an IgGl are considered to be in a unique epitope bin from that particular IgGl. Individual IgGZa isotype antibodies that were unable to bind in the presence of an IgGl are considered to be in the same epitope bin as that particular IgGl. In this way multiple epitope bins were defined for huCD98 binding dies, as illustrated in Fig. 2.

EXAMPLE 5: BINDING TY

[0315] Purified anti-CD98 onal antibodies are tested for affinity by the general method published by Carderelli et al. (2002) Cancer Immunol Immunother 51; 15—24. Briefly, CD98 expressing cells are incubated with different s of anti—CD98 monoclonal antibodies overnight, then assessed by FACS with a goat anti—human Fc c or anti—mouse Fc specific PIE-conjugated secondary antibody (Jackson Immunologicals). FACS data is analyzed using Graphpad Prism software and de are determined using the Graphpad Prism Kd calculation tool.

EXAMPLE 6: PRODUCTION AND CHARACTERIZATION OF CHIMERIC ANTIBODIES Total RNA was ted from hybridomas producing anti-CD98 monoclonal antibodies using the Qiagen RNeasy Mini kit (Cat No. 74104), ed by the Qiagen OneStep RT~PCR Kit (Cat No. 210210). RT—PCR was performed with primer sets c for murine heavy and light chain sequences. For each RNA sample, 12 individual heavy chain and 11 light chain RT-PCR reactions were set up using degenerate forward primer mixtures covering the leader sequences of murine variable regions. Reverse primers were located in the constant regions of murine heavy and light chains. The RT- PCR products from the first-round reactions were further amplified in the second—round PCR. 12 individual heavy chain and 11 light chain RT-PCR reactions were set up using semi-nested primer sets specific for antibody variable regions. PCR reactions were run on agarose gels and heavy and light chain PCR products were cut from the gel and cloned into sequencing vectors. 10—20 clones per hybridoma were ced to determine the anti-CD98 monoclonal antibody variable region. Heavy chain variable regions were then cloned in—frame into a vector containing human IgG1 heavy chain constant region sequence and light chain variable regions were cloned in—frame into a vector containing human kappa light chain constant region sequence. Chimeric antibodies were generated from transient transfection of HEK293 Freestyle cells and d using the methodology described in Example 3.

Purified chimeric antibodies were subjected to binding analysis as described in Example 4 for the mouse monoclonal anti—CD98 antibodies. Fig. 3A shows the results of competitive binding assays for the 8—34B, 18-2A 2.1, 18—2A 2.2 and 18-2A 7.1 chimeric antibodies. Reference antibodies 1—4 are as in Fig. 2, while “isotype” is a control IgGZa antibody that does not bind to CD98.

The s shown in Fig. 3A trate that the ic antibodies retain the epitope g specificities of the murine antibodies from which they were d. The binding affinities of the murine and chimeric anti-CD98 monoclonal antibodies were determined by FACS analysis as described in Example 5 with colon cancer cell line DLDl. The Kd values (ranging from 0.9nM to 4.5 nM) are shown in Fig. 3B, indicating that all these recombinant antibodies retain high-affinity binding to CD98, comparable to their parental murine antibodies. Purified ic monoclonal antibodies were also ted to FACS analysis as described in Example 5 with three AML primary tumor samples and a cell line expressing cynomolgus monkey CD98 (cynCD98). The results shown in Fig. 3C demonstrate that all chimeric antibodies retained the binding to human CD98 on AML cells and cynCD98.

The binding affinities of the murine and chimeric D98 monoclonal antibodies were determined by on/off rate determination using a BIACORE system (reviewed in Lipschultz et al., Methods 20: 310-318, 2000). As shown in Table 2, the affinities of the ic antibodies were similar to those of the parent murine antibodies. Data for the humanized antibody 8—34B H2 L1 (see Example 7) is also shown. 2012/066347 Table 2 Antibody ka kd Kd [nM] (on-rate) (off-rate) [M-1‘s-1] [s-1] 18—2A 6.2 x105 3.5 x105 0.56 18—2A ch7.1 J2 x105 2.6 x1035 0.22 18—2A ch22 22-5 x105 3.5 x104 i 1.4 8-34B 8.2 x 104 1.6 x10'4 2.0 8 348 ch 1.7x 165" 1.6 x10" 0.94 8-34B H2 L1 2.5 x 10‘5 ‘ 3.2 x10“4 1.3 EXAMPLE 7: PREPARATION OF HUMANIZED ANTIBODIES A humanized form of the murine monoclonal antibody 8-34B was made by grafting the CDRs of the murine heavy chain and light chain variable domains into the human acceptor framework regions as shown in Fig. 4. The humanized 8-34B light chain variable domain L1 (SEQ ID NO:15) was constructed by grafting the CDRs of the murine light chain into the human or sequence (GenBank Accession No. ACJ71709.1). The 8-34B humanized light chain variable domain L2 (SEQ ID NO:16) was constructed by replacing two residues in FR3 of the human acceptor light chain with the corresponding residues of the murine monoclonal antibody (amino acid substitutions S63T andD70E by Kabat numbering; see Fig. 4A). The 8—34B humanized heavy chain H1 (SEQ ID NO: 17) was constructed by ng the CDRs of the murine heavy chain into the human acceptor sequence (GenBank Accession No. 137782). The 8-34B humanized heavy chain variable domain H2 (SEQ ID NO:18) was ucted by ing one residue in FR2 and two residues in FR3 of the human acceptor heavy chain with the corresponding es of the murine monoclonal antibody, resulting in the amino acid substitutions I48L, V71K and F78V by Kabat numbering (see Fig. 4B). The 8-34B humanized heavy chain variable domain H3 (SEQ ID NO:19) added two onal substitutions back to the murine e in FR3, V67L and T73N, as shown in Fig. 4B.

The nucleic acid and amino acid sequences for the H2 heavy chain and the L1 light chain variable regions of the humanized antibody 8-34B are shown below: Humanized 8—34B heavy chain variable region H2 CAGGTGCAGCTGCAGGAGTCCGGCCCCGGCCTGGTGAAGCCCTCCGAGACCCT GTCCCTGACCTGCACCGTGTCCGGCTTCTCCCTGACCTCCTACGGCGTGCACTGGATCCGCCAG CCCCCCGGCAAGGGCCTGGAGTGGCTGGGCCTGATCTGGGCCGGCGGCTCCATCAACTACAACT CCGCCCTGATGTCCCGCGTGACCATCTCCAAGGACACCTCCAAGAACCAGGTGTCCCTGAAGCT GTCCTCCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCAAGGGCCACATGTACTCC TACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC (SEQ ID NO: 25) HBZM QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVHWIRQPPGKGLEWLGLIWA GGSINYNSALMSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARKGHMYSYAMDYWGQGTLVTV U. SS (SEQ ID NO: 18; CDRs are underlined) “B25 Hummumd864Bhghchmnvmmwemganl GTGATGACCCAGTCCCCCGACTCCCTGGCCGTGTCCCTGGGCGAGCG CGCCACCATCAACTGCAAGTCCTCCCAGTCCCTGCTGAACTCCGGCAACCAGAAGACCTACCTG ACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCG AGTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTC CTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAACGACTACTCCTACCCCCCCTGG GGCCAGGGCACCAAGGTGGAGATCAAG (SEQ ID NO: 24) HBZN DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKTYLTWYQQKPGQPPKLL :YWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPPWTFGQGTKVEIK l5 SEQ ID NO: 15; CDRS are underlined) A humanized form of the murine monoclonal antibody 18~2A was made by grafting the CDRs of the murine heavy chain and light chain variable domains from the chimeric antibody 18-2A 7.1 into the human acceptor framework regions. The humanized 18—2A 7.1 light chain variable domain L1 (SEQ ID NO:20) was ucted by grafting the CDRs of the murine light chain into the human acceptor sequence (GenBank Accession No. ACJ71709.1). The 18- 2A 7.1 light chain variable domain L2 (SEQ ID NO:21) was constructed by ing certain human framework residues with the corresponding residues from the murine monoclonal antibody. The 18~2A 7.1 humanized heavy chain variable domain H1 (SEQ ID NO: 22) was constructed by grafting the CDRs of the murine heavy chain into the human or sequence.

The 18-2A 7.1 humanized heavy chain variable domain H2 (SEQ ID NO: 23) was constructed by replacing certain human framework residues with the corresponding residues from the murine onal antibody.

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

[0330] Humanized 18-2A 7.] heavy chain variable region H1 CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGT GAAGGTGTCCTGCAAGGCCTCCGGCAACGCCTTCACCAACTACCTGATCGAGTGGGTGCGCCAG GCCCCCGGCCAGGGCCTGGAGTGGATGGGCGTGATCAACCCCGGCTCCGGCATCACCAACTACA ACGAGAAGTTCAAGGGCAAGGCCACCATCACCGCCGACAAGTCCACCTCCACCGCCTACATGGA GCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTACTGCTCCGGCTCCGCCAACTGGTTC GCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC (SEQ ID NO: 26) PCT/U82012/066347 HB3H QVQLVQSGAEVKKPGSSVKVSCKASGNAFTNYLIEWVRQAPGQGLEWMGVINP GSGITNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCSGSANWFAYWGQGTLVTVSS HB3M (SEQ ID NO: 22; CDRs are underlined) Humanized 18—2A 7.1 light chain variable region Ll "B3fl GACATCGTGATGACCCAGTCCCCCGACTCCCTGGCCGTGTCCCTGGGCGAGCG CGCCACCATCAACTGCAAGTCCTCCCAGTCCCTGCTGTACTCCTCCAACCAGAAGAACTACCTG GCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCG ACTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTC CTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCGCTACTACGGCTACCCCTGGACC TTCGGCGGCGGCACCAAGGTGGAGATCAAG (SEQ ID NO: 27) HB3Q DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLL IYWASTRDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQRYYGYPWTFGGGTKVEIK (SEQ ID NO: 20; CDRS are underlined) Humanized 18—2A 7.1 heavy chain variable region H2 HBSM CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGT GAAGGTGTCCTGCAAGGCCTCCGGCAACGCCTTCACCAACTACCTGATCGAGTGGATCCGCCAG GCCCCCGGCCAGGGCCTGGAGTGGATCGGCGTGATCAACCCCGGCTCCGGCATCACCAACTACA ACGAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCCACCTCCACCGCCTACATGGA GCTGTCCTCCCTGCGCTCCGAGGACACCGCCGTGTACTACTGCTCCGGCTCCGCCAACTGGTTC GCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC (SEQ ID NO: 28) “B3% QVQLVQSGAEVKKPGSSVKVSCKASGNAFTNYLIEWIRQAPGQGLEWIGVINP YNEKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCSGSANWFAYWGQGTLVTVSS (SEQ ID NO: 23; CDRS are underlined) Humanized 18—2A 7.] light chain variable region L2 HB4H GACATCGTGATGACCCAGTCCCCCGACTCCCTGGCCGTGTCCCTGGGCGAGCG CATCAACTGCAAGTCCTCCCAGTCCCTGCTGTACTCCTCCAACCAGAAGAACTACCTG GCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCTCCACCCGCG ACTCCGGCGTGCCCGACCGCTTCACCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTC CTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCGCTACTACGGCTACCCCTGGACC TTCGGCGGCGGCACCAAGGTGGAGATCAAG (SEQ ID NO: 29) “B4” DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLL IYWASTRDSGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQRYYGYPWTFGGGTKVEIK (SEQ ID NO: 21; CDRs are underlined) EXAMPLE 8: ANTI-CD-98 MONOCLONAL ANTIBODY-MEDIATED INHIBITION OF TUMORS IN VIVO dy efficacy on tumor growth and metastasis formation is studied, e.g., in mouse subcutaneous or orthotopic cancer xenograft models. The antibodies can be ugated, or can be conjugated to a therapeutic agent, as appreciated in the art.

Monoclonal antibodies are raised against CD-98 as described in Example 1, and purified and terized as described above. Chimeric or humanized antibodies as described above may also be used. A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of tumor xenografis.

Subcutaneous tumors are generated by injection of 1 x 107 cancer cells in a mixture of PBS (without magnesium or calcium) and BD Matrigel (BD Biosciences) at a 1:1 ratio in the right flank of female SClD or nu mice. The injected total volume per mouse is 200ml with 50% being Matrigel (BD Biosciences). Mice are randomized once tumors reach a size between 65—200mm3. Antibodies are administered weekly, and body weights and tumors are measured once and twice weekly, respectively.

Tumor volume is calculated as described (van der Horst et al. (2009) Neoplasia 11: 355—364). As a negative control, mice are injected with either purified mouse lgG or PBS; or a purified monoclonal antibody that recognizes an antigen other than CD98.

EXAMPLE 9: EFFECT OF CD-98 MONOCLONAL DIES ON THE GROWTH OF B- CELL LYMPHOMA XENOGRAFTS IN MICE The Ramos (B—cell lymphoma) cell line was ed from ATCC and cultured according to the suppliers’ protocols. Animals were obtained from Charles River Laboratories. 4-6 ld immunodeficient SClD female mice on a CB. 1 7 background were subcutaneously injected on the right flank with 1x107 viable cells in a mixture of PBS ut magnesium or calcium) and BD Matrigel (BD Biosciences) at a 1:1 ratio. The ed total volume per mouse was 200p] with 50% being Matrigel (BD Biosciences). Once the tumor reached a size between 65—200mm3 mice were randomized. dies were administered , and body weights and tumors were measured once and twice , tively. Tumor volume was calculated as described (van der Horst et al. (2009) Neoplasia 11: 355—364). All the experiments were performed on groups of at least 7 animals per experimental point. Animal ments were med in accordance with protocols approved by the Igenica lnc. utional Review Board - Animal Care and Use Committee.

Statistical significance between treatment and control groups was calculated using the Graphpad Prism software package and applying Student’s two—tailed t—test. A p—value of less than 0.05 was considered significant. Doubling time and time to ssion analysis was calculated as described in Daniel et al. (2007) Blood 1 10:4037—4046.

Rituximab CD20 antibody) was used as a positive therapeutic control antibody.

Antibody H8121 was an IgG2a negative control. Rituximab and the anti—CD98 antibodies 8-93A and 18- 3A are lgGl antibodies, s all other anti-CD98 antibodies are IgG2a antibodies.

Treatment with anti-CD98 antibodies was shown to induce strong tumor growth inhibitiion in established B cell lymphoma (Ramos) tumors. Notably, anti-CD98 antibody treatment was superior to rituximab (see Fig. 5A, Fig. 5B) in inducing tumor growth inhibition.

Anti-CD98 antibodies were also shown to significantly prolong time to ssion of treated RAMOS tumors. The tumor doubling time of the tumor regrowth data from Figs. 5A—C was calculated as described above and used for further prediction of time to ssion (TTP). TTP was then extrapolated for each animal within the treatment , until 3 would have been reached and graphed as a Kaplan-Meier curve, as shown in Figs. 6A-C. As shown in Figs. 6A—C, various anti-CD98 antibodies are superior to rituximab in prolonging time to progression in Ramos tumors. [0349} The starting tumor volumes of established Ramos tumors were increased in order to assess the potential maximum therapeutic efficacy of D98 antibodies. Table 3 shows the therapeutic efficacy of anti—CD98 antibodies in Ramos xenograft tumor models with sing tumor volume starting size. As can be seen, D98 antibodies retain tumor growth inhibition (TGl) even at sing tumor volumes.

Table 3 Antibody [V0=75.6mm3] {V0=144.3mm3] [V0=249.6mm3] TGI [%} p—Value TGI [%} p-Value TGI {0A,} p-Value 0.000127 0.000009 % 0.000022 21 0.000004 0.000009 —65.26% 733000046 0.000006 ~51 29% 0.000665 0.000015 0.000022 8-300B —63.52% 0.000105 l8—4A % 0.000072 8—361A6 —60.59% 0.000141 8—25C —57.94% 0.000140 456—83A -57.08% 0.000151 8-162A4 -55.89% 0.000236 l—47C —54.23% 0.000874 456—26A —53.07°/o 0.000277 8—319A2 ~52.55% 0.000307 -51.97% 0.000576 Rituximab 0.005564 8-93A 0.016095 18—3A 0.066085 E 10: EFFECT OF ANTI-CD—98 MONOCLONAL ANTIBODIES ON THE INHIBITION OF TUMOR GROWTH IN VIVO The effects of the anti—CD98 monoclonal antibodies 8-348 and 18—2A were tested in several xenograft models the protocol described in e 7. , using The DLD—l (colorectal carcinoma), A549 (non small cell lung carcinoma), Ramos (B- cell lymphoma), and OCl-AML-3 (acute myeloid leukemia) cell lines were obtained from ATCC and cultured according to the suppliers’ protocols. Animals were ed from Charles River tories. 4—6 week-old immunodeficient NOD.SCID female mice on a CB. 17 background were used for the sarcoma tumor model, 46 week-old immunodeficient SCID female mice on a CB.l7 background were '/' female mice used for the Ramos and DLD-l tumor models, and 4-6 ld immunodeficient nu were used for the A549 and the OCl-AML—3 tumor model. Either rituximab (an IgGl anti-CD20 antibody) or Erbitux (an IgGl anti—EGFR antibody) was used as a positive control antibody, and an IgGga antibody to an irrelevant antigen was used as a negative l. DC101 is a rat anti-mouse VEGFRZ/KDR lgGl mAb (ATCC No. HB-11534) and serves as a positive control. ions, antibody treatment and statistical calculations were performed as described in Example 9. {0352] As shown in Figs. 6—9, the anti—CD98 monoclonal antibody l8-2A is a potent inhibitor of tumor growth in the colorectal cancer (DLD—l), non-small cell lung cancer (A549), Burkitts lymphoma ) andAML (OCl-AML-3) xenografts. The effect of 18-2A compared bly to that of Rituxan (Fig. 7) and Erbitux (Fig. 9 and Fig. 10).

The anti—CD98 monoclonal antibody 8-34B inhibited tumor growth in the Ramos and AML ML-3) xenografts, as shown in Fig. 7 and Fig. 8.

EXAMPLE 11: ANTI—CD98 MONOCLONAL ANTIBODIES IN MOUSE STRAINS WITH DIFFERENT IMMUNODEFICIENT BACKGROUNDS Three anti—CD98 onal antibodies, 8-300B, 8-34B and l8—2A, were tested in the RAMOS xenograft model as described in Example 9, using different mouse strains. An lgGga antibody was used as a negative control. The three immunodeficient mouse strains ranged from less to highly immunocompromised. SCID mice lack functional B and T cells, but retain natural killer (NK) cell function and some complement function. NOD.SCID mice lack complement function and have only partial NK function, while NSG (NOD/SClD/gamma) mice lack NK function.

As shown in Fig. l l, the growth inhibitory effect of anti—CD98 antibodies in the RAMOS xenograft model increases when assessed in more immuno—competent mouse strains, indicating that the in vivo umor ty of the anti-CD98 antibodies is due to a combination of immunoeffector function and inhibition of CD98 activity. The results in the NSG mice, which lack all ADCC and CDC function, suggests that anti—CD98 antibodies may also inhibit the biological function of CD98 in the RAMOS xenograft, and that CD98 may be al for RAMOS tumor growth and/or maintenance in vivo.

EXAMPLE 12: IN VIVO EFFICACY OF MURINE AND CHIMERIC ANTI-CD98 MONCLONAL ANTIBODIES The in vivo efficacy of the murine anti—CD98 dies 8-34B and l8-2A were compared to that of the chimeric dies 8-34B~ch and 18-2A-ch7.1 in the RAMOS xenograft model, as described in Example 9. The antibodies were tested at 0.5 mg/kg dose (~30 nm). The s shown in Fig. 12 confirm that the ic anti—CD98 antibodies inhibit in vivo tumor growth at an effectiveness similar to their parental murine counterparts.

EXAMPLE 13: EPITOPE MAPPING OF HUMANIZED MONOCLONAL ANTIBODY IGN523 Materials and methods

[0357] ts. FREESTYLETM CHO—S cells (Invitrogen) were maintained in FREESTYLETM CHO expression medium (Invitrogen) supplemented with GlutaMAXTM. Control antibodies 4F2 and MEM108 were obtained from Santa Cruz Biotechnology and Thermo Scientific, respectively. dies were labeled with Alexa Fluor 647 using the appropriate Zenon Antibody Labeling Kit (lnvitrogen).

[0358] Plasmid constructions. Full length CD98, CD98 point mutants and mouse and human CD98 chimeras were ucted by gene synthesis (GeneWiz) and cloned into the pCDNA3.1 vector (Invitrogen). CD98 ECD, CD98 ECD point mutants and CD98 ECD chimeras were constructed by gene sis and cloned into the pDisplay vector (Invitrogen). All constructs were confirmed by DNA sequencing.

[0359] Fluorescence—activated cell sorter (FACS) analysis ofIGN523 binding to CD98.

CD98 chimeras, CD98 point mutation constructs and human wild type control constructs were transfected by electroporation with a Nucleofector 4D unit ). Constructs were mixed with the transfection solution and then transiently transfected into FREESTYLETM CHO-S cells. ected CHO-S cells were harvested 24 hours after transfection. Cells were quantitated and then stained with lGN523, MEM108 or 4F2 antibodies. Before ng, the antibodies were labeled with Alexa Fluor 647 using the appropriate Zenon Antibody Labeling Kit (lnvitrogen). Flow data were acquired on a yi MACSQuant Analyzer (Miltenyi Biotec) and data analysis was performed using FlowJo software version 9.5.3 (Tree Star, Inc).

Determining the IGN523 bindingreguon on CD98

[0360] To determine the region of CD98 that is involved in IGN523 binding, regions of approximately 40 contiguous amino acids of human CD98 (SEQ ID NO: 1) were substituted with the corresponding regions of mouse CD98 (SEQ ID NO: 96), and the effects of these substitutions on the overall binding of IGN523were monitored using FACS analysis. Fig. 13 shows the regions of the mouse WO 78377 sequence that were substituted into the human sequence to form the 13 mouse-human CD98 chimera ucts. Fig. 14 shows the binding of IGN523 and a control antibody to murine CD98 (Mu), human CD98 (Hu) and the 13 mouse-human chimeras. The s demonstrate that the region substituted in chimera construct 10 is required for IGN523 binding to CD98. In contrast, it can be seen that the region U! sible for binding of the control antibody is found in the regions substituted in chimeras 11 and 12.

Fig. 15 shows the sequence of the region of human CD98 within which IGN523 binds, as identified using the mouse-human CD98 chimera constructs, and the location of this sequence within the three-dimensional structure of CD98. The region defined by chimera 10 consists of amino acid residues T358-N405 of human CD98. Amino acids T358—G368 lined) are buried in the crystal structure and are unlikely to be part of the binding interface. Non-conserved residues between the human and mouse ces are shown in bold. The substitution ofN at site D391 results in an extra glycosylation site in the mouse sequence as compared to human.

Fine mapping of IGN523 with single and multiple mutations in CD98 To further define the IGN523 epitope, single or multiple amino acid changes were introduced in the region defined by mouse—human a 10. Four constructs were made, each introducing the nonhomologous residues from a portion of the mouse CD98 sequence into the human CD98 sequence. uct 4.1 contained mutations l371L, D3 74Q, A3 75G and a deletion of A3 76.

Construct 42 contained mutations M3 83A and E3 84K. Construct 4.3 contained mutations D391N, F3951, P396F and D397H. Construct 4.4 contained mutations G400R, A401P and A404L. FACS analysis of CHO cells transfected with the respective constructs showed that the mutations contained in construct 4.1 prevented IGN523 from binding to CD98 (Fig. 16). The mutations contained in construct 4.4 also affected binding of IGN523 to CD98, to a lesser degree (Fig. 16).

To determine which hydrophobic residues are involved in the g interface of IGN523 to human CD98, single mutation constructs of hydrophobic es in the targeted loop region were created by substituting these hydrophobic residues with highly charged amino acids such as aspartic acid or asparagine. The single mutant constructs were individually transfected into CHO cells and analyzed by FACS to determine g to IGN523. As shown in Fig. 17, on of hydrophobic residues A377 and L378 to a charged amino acid negatively affected IGN523 binding. To a lesser extent, mutation of residues 1398 and A401 showed a similar negative effect on binding of IGN523.

Further constructs containing multiple ons were made to identify additional important residues for IGN523 binding to CD98. As shown in Fig. 18, the multiple mutations in construct M1 (D374Q, D397H, G400R and A401P) completely prevented IGN523 from binding, indicating the significance of these residues. Construct M2 (D374E and A3 75E) and M3 (D3978 and l398T) also resulted in reduced binding. onal experiments with constructs comprising a deletion of A376 (not shown) indicated that the ce of this residue appears to be required for correct folding of the epitope loop region.

Based upon the binding studies using the mouse—human chimeras, mutations of hydrophobic residues, and multiple mutations, the following amino acids were determined to be part of the lGN523 epitope: D374, A377, L378, D397, 1398, G400 and A401.

Peptide scanning analysis As a complement to these mutational studies, the binding epitope of lGN523 was r analyzed using a peptide scanning analysis an).

Synthesis ofpeptides: To reconstruct discontinuous epitopes of the target molecule, a library of structured peptides was sized using Pepscan’s proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology (Pepscan). Chemical linkage of peptides onto lds was carried out essentially as follows: The side—chains of multiple cysteines in the peptides were coupled to one or two CLIPS templates. A 0.5 mM solution of the T2 CLIPS template 1,3—bis (bromomethyl) benzene was dissolved in ammonium bicarbonate (20 mM, pH 7.9)/acetonitrile (1 : 1(v/v). This solution was added onto the peptide arrays, causing the CLIPS template to bind to side—chains of two cysteines as present in the solid—phase bound es of the peptide—arrays (455 wells plate with 3 ul wells). The peptide arrays were gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrayswere washed extensively with excess of H20 and sonicated in disrupt—buffer containing 1 percent SDS/0.1 percent beta-mercaptoethanol in PBS (pH 7.2) at 70°C for 30 minutes, followed by sonication in H20 for another 45 minutes. See also the methods described in Timmerman et a]. (2007), J.

Mol. it. 202283-99; and Slootstra et a1. (1996), Molecular Diversity 1: 87-96.

[0369] ELISA screening: The binding of antibody to each of the synthesized es was tested in a PEPSCAN—based ELISA. The peptide arrays were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of an antibody peroxidase ate (SBA, catalog no. 2010-05) for one hour at 25°C. After washing, the dase substrate 2,2’-azino—di—3—ethylbenzthiazoline sulfonate (ABTS) and 2 ul/ml of 3 percent H202 were added. After one hour, the color development was ed. The color development was fied with a charge coupled device (CCD)-camera and an image processing system.

Data processing: The values obtained from the CCD camera range from 0 to 3000 mAU, r to a standard 96-well plate ELISA-reader. The results were quantified and stored into the Peplab database. Occasionally a well contains an air-bubble resulting in a false—positive value. The cards were ly ted and any values caused by an air—bubble are scored as 0.

Synthesis quality control: To verify the quality of the sized peptides, a separate set of positive and ve control peptides was synthesized in parallel. These were screened with antibody 57.9 umus et al., J. Virology, 1990, 64:3304-3309).

The results of a variable-length peptide screen are shown in Fig. 19. ELISA results for each peptide are shown as a horizontal line. Start and end points of the lines indicate which residues are included in the peptide, and the Y-value of the line shows the ELISA result obtained for that peptide. The ELISA results for the peptides show dominant binding for 395FPDIPGA401 (SEQ ID NO: 42) and secondary binding for 37()PGQPm (SEQ ID NO: 43). A global analysis of 29 -positions alanine replacement sets showed strongest binding for 394sFDIPGAVASAixnvrrv‘m (SEQ ID NO: 44). Fig. 20 shows the results of a best-binding single—positions alanine-replacement peptide set, in which each residue of peptide SEQ ID NO:44 was replaced by alanine, or by glycine if the original residue was alanine. The analysis shows strongest dependency for residues F395, P396, D397 and I398, which appear to form the core of this epitope.

Next, CLIPS conformational matrix structures were used to to bring together pairs of peptides from the two regions fied in the peptide screen, so as to assay antibody binding to conformational epitopes. Fig. 21 shows heat maps representing the data obtained from CLIPS conformational matrix structures that combined two partial sequences of human CD98 (shown on X axis and Y axis). The results indicated a high dependency on secondary ure. The best binding was observed for peptides that ed 395FPDIPGAVSAN405 (SEQ ID NO: 70) and 372GLDAAALPGQP382 (SEQ ID NO: 50). These two peptides were used as the basis for a nesis screen as shown in Fig. 22. SEQl shows the sequence of the e and DlFl indicates where the mutation is located in the peptide. Grey fields indicate peptides having non-mutated sequences. The last column shows the difference in ELISA value between wild—type and mutated peptide. High values indicate that the mutation has a strong negative effect on binding. The mutagenesis screen identified P379, G380, D397 and I398 as important binding residues.

Fig. 23 shows the location on the surface of human CD98 of the amino acid residues determined to be important for binding of humanized monoclonal antibody . Fig. 23A shows the location of the residues identified by the a and mutagenesis studies, while Fig. 23B shows the location of residues determined by n analysis. Fig. 23C shows that both sets of residues substantially overlap, confirming that the highlighted loop region is the g epitope for lGN523.

EXAMPLE 14: IN VIV0 ANTI—TUMOR ACTIVITY OF HUMANIZED ANTI-CD98 MONOCLONAL ANTIBODY IGN523 The effects of the anti-CD98 humanized monoclonal antibody IGN523 were tested in several xenograft models the protocol described in Example 8. Injections, antibody ent and , using statistical calculations were performed as described in Example 9. IGN523 was compared to the standard of care drug rituximab in the RAMOS (RA.1) and DAU Burkitt lymphoma models (Fig. 24 and Fig. 25).

IGN523 was then ed to carboplatin, at its maximum tolerated dose, in the patient—derived NSCLC aft model IGN—LNG—12 (Fig. 26). IGN523 was also tested in the AML xenograft model [(6—1 e 27). The tumors used were minimally passaged in ID mice, t any ening cell culture, in order to preserve the heterogeneity of the original tumors.

In all cases IGN523 showed significant tumor growth inhibition. Interestingly, IGN— LNG—12 patient~derived tumors lead to weight-loss in NOD-SCID mice, which correlated with tumor burden (Fig. 26A and 26B). Although carboplatin at its maximal tolerated dose induced a significant tumor growth inhibition, it also displayed an increase in body weight loss. On the other hand, IGN523 ent exerted a similar anti-tumor effect as carboplatin, but d IGN—LNG—l2 induced body WO 78377 weight loss. These data demonstrate that IGN523 treatment is as effective as carboplatin in this NOD- SCID model without inducing body weight loss. {0377] The data demonstrate significant tumor growth tion in Burkitt lymphoma models RAMOS (RA.1) and DAU, in the t-derived lung tumor xenograft lGN-LNG-lZ, and in the AML model KG-l. Moreover, the tumor growth inhibition of IGN523 is comparable to the anti—cancer agents carboplatin or rituximab, tively (Table 4).

Table 1 T6! [%1 RAMOS Treatment (RA.1) DAU IGN-LNG-12 KG—1 IGN523 92 76 44 | 53 Rituximab 84 67 N/A 1 N/A Carboplatin N/A N/A 55 1 N/A The dose response relationship of IGN523 in lGN-LNG-lZ tumors was also investigated. lGN-LNG—IZ was chosen to determine the dose response to the antibody using a “therapeutic dosing regimen”. IGN523 was dosed on days 12 and 19 between 1 mg/kg and 30 mg/kg (Figure 15). A dose of 10 and 30 mg/kg produced the maximum tumor growth reduction of 50—66% relative to the control group in lGN-LNG—12 lung tumors.

Taken together, in vivo efficacy data demonstrate that lGN523 induces significant tumor growth inhibition in various xenograft models, which is at least comparable to that of standard clinical agents.

E 15: RECEPTOR BINDING SPECIFICITY FOR HUMANIZED ANTI—CD98 ONAL ANTIBODY IGN523

[0380] Table 5 shows the percentage of sequence homology of the extracellular domain (ECD) of CD98 between the ted species and of the epitope of CD98 to which IGN523 binds. As shown in Table 5, the homology between the human and cynomolgus monkey epitope of CD98 to which IGN523 binds is 96%. Receptor binding specificity studies utilizing various methodologies, such as surface plasmon resonance (SPR, Biacore), bio-layer interferometry ) or ELISA, determined that IGN523 binds with high affinity to human and cynomolgus monkey CD98, but does not bind to other s ing the murine, rat, rabbit, dog, and pig homologs of CD98, due to decreasing homology (Table 5). Biacore and Octet data demonstrate that the KB of IGN523 ranges n 2 and 6 nM for human CD98, and between 8 and 14 nM for cynomolgus monkey CD98. ELISA binding data show that the EC50 for IGN523 is 9 ng for human and 39 ng for cynomolgus monkey CD98.

PCT/U82012/066347 Table 5 % ty to "/0 Identity to lGN523 Biacore Human Epitope Region of Species CD98 ECD Human CD98 Cynomolgus monkeyfiL 13 — 14 39 Rabbit ve negative Dog negative negative Rat negative negative Mouse negative negative Pig negative negative ND = Not Determined.

In order to corroborate that cynomolgus monkey is the relevant species for toxicology studies, human and cynomolgus frozen tissue sections of kidney, placenta and cerebrum were stained with lGN523 (Fig. 29). In cryosections of human kidney and placenta (positive control), moderate to e membrane and cytoplasmic staining of renal tubular epithelial (kidney) and trophoblastic epithelium cells (placenta) was observed, which was similar, if not identical, to the staining ity in counterpart cynomolgus monkey tissue sections. In the case of human and cynomolgus monkey cerebrum, similar weak~to—moderate cytoplasmic staining of neuropil and cytoplasmic granule epithelium l0 was observed. Taken together, it appears that staining in lgus monkey tissue is comparable to that in human tissues. Based on the affinity and staining data the cynomolgus monkey is considered to be the appropriate species to evaluate the safety of IGN523.

EXAMPLE 16: SINGLE-DOSE PHARMACOKINETIC STUDY IN LGUS MONKEYS An exploratory non-GLP single dose intravenous cokinetic study of IGN523 was performed in male and female cynomolgus monkeys at doses of l, 3, 10, and 100 mg/kg (N = 2 per sex/group). Pronounced dose—dependent kinetics of IGN523 was observed following a single l—h lV infusion of l, 3, 10 or 100 mg of IGN523 per kg of body weight in the monkey. Therefore, the basic tion regarding linearity that is implicit in the application of the non-compartmental analysis methods used does not apply to IGN523 over the entire dose-range studied, and the ters are displayed for making descriptive comparisons among the dose groups. gh only 2 animals per sex were evaluated at each dose level, there were no apparent differences in PK profiles of male versus female animals. The mean concentration-time profiles of male and female s were similar within each dose group at doses ranging from 1 to 100 mg of IGN523 per kg of body weight (Table 6).

W0 78377 PCT/U82012/066347 Table 6: PK Parameters of lGN523 Dose TWA (hours) CL, (mL/h/kg) V55, (mL/kg) (mg/kg) Mean Range Mean Range Mean Range 1 4.7 3.7-5.9 4.30 3.54-4.82 32.1 i 27.4—39.8 3 12.4 102-135 1.7 1.58-1.88 31.5 i 282-351 i T I 10 12.0 63.4-15.8 1.03 1.00-1.10 29.3 i 257-317 | T 100 60.9 44.9—96.9 0.44 034—049 41.1 } 37.8—44 TWA; terminal half-life, CL; clearance, V33; Volume of distribution Taken together, there was a 10-fold reduction in the “apparent” plasma CL of lGN523 over the dose range of l to 100 mg/kg. Mean “apparent” MRT and mean “apparent” T1Q increased 12 to 13-fold over the dose range of 1 to 100 mg/kg. There were no ences in mean “apparent” VSS at doses of l, 3 or 10 mg/kg, but mean “apparent” VSS in the 100 mg/kg dose cohort was approximately 30 to 40% higher compared to the l, 3 and 10 mg/kg cohorts. All of these findings suggest that the nonlinear disposition characteristics of IGN523 in the monkey may be due to target-mediated drug disposition (TMDD). TMDD models have previously been used to describe the ear disposition of other monoclonal antibodies. TMDD arises when the antibody has specificity for densely populated cell— surface targets that are abundantly sed so that the target-antibody interactions represent a quantitatively important clearance pathway at low doses (Mager 2001, Mager 2003, Luu 2012).

EXAMPLE 17: REPEAT DOSE GLP STUDY IN CYNOMOLGUS MONKEY

[0385] The toxicology, PK and immunogenicity of IGN523 are studied in a GLP multi-dose IV administration toxicology study in cynomolgus monkeys. This study provides hensive data on clinical endpoints, toxicokinetics, immunogenicity opment of anti—IGN523 antibodies), and histopathology involving a broad list of s (including the injection site) and is ted with formulated al representative of that which will be administered in the clinical trial. In addition, selected safety pharmacology endpoints (neurobehavioral, electrocardiography, respiratory behavior) are also evaluated.

The closing n is a once per week intravenous 60-min infusion for 8 weeks. The doses employed are 10, 30, and 100 mg/kg of IGN523 once weekly (total of 9 doses) followed by a 4— week treatment—free recovery period (Table 5). The 4—week recovery period is considered sufficient to allow for complete clearance of lGN523 and to assess the reversibility of any potential toxicity. The highest dose is anticipated to approximate a MTD and provide for a significant exposure multiple beyond that anticipated in patients. genicity IGN523 antibodies) and toxicokinetics are monitored.

WO 78377 Table 7: Treatment Groups for Proposed lgus GLP Toxicology Study Group Treatment in mglkg No. of Male? No. of Females 1 Treatment Schedule 1 O 5 5 l Once/Week for 9 doses 2 10 5 l 5 OnceNVeek for 9 doses 3 30 5 i 5 Once/Week for 9 doses 4 100 i 5 1 5 l OnceNVeek for 9 doses Table 8 contains a detailed summary of the study design for the multi—dose lgus GLP toxicology study. Selected safety pharmacology endpoints (neurobehavioral, electrocardiography, respiratory behavior) will be evaluated in the context of the GLP repeat dose study in cynomolgus monkeys.

Table 8: Tabular Overview of Cynomolgus GLP Toxicology Study Eight Week Intermittent (9 Weekly Doses) Intravenous Toxicity and Toxicokinetic Study with IGN523 in Cynomolgus Monkeys with a 4-Week Recovery Period No. of Animals: 40 (20M, 20F): 3 animals per sex per Main Group (0, 10, 30, 100 mg/kg) plus 2M and 2F per Recovery Group (all dose groups) Dose: 0, 10, 30, 100 mg/kg, once weekly : 2—4 kg, 2 to 4 years of age Route: Intravenous 60 min infusion Recovery: M4 weeks (2M and 2F Control; 2M and 2F 10, 30, 100 mg/kg) Toxicokinetic (TK) Sampling: Days 0, 56: pre-dose, 0.083, 1, 3, 6, 12, 24, 48, 72, 96, 120 hr post dose.

Days 7, 14, 21,28, 35,42, and 49: pre and 0.083 hr post dose.

Days 56: pre-dose, 0.083, 1, 3, 6, 12, 24, 48 hr post dose.

Additional (TK) Recovery Relative to dosing on Day 56, at imately 0.083, 1, 3, 6, 12, 24, Samples: and 48 hr following .

During the recovery period, 3, 4, 5, 7, 14, and 28 days following the final dose. lmmunogenicity: Days 0, 14, 28, 42, 56: pre—dose (Anti—drug antibody) Days 70, 84 (recovery animals) ment Activation Day 56: Approximately 0.25 hr following dosing.

Evaluation Clinical Pathology: Samples collected from all animals during pretest and on days 2, 58 and 84 are evaluated for hematology, coagulation (including fibrinogen), serum chemistry (including C—reactive protein) and urinalysis. ity/Mortality: Twice daily Peripheral Blood Phenotyping: Blood collected and analyzed from all animals once during pretest and on Days 7, 14, 58 and once near the end of the recovery period Detailed Observations: Once during pre-dose phase, Pre—dose on Day 1, weekly during the dosing and recovery phase, and on day of led sacrifice ing neurobehaviour (e.g tremor, convulsions, hyper—, hypoactivity) Cageside ations: 1—2 hours post each dose (time recorded) and once daily during non- dose days and recovery period including neurobehaviour (e.g tremor, convulsions, hyper—, hypoactivity) Qualitative Food Consumption: Once daily during dosing phase and recovery phase Physical Examination: Once prior to treatment, on the days of dosing, once weekly during the remainder of the study period Ophthalmic Examination: Once prior to treatment, during the last week of the treatment period and during the last week of recovery phase ECGs, Respiratory Rate and Once prior to ent, 2—3 hr nd infusion on day 0 and day 56, Blood pressure measurements: near the end of the recovery period E 18: HEMOLYTIC POTENTIAL EVALUATION OF IGN523 The hemolytic potential of IGN523 Will be assessed in vitro using cynomolgus whole blood. The s of this test will be used to identify any potential effects on hemoglobin. The study will be performed using non-human e whole blood on the day of whole blood collection. Table 8 contains a summary of the GLP study design for the hemolytic potential evaluation of IGN523.

Table 2: Outline of ed Hemolytic Potential tion GLP Study Number of Blood Treatment Group Treatment Formulation Samples Volume Volume 1 Test Article 1 x Projected 3 0.5 mL 0.5 mL Concentration 2 Test Article 2 x Projected 3 0.5 ml. 0.5 mL Concentration 3 Test e 4 x Projected 3 05 mL 0.5 mL Concentration 4 Negative Saline 3 0.5 mL 0.5 mL Control Positive Control Distilled Water 3 0.5 mL 0.5 mL EXAMPLE 19:ACUTE INTRAVENOUS AND PERIVASCULAR IRRITATION The irritation and local tissue tolerance study is designed to assess the term toxicities of compounds in the immediate area of injection at high concentrations. As part of this study the compound is administered intravenously and perivascularly to fy effects on tissues expected to ter the initial exposure. The rabbit is the standard animal model for this evaluation.

EXAMPLE 20:TISSUE CROSS—REACTIVITY STUDY OF IGN523 The tissue cross—reactivity profile of IGN523 is characterized using a full range of human tissues as detailed in the FDA guidance, Points to Consider in the Manufacture and Testing of Monoclonal Antibody Productsfor Human Use (February, 1997). A comprehensive list of s to be studied is included in Table 9.

Table 3: Outline of Proposed GLP Tissue Reactivity Study 1. Adrenal 17. Lymph Node 2. Bladder 18. Ovary L3; Blood Cells 19. Pancreas 4. Bone Marrow 20. Parathyroid r .

. Breast 21. Pituitary 6. Cerebellum 22. Placenta (if ble) ‘ 1 7. Cerebral Cortex 23. Prostate 8. Colon 24. Skin 9. Endothelium 25, Spinal Cord . Eye 26. Spleen 11. Fallopian Tube 27. Striated Muscle 12. Gastrointestinal Tract 28. Testis 13. Heart 29. Thymus 14. Kidney (glomerulus, ) 30. Thyroid . Liver 31. Ureter 16. Lung 32. Uterus (cervix, endometrium) EXAMPLE 21: PHASE I CLINICAL STUDY TO EVALUATE THE SAFETY AND PHARMACOKINETICS OF IGN523 IN PATIENTS WITH RELAPSED OR TORY ACUTE MYELOID LEUKEMIA Primatgy objectives The primary objectives of this study are 0 To te the safety and tolerability of IGN523 administered on a le starting with weekly dosing to patients with ed or refractory acute myeloid leukemia (AML) 0 To determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of IGN523 when administered weekly x 4 0 To identify a recommended Phase 2 dose (RPZD) of IGN523 on the basis of safety, pharmacokinetic, and pharmacodynamics data Secondary objectives 0 To assess the incidence of antibody formation to IGN523 0 To characterize the pharmacokinetics of IGN523 in patients with relapsed or refractory AML 0 To make a preliminary assessment of the anti—leukemic ty of IGN523 in patients with relapsed or tory AML - To make a preliminary assessment of biologic markers that might predict IGN523 anti-leukemic activity {0393] Methodology Open~label, dose-escalation study of approximately 6 dose cohorts using standard 3+3 design, plus expansion cohort at MTD or RPZD Number of Subjects Dose Escalation: approximately 21—30 Dose Expansion: 20 lnvestigational drug 1GN523 drug product will be supplied as 20 mL of a 10 mg/mL solution in a 25—mL single-use glass vial. The appropriate volume of IGN523 drug product will be diluted to 250 mL and infused intravenously (IV) over 1 hour. In the Phase I dose-escalation study (see Protocol Synopsis in Section 10.2.1), cohorts of patients may be treated at escalating doses up to 30 mg/kg weekly for 8 doses.

Continued treatment beyond 8 weeks will be offered to patients with ongoing clinical benefit (i.e., lack of disease progression and no unacceptable toxicity). lntra~patient dose escalation may be permitted under specific ions (described in Section ) in order to maximize the accumulation of data at relevant doses and to ze treatment of patients at potentially sub-therapeutic doses.

Diagnosis and Main Criteria for Inclusion ed or Treatment Refractory AML for which no effective standard therapy exists. 0 Must have measurable disease 0 Age _>_18 years, 0 n Cooperative Oncology Group (ECOG) performance status 0-2 0 Life expectancy of at least 12 weeks 0 Platelet count 2 25,000/mm3 (may be maintained by transfusion) 0 AST(SGOT)/ALT(SGPT) S 2.5x institutional upper limit of normal (ULN) 0 Total bilirubin S 1.5x institutional ULN 0 Creatinine _<_ 2x institutional ULN or calculated or measured creatinine clearance Z 50 mL/min - For women of childbearing potential and men, ent to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry and for the duration of study participation. 0 Ability to understand and the willingness to sign a n informed t document Exclusion Criteria Use of monoclonal antibody therapy within 4 weeks, or chemotherapy or radiotherapy within 2 weeks, before Cycle 1 Day 1 (hydroxyurea given to control peripheral blast counts up to 72 hours before Cycle 1, Day 1 is allowed) Unresolved acute toxicity ofNCI CTCAE v4.0 Grade >1 from prior ancer therapy Prior neic stem cell transplant currently requiring immunosuppressive therapy History of severe allergic or anaphylactic reactions to onal antibody therapy Known eningeal or CNS involvement of leukemia Uncontrolled intercurrent illness, including, but not limited to, ongoing or active infection, symptomatic tive heart failure, unstable angina/pectoris, cardiac arrhythmia, or psychiatric illness/social ions that would limit compliance with study requirements Recent major surgery within 4 weeks prior to Cycle 1, Day I Pregnant or lactating women Diagnostic Plan t AML samples from peripheral blood (or bone marrow) will be analyzed retrospectively after study enrollment for expression of CD98 (and not analyzed as part of patient screening and used as a condition for study eligibility), based upon the following rationale: CD98 is differentially overexpressed in the CD34T/CD33I and CD34+/CD33' subpopulations of the ty (~94%) of AML patients compared to similarly “gated” cells from normal bone marrow samples.

There is no clinically validated method of detecting and quantitating CD98 sion on patient samples that would be adequate to select patients for participation (or exclude patients from participating) in the proposed Phase I study.

Qualification of assays and analysis of Phase I results will be used to inform the design of future clinical studies that could require patient ion.

[0401] Test ProductLMode of Administration, Starting Dose lGN523 Intravenous Infusion Starting Dose: No greater than 1/6 the human-equivalent dose (HED) of the No Observed Adverse Effect Level ) observed in the GLP multi—dose cynomolgus toxicity study

[0402] Cohort Initiation and Duration of Treatment The first patient in each new dose cohort will be dosed at least 1 day prior to any other patients in that cohort, to allow for observation of possible severe and/or serious acute (e.g. infusion- related) toxicities that might affect subsequent patient enrollment or dosing decisions.

Patients will e weekly intravenous doses of lGN523 for 8 weeks (two 28-day cycles). Dosing beyond 8 weeks will be ted for patients meeting criteria for ongoing clinical benefit (i.e. lack of disease progression) and acceptable safety for up to 1 year.

Intra-Patient Dose Escalation To maximize the collection of information at relevant doses and to minimize the exposure of patients to potentially sub—optimal doses, intra-patient dose escalation may be permitted under the following conditions: Patients must complete at least two 28~day cycles at their originally assigned dose level prior to any dose escalation Patients may only escalate their dose to the highest dose level cleared by a completed 3-6 patient dose cohort through at least one 28-day cycle of lGN523 administration All intra-patient dose escalation decisions will be based on the igator’s judgment of whether it is felt to be in the best interest of the patient, in coordination with the Medical Monitor Definition of DLT A DLT will be any of the ing adverse events considered by the investigator to be related to lGN523 (and not attributable to another clearly identifiable cause) occurring during Days 1-28 onycle l: 0 Grade 3 or 4 non—hematologic toxicity, except for Reversible Grade 3 nomallergic infusion toxicities (including symptoms such as fever, /rigors, nausea, vomiting, pruritis, headache, rhinitis, rash, ia, and/or hypoxia (in the absence of signs/symptoms of respiratory distress) ing during or within 24 hours after completing an on and resolving within 24 hours with a reduced infusion rate, supportive care, and/or administration or corticosteroids Grade 3 or 4 hyperuricemia, hyperphosphatemia, or hypocalcemia, or Grade 3 hyperkalemia, if transient (i.e. lasting <48 hours) and without manifestations of clinical tumor lysis syndrome (i.e. creatinine 2 1.5 x ULN, cardiac arrhythmias, sudden death, or seizures) - Grade 3 or 4 thrombocytopenia (in a t without isting thrombocytopenia requiring transfusion support) that either results in bleeding, or does not improve to 2 80% of baseline value within 2 weeks without platelet transfusion 0 Grade 3 or 4 neutropenia (in a patient without pre-existing neutropenia requiring growth factor support) that either is ated with a fever (oral or tympanic ature of F/380C) or does not improve to 2 80% of baseline value within 2 weeks without growth factor support

[0409] DLT Window Days 1—28 of Cycle 1 Dose Escalation Scheme Dose escalation may only take place after each individual in a given cohort has reached Day 28. Patients who experience disease progression and withdraw from the study prior to Day 28 without DLT will not be ble for DLT and will be replaced. Dose escalation will proceed n cohorts at up to 100% increments (or less if significant AEs are observed) according to the following scheme: If 0/3 patients have DLT at a given dose level, 3 patients may be enrolled at the next dose level If 2 2/3 patients experience a DLT at a given dose level, dose escalation will be stopped, and this dose will be declared to exceed the MTD. lf l/3 patients experience DLT at a given dose level, at least 3 more patients will be enrolled at the same dose level. If 0 of these 3 patients ence DLT, proceed to the next dose level (which may be at a <100% dose increment). If 1 or more of this group experience DLT, then dose escalation is stopped, and this dose will be declared to exceed the MTD.

Once MTD has been exceeded, if the preceding dose escalation increment was S 30%, then a minimum of 6 evaluable patients may be enrolled at the previous dose level to te it as an MTD. If the preceding dose escalation increment was > 30%, then at least one dose level intermediate between the two highest dose levels may be evaluated.

The t dose level resulting in DLTs in less than one-third of a minimum of 6 patients will be declared the MTD.

Dose Escalation tee

[0413] Agreement to proceed with dose escalation, modify dose escalation , or stop the study will be made by an internal Dose Escalation Committee made up of the Medical Monitor, Drug Safety representative, and additional ad hoc study team members, in consultation with the study investigators at the conclusion of each study cohort. This committee will review all available study data from the current cohort and all ble safety data in the us cohorts before deciding on dose escalation for the subsequent cohort. All study subjects will receive study therapy until e progression, the development of ptable toxicity, noncompliance, or withdrawal of consent by the subject, or by investigator decision.

Criteria for Evaluation Efficacy Peripheral blood counts Bone marrow aspirate and biopsy Safety Safety outcome measures are as follows: nce and nature of DLTs, Incidence and ty of adverse events

[0415] PK Sampling PK assessment will be done on the following schedule: Day 1 dose: pre-dose, and 30 minutes, 4 hours, 24 hours, and 48 (or 72 hours) post-dose Days 8, 15, and 21 doses: se, and 30 minutes, 4 hours, and 48 (or 72 hours) post—dose Subsequent doses: pre—dose and 30 minutes post-dose

[0416] Expansion Cohort In order to obtain additional safety, bility, and pharmacokinetic data, and preliminary evidence of clinical activity, up to an additional 20 patients with relapsed or refractory AML will be enrolled into an expansion cohort at the MTD or RP2D. While the MTD will be determined primarily by the safety (DLT) data observed during the DLT observation s of Phase I, the RP2D will also take into account additional safety data beyond the DLT windows, and may also include information gathered during Phase I dose escalation, including PK and target occupancy data.

Claims (25)

1. An isolated antibody or a functional fragment f comprising all three heavy chain CDRs from a heavy chain variable domain having an amino acid sequence set forth in SEQ ID NO: 12, and all three light chain CDRs from a light chain variable domain having an amino acid sequence set forth in SEQ ID NO: 14.

2. The antibody of claim 1, wherein the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 12 and the light chain variable domain sequence of SEQ ID NO: 14.

3. A binding agent capable of binding to CD98 which is an antibody or a functional fragment thereof, wherein the antibody of claim 1 or claim 2 displaces the binding agent in a competitive binding assay, or n the binding agent displaces the antibody of claim 1 or claim 2 in a competitive binding assay.

4. The antibody, functional fragment or binding agent of any one of claims 1 to 3, n the antibody, functional nt or g agent is conjugated to a cytotoxic agent.

5. The antibody, functional fragment or binding agent of claim 4, n the cytotoxic agent is selected from a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, or a ctive isotope.

6. The antibody, functional fragment or binding agent of any one of claims 1 to 3, wherein the antibody, functional nt or binding agent is conjugated to a detectable marker.

7. The antibody, functional fragment or binding agent of claim 6, wherein the detectable marker is selected from a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound and a chemiluminescent compound.

8. A vector comprising a polynucleotide encoding the antibody, fragment or binding agent of any one of claims 1 to 3.

9. A pharmaceutical ition that comprises the antibody, functional fragment or binding agent of any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

10. Use of the antibody, functional fragment or binding agent of any one of claims 1 to 3 in the manufacture of a medicament for inhibiting growth of a cancer cell that expresses CD98.

11. The use of claim 10, wherein the cancer cells are from a cancer selected from r, breast, colon, , gastric, esophageal, lung, , kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, lymphoma or ia, optionally acute myeloid leukemia, or a metastasis of any of these cancers.

12. Use of the antibody, functional fragment or binding agent of any one of claims 1 to 3, or the pharmaceutical composition of claim 11 in the manufacture of a medicament for treating a cancer in a subject.

13. The use of claim 12, wherein the cancer is selected from bladder, breast, colon, rectal, gastric, esophageal, lung, larynx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, , lymphoma or leukemia, optionally acute myeloid leukemia, or a metastasis of any of these cancers.

14. The use of claim 13, wherein the subject has relapsed or refractory acute myeloid leukemia.

15. The use of claim 13, wherein the medicament is adapted for administration with one or more chemotherapeutic compound, wherein the chemotherapeutic compound is selected from bendamustine hloride, cyclophosphamide, ifosfamide, fludurabine, cytarabine, gemcitabine, prednisone, prednisolone, methylprednisolone, axel, docetaxel, vinorelbine, vincristine, etoposide, ecan, anthracycline, adriamycin, cisplatin, carboplatin and rituximab.

16. The use of claim 13, wherein the cancer is associated with increased expression of CD98 on the surface of a cell.

17. A method of detecting the presence of CD98 in a biological sample, comprising contacting the biological sample with the antibody, onal nt or binding agent of any one of claims 1 to 3 under conditions permissive for binding of the antibody, functional nt or g agent to CD98, and detecting whether a complex is formed between the antibody, functional fragment or binding agent and CD98.

18. A method of diagnosing a cancer associated with increased expression of CD98, comprising contacting an in vitro or ex vivo test cell with the antibody, functional fragment or binding agent of any one of claims 1 to 3; determining the level of expression of CD98 by detecting binding of the antibody, functional nt or binding agent to CD98; and comparing the level of expression of CD98 in the test cell with the level of sion of CD98 in a control cell, n a higher level of expression of CD98 in the test cell as compared to the control cell indicates the presence of a cancer associated with increased expression of CD98.

19. The method of claim 17 or claim 18, wherein the biological sample or in vitro or ex vivo test cell is from a mammal or patient suspected of having a cancer selected from bladder, breast, colon, rectal, gastric, esophageal, lung, larynx, kidney, oral, ovarian, or prostate cancer, or a sarcoma, melanoma, glioma, ma or leukemia, or a metastasis of any of these cancers.

20. The isolated dy or onal fragment thereof of claim 1, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.

21. The binding agent of claim 3, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.

22. The vector of claim 8, substantially as herein described with reference to any one or more of the examples but ing comparative examples.

23. The pharmaceutical composition of claim 9, ntially as herein described with reference to any one or more of the examples but excluding comparative examples.

24. The use of claim 10 or claim 12, substantially as herein described with reference to any one or more of the examples but excluding comparative es.

25. The method of claim 17 or claim 18, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.