Anti-CD94 antibodies and methods of use thereof

Abstract

The present disclosure relates to an antibody that binds human CD94, as well as methods, uses, polynucleotides, vectors, host cells, and pharmaceutical compositions related thereto. In some embodiments, the antibody is a human or humanized antibody that binds human CD94 and does not block the binding between CD94 and HLA-E. In some embodiments, the antibody cross-reacts with cynomolgus monkey CD94. In some embodiments, the antibody does not promote internalization of surface-expressed CD94 as well as existing antibodies. In some embodiments, the antibody promotes ADCC targeting cells that express human CD94, for example, on the cell surface.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 323,959, filed March 25, 2022, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Reference to electronic sequence listing The contents of the electronic format of the sequence listing (186542000740SEQLIST.xml, size: 69,347 bytes, and creation date: March 20, 2023) are incorporated by reference in their entirety into this specification.

[0003] The present disclosure relates to antibodies that bind to CD94 and their uses for treating diseases and disorders associated with NK cells and / or T cells. [Background technology]

[0004] Natural killer (NK) / T-cell lymphomas and leukemias are characterized by the clonal expansion of NK cells and / or CD8+ and CD4+ T cells. NK / T-cell lymphomas account for a small proportion of non-Hodgkin's lymphomas (NHLs) with a 5-year survival rate of less than 50% (Kwong, 2012). There are approximately 4,000-7,000 new cases per year in the United States alone, with a higher frequency in Asian populations (Bajaj, A. (2019) Int. J. Hemat.. Ther 5:1-7). NK / T-cell lymphomas can occur at any age, but more than half of NHL patients are 65 years of age or older (Bajaj, 2019). There are also NK-cell and T-cell based leukemias, such as LGL leukemia and aggressive NK leukemia. Other examples of diseases and disorders in which NK cells play a role include LGL leukemia (e.g., T-cell LGL leukemia), chronic NK cell lymphoproliferative disorder (CLPD-NK, formerly known as NK-LGL), rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia (e.g., aggressive natural killer leukemia (ANKL) and extranodal NKL nasal type (ENKL)), IBM, and IBD.

[0005] There are 13 distinct diseases involving NK / T-cell lymphoma: extranodal NK / T-cell lymphoma, hepatosplenic T-cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, and subcutaneous panniculitis TCL (Bajaj, 2019). The major subtypes of NK / T cell lymphoma, mostly driven by cytotoxic cells (NK / CD8+ T cells), include extranodal NK / T cell lymphoma, hepatosplenic TCL, enteropathy-associated TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, and subcutaneous panniculitis TCL. NK / T cell lymphomas affect various organs, such as the skin, gastrointestinal (GI) tract, liver, spleen, and bone marrow. Symptoms include lymphadenopathy in the neck. Most NK / T cell lymphoma subtypes are driven by Epstein-Barr virus infection.

[0006] Current treatments include chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone) followed by stem cell transplantation, but progression-free survival rates remain at 40-50%. Current treatments under investigation include alemtuzumab, carfilzomib (proteasome inhibitor), romidepsin (HDAC inhibitor), bevacizumab, brentuximab vedotin (antibody-drug conjugate), bortezomib (proteasome inhibitor), belinostat (HDAC inhibitor), pralatrexate, vorinostat (HDAC inhibitor), and avelumab. Despite the diversity of drugs in development, many of them are often used off-label and have produced mixed results.

[0007] Currently, there is no effective therapy for the treatment of NK / T cell lymphoma, and no therapy has been developed that selectively targets NK / T cell lymphoma. Current therapies for NK / T cell lymphoma may have off-target effects and are not completely effective. The high mortality rate of the disease combined with the lack of effective treatment highlights the need for therapeutic advances in NK / T cell lymphoma.

[0008] Thus, there is a need in the art to develop safe and effective therapies for treating diseases mediated by NK cells and / or T cells that express CD94, such as NK / T cell lymphoma. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Bajaj, A. (2019) Int. J. Hemat.. Ther 5:1-7 Summary of the Invention [Means for solving the problem]

[0010] To meet these and other needs, the present disclosure provides, inter alia, antibodies that specifically bind CD94 (e.g., human CD94), methods of treating diseases or injuries associated with NK cells and / or T cells expressing CD94, such as NK / T cell lymphomas and leukemias (e.g., LGL leukemia), and methods of depleting or reducing the number of NK cells and / or T cells expressing CD94 in a subject upon administration of an antibody that specifically binds CD94. These antibodies of the present disclosure may have one or more of the following properties: high affinity binding to human CD94 (e.g., cells expressing human CD94 on their surface), cross-reactivity to cynomolgus monkey CD94 (useful for preclinical studies), ability to bind human CD94 without blocking the interaction of human CD94 with HLA-E, minimal internalization following binding to CD94, and / or induction of ADCC against cells expressing CD94, such as leukemia cells. These properties are believed to be advantageous for testing, development, and use in treating NK / T cell-based diseases such as, for example, lymphomas and leukemias. For example, without wishing to be bound by theory, it is believed that targeting CD94 on NK cells by ADCC may induce cancer cells (e.g., LGL leukemia or lymphoma cells) to kill other cancer cells.

[0011]

[0013] In some aspects, provided herein is an antibody that binds (e.g., specifically binds) human CD94, the antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain having the amino acid sequence: EVQLX1ESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISX2SSX3X4IYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGR YYYYMDVWGKGTTVTVSS, in which X1 is L or V, X2 is T or S, X3 is N or S, and X4 is F or Y (SEQ ID NO: 69), and the VL domain comprises the amino acid sequence DIX1MTQSPSSLSASVGDRVTITCRASQSISSWLAWX2QQKPX3KX4PKX5LIYAASSLQSGVPSX6FSGSGSGTDFTLTISSLQPED X7ATYYCQX8YNSX9PFTFGPGTKVDIK, in which X1 is V or Q, X2 is Y or F, X3 is G or E, X4 is A or V, X5 is S or L, X6 is K or R, X7 is V or F, X8 is K or Q, and X9 is A or Y (SEQ ID NO: 70), The antibody does not comprise a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO:2), a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3), a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:6). In some embodiments, the antibody is a human antibody. In some embodiments, the antibody does not comprise a VH domain comprising the amino acid sequence of SEQ ID NO:16 and a VL domain comprising the amino acid sequence of SEQ ID NO:17.

[0012] In some aspects, provided herein is an antibody that binds (e.g., specifically binds) human CD94, the antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1); a CDR-H2 comprising an amino acid sequence selected from the group consisting of SISTSSNFIYYADSVKG (SEQ ID NO:2), SISSSSNFIYYADSVKG (SEQ ID NO:18), SISTSSSFIYYADSVKG (SEQ ID NO:21), and SISTSSNYIYYADSVKG (SEQ ID NO:24); and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3), and wherein the VL domain comprises a CDR-H4 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4). L1; CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5); and CDR-L3 comprising an amino acid sequence selected from the group consisting of QKYNSAPFT (SEQ ID NO:6), QQYNSAPFT (SEQ ID NO:27), and QKYNSYPFT (SEQ ID NO:28), wherein the antibody does not comprise CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO:2), CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3), CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:6). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO:2), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISSSSNFIYYADSVKG (SEQ ID NO:18), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3).In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISTSSSFIYYADSVKG (SEQ ID NO:21), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISTSSNYIYYADSVKG (SEQ ID NO:24), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3). In some embodiments, the VH domain further comprises: (a) a FR1 comprising an amino acid sequence selected from the group consisting of EVQLLESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 48) and EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 49); (b) a FR2 comprising an amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO: 50); (c) a FR3 comprising an amino acid sequence of RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 51); and (d) a FR4 comprising the amino acid sequence of WGKGTTVTVSS (SEQ ID NO: 52). In some embodiments, the VH domain further comprises (a) an FR1 comprising the amino acid sequence of EVQLLESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 48), (b) an FR2 comprising the amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO: 50), (c) an FR3 comprising the amino acid sequence of RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 51), and (d) an FR4 comprising the amino acid sequence of WGKGTTVTVSS (SEQ ID NO: 52). In some embodiments, the VH domain further comprises (a) an FR1 comprising the amino acid sequence of EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 49), (b) an FR2 comprising the amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO: 50), (c) an FR3 comprising the amino acid sequence of RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 51), and (d) an FR4 comprising the amino acid sequence of WGKGTTVTVSS (SEQ ID NO: 52).In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:6). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QQYNSAPFT (SEQ ID NO:27). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QKYNSYPFT (SEQ ID NO:28). In some embodiments, the VL domain comprises (a) a FR1 comprising an amino acid sequence selected from the group consisting of DIVMTQSPSSLSASVGDRVTITC (SEQ ID NO: 53) and DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 54); (b) a FR2 comprising an amino acid sequence selected from the group consisting of WYQQKPGKAPKSLIY (SEQ ID NO: 55), WFQQKPGKAPKSLIY (SEQ ID NO: 56), WYQQKPEKAPKSLIY (SEQ ID NO: 57), WYQQKPGKVPKSLIY (SEQ ID NO: 58), and WYQQKPGKAPKLLIY (SEQ ID NO: 59). SEQ ID NO:59), (c) a FR3 comprising an amino acid sequence selected from the group consisting of GVPSKFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO:60), GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO:61), and GVPSKFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:62), and (d) a FR4 comprising the amino acid sequence of FGPGTKVDIK (SEQ ID NO:63). In some embodiments, the antibody is a human antibody.

[0013] In some aspects, provided herein is an antibody that binds (e.g., specifically binds) human CD94, the antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7); CDR-H2 comprising an amino acid sequence selected from the group consisting of ISTSSNFI (SEQ ID NO:8), ISSSSNFI (SEQ ID NO:19), ISTSSSFI (SEQ ID NO:22), and ISTSSNYI (SEQ ID NO:25); and CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9), wherein the VL domain comprises CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO:10); (SEQ ID NO:11); and a CDR-L3 comprising an amino acid sequence selected from the group consisting of QKYNSAPFT (SEQ ID NO:12), QQYNSAPFT (SEQ ID NO:27), and QKYNSYPFT (SEQ ID NO:28), wherein the antibody does not comprise a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISTSSNFI (SEQ ID NO:8), a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9), a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO:10), a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO:11), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:12). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISTSSNFI (SEQ ID NO:8), and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISSSSNFI (SEQ ID NO:19), and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9).In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISTSSSFI (SEQ ID NO:22), and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISTSSNYI (SEQ ID NO:25), and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO:10), a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO:11), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:12). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO: 10), a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO: 11), and a CDR-L3 comprising the amino acid sequence QQYNSAPFT (SEQ ID NO: 27). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO: 10), a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO: 11), and a CDR-L3 comprising the amino acid sequence QKYNSYPFT (SEQ ID NO: 28). In some embodiments, the antibody is a human antibody.

[0014] In some aspects, provided herein is an antibody that binds (e.g., specifically binds) human CD94, the antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13); a CDR-H2 comprising an amino acid sequence selected from the group consisting of STSSNF (SEQ ID NO: 14), SSSSNF (SEQ ID NO: 20), STSSSF (SEQ ID NO: 23), and STSSNY (SEQ ID NO: 26); and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15), wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4); and a CDR-L3 comprising an amino acid sequence selected from the group consisting of QKYNSAPFT (SEQ ID NO: 6), QQYNSAPFT (SEQ ID NO: 27), and QKYNSYPFT (SEQ ID NO: 28), wherein the antibody does not comprise a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence STSSNF (SEQ ID NO: 14), a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15), a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence STSSNF (SEQ ID NO: 14), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence SSSSNF (SEQ ID NO: 20), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence STSSSF (SEQ ID NO: 23), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15).In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence STSSNY (SEQ ID NO: 26), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QQYNSAPFT (SEQ ID NO: 27). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSYPFT (SEQ ID NO: 28). In some embodiments, the antibody is a human antibody.

[0015] In some embodiments according to any of the embodiments described herein, the VH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 29-32, and / or the VL domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17 and 33-41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 36.In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30 and the VL domain comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30 and the VL domain comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30 and the VL domain comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:17.In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:38.In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the antibody does not comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a VL domain comprising the amino acid sequence of SEQ ID NO: 17.

[0016] In some embodiments according to any of the embodiments described herein, the antibody is an antigen-binding antibody fragment or a single chain antibody. In some embodiments, the antibody further comprises an Fc region, e.g., a human IgG1 Fc region. In some embodiments, the antibody comprises a non-fucosylated human Fc region. In some embodiments, the antibody is produced in a cell line (e.g., a CHO cell line) that is deficient in alpha-1,6-fucosyltransferase gene, such as FUT8. In some embodiments, the antibody binds to human cell Fc gamma receptor IIIA to a greater extent than an antibody that comprises a wild-type human IgG1 Fc region. In some embodiments, the antibody can induce antibody-dependent cellular cytotoxicity (ADCC) against cells expressing human CD94 on the surface.

[0017] In a further aspect, provided herein is a polynucleotide(s) encoding an antibody according to any one of the above embodiments. Further provided herein is a vector (e.g., an expression vector) comprising a polynucleotide(s) according to any one of the above embodiments. Further provided herein is a host cell (e.g., an isolated host cell or cell line) comprising a polynucleotide(s) or vector according to any one of the above embodiments. Further provided herein is a method for producing an antibody, comprising culturing a host cell according to any one of the above embodiments under conditions suitable for producing the antibody. In some embodiments, the method further comprises recovering the antibody from the host cell.

[0018] Further provided herein is a composition (eg, a pharmaceutical composition) comprising an antibody according to any one of the above embodiments and a pharma- ceutically acceptable carrier.

[0019] In a further aspect, a method for treating a disease or disorder in a subject is provided herein, comprising administering to the subject an effective amount of an antibody or composition according to any one of the above embodiments. In some embodiments, administering the antibody results in a reduction in the number of peripheral blood LGL cells or NK cells in the subject. In some embodiments, the disease or disorder is Felty syndrome, and administering the antibody to the subject results in a reduction in one or more symptoms of Felty syndrome in the subject. In some embodiments, the disease or disorder is inclusion body myositis, and administering the antibody to the subject results in a reduction in one or more symptoms of inclusion body myositis in the subject. In some embodiments, the disease or disorder is aggressive NK leukemia, and administering the antibody to the subject results in a reduction in one or more symptoms of aggressive NK leukemia in the subject. In some embodiments, the disease or disorder is rheumatoid arthritis, and administering the antibody to the subject results in a reduction in one or more symptoms of rheumatoid arthritis in the subject. In some embodiments, the disease or disorder is LGL leukemia, and administering the antibody to the subject results in a reduction in one or more symptoms of LGL leukemia in the subject. In some embodiments, the disease or disorder is CLPD-NK, and administering the antibody to the subject results in a reduction in one or more symptoms of CLPD-NK in the subject. In some embodiments, the disease or disorder is natural killer (NK) cell lymphoma or T cell lymphoma, and administering the antibody to the subject results in a reduction in one or more symptoms of lymphoma in the subject. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic enteric TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.In some embodiments, the disease or disorder is microscopic colitis, and administration of the antibody to a subject results in a reduction of one or more symptoms of microscopic colitis in the subject.

[0020] In a further aspect, provided herein is a method for reducing the number of peripheral blood LGL cells and / or NK cells in a subject, comprising administering to the subject an effective amount of an antibody or composition according to any one of the above embodiments. In a further aspect, provided herein is a method for inducing ADCC activity in a subject, comprising administering to the subject an effective amount of an antibody or composition according to any one of the above embodiments. In a further aspect, provided herein is a method for treating CLPD-NK in a human subject in need of such treatment, comprising administering to the subject an effective amount of an antibody or composition according to any one of the above embodiments. In some embodiments, administration of the antibody results in amelioration of one or more symptoms of CLPD-NK in the subject.

[0021] In a further aspect, provided herein is a method of treating natural killer (NK) cell lymphoma or T cell lymphoma, comprising administering to a subject in need thereof an effective amount of an antibody or composition according to any one of the above embodiments. In some embodiments, the antibody does not bind to the same epitope on human CD94 as anti-CD94 antibody clones HP-3D9, HP-3B1, DX22, 131412, or 12K45. In some embodiments, the antibody binds to human CD94 with a higher affinity than anti-CD94 antibody clones HP-3D9, HP-3B1, DX22, 131412, and 12K45. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, or adult TCL. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

[0022] In a further aspect, provided herein is a method of enhancing chimeric antigen receptor T cell (CAR-T) therapy in a human subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or composition according to any one of the above embodiments prior to administration of CAR-T treatment to the subject. In some embodiments, administration of the antibody or composition results in depletion of NK cells in the subject prior to administration of CAR-T treatment.

[0023] In a further aspect, provided herein is a method of depleting CD8+ CD94+ T cells in a human subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or composition according to any one of the above embodiments. In some embodiments, administration of the antibody or composition results in depletion of CD8+ CD94+ T cells in the subject.

[0024] In some embodiments according to any of the embodiments described herein, the method further comprises administering an IL-2 polypeptide to the subject.

[0025] In some embodiments according to any of the embodiments described herein, the subject is a human.

[0026] In a further aspect, there is provided herein an antibody or composition according to any one of the above embodiments for use in treating a disease or disorder in a subject, reducing the number of peripheral blood LGL cells and / or NK cells in a subject, treating CLPD-NK in a human subject in need of such treatment, treating natural killer (NK) cell lymphoma or T cell lymphoma, treating microscopic colitis in a subject, or augmenting CAR-T therapy in a subject in need of such augmentation. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic enteric TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

[0027] In a further aspect, provided herein is the use of an antibody or composition according to any one of the above embodiments for the manufacture of a medicament for use in, for example, treating a disease or disorder in a subject, reducing the number of peripheral blood LGL cells and / or NK cells in a subject, treating CLPD-NK in a human subject in need of CLPD-NK treatment, treating natural killer (NK) cell lymphoma or T cell lymphoma, treating microscopic colitis in a subject, or enhancing CAR-T therapy in a subject in need of CAR-T therapy enhancement. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, or adult TCL. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

[0028] In a further aspect, a kit or article of manufacture is provided herein, comprising the antibody or composition according to any one of the above embodiments.In some embodiments, the kit further comprises instructions for use of the kit, for example, in treating a disease or disorder in a subject, in reducing the number of peripheral blood LGL cells and / or NK cells in a subject, in treating CLPD-NK in a human subject in need of CLPD-NK treatment, in treating natural killer (NK) cell lymphoma or T cell lymphoma, in treating microscopic colitis in a subject, or in enhancing CAR-T therapy in a subject in need of CAR-T therapy enhancement. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic enteric TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

[0029] It should be understood that one, some, or all of the specifics of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in the following detailed description.

[0030] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief description of the drawings]

[0031] [Figure 1]Figure 1 shows the affinity of anti-CD94 antibody 18H3 to human primary natural killer (NK) cells as measured by flow cytometry. Peripheral blood mononuclear cells (PBMCs) from healthy donor HD-40 were used for antibody staining. 18H3 antibody was titrated from 100 nM to 0.046 nM at 1:3 dilution and incubated with PBMCs. CD3 and CD56 antibodies were used to identify NK cells based on flow scatter. Binding of 18H3 antibody on CD3+ and CD56 bright NK cells was used to assess the affinity of 18H3. Titration curves and EC50 were generated using Graphpad Prism. 18H3 bound to CD3+CD56 bright NK cells with an affinity of 2.6 nM. Human IgG1 isotype control with secondary antibody (hIgG1) and goat (Fab)2 fragment anti-human Fcy specific secondary antibody AF647 alone (secondary Ab) were used as controls. [Diagram 2] Figure 1 shows binding of anti-CD94 antibodies to human primary NK cells as measured by flow cytometry. Anti-CD94 hybridoma supernatants were screened on human primary NK cells by flow cytometry. HP-3D9 is a commercial anti-CD94 antibody used as a positive control. Mouse IgG1, IgG2a, IgG2b, and IgG3 were used as negative controls. Anti-CD94 antibodies 18H3, 1M4, and 1E4 bound to CD94 expressed on human primary NK cells. [Diagram 3]A-B show the cross-reactivity of anti-CD94 antibodies to cynomolgus CD94. A shows the cross-reactivity of hybridoma antibodies to cynomolgus CD94. Anti-CD94 hybridoma supernatants were screened for cross-reactivity to cynomolgus CD94 by flow cytometry in cynomolgus CD94-expressing HEK293 cells. HP-3D9 is a commercial anti-CD94 antibody used as a negative control. Mouse IgG1, IgG2a, IgG2b, and IgG3 were used as negative controls. 18H3, 1M4, and 1E4 cross-reacted with cynomolgus CD94, while 20F2 is an example of another CD94 antibody clone that reacted with human CD94 but did not cross-react with cynomolgus CD94. B shows the cross-reactivity of commercial antibodies to cynomolgus CD94. Cynomolgus cross-reactivity was tested using a commercial anti-CD94 antibody. 18H3 was used as a positive control. The MFI of each antibody was normalized to the MFI of its respective isotype. Unlike 18H3, HP-3B1, 131412, 12K45, DX22, and HP-3D9 did not cross-react with cynomolgus CD94. [Figure 4] Figure 1 shows the results of an HLA-E tetramer blocking assay performed with anti-CD94 and a commercial anti-CD94 antibody using flow cytometry. PBMCs from healthy donors were incubated with anti-CD94 antibody. PE-labeled HLA-E tetramers were then incubated with the cell and antibody mixture and detected using flow cytometry. For HP-3B1, 131412, 12K45, DX22, 1M4, and 1E4, 2.5 μl of HLA-E tetramer reagent was used, while for 18H3 and HP-3D9, 5 μl was used. Saturating concentrations of each antibody were used in the assay. Percent blocking was calculated as 100-((percent HLA-E positive for anti-CD94 antibody) / (percent HLA-E positive for isotype)×100). 18H3 and 1E4 antibodies did not block HLA-E binding to CD94. [Figure 5A]Figure 1 shows the results of a competition assay performed to evaluate the 18H3 anti-CD94 antibody. For all competition assays, 18H3 was incubated with PBMCs at a concentration of 1.3 μg / ml. Figure 2 shows a competition assay between 18H3 and a commercial anti-CD94 antibody. The commercial anti-CD94 antibody was titrated and incubated with PBMCs in parallel with 18H3. 18H3 was fluorescently tagged with AF647 and incubated with cells in parallel with 18H3-AF647. 18H3 only partially competed with HP-3D9 and did not compete with DX22, HP-3B1, 131412, and 12K45. These results suggest that the 18H3 antibody binds to an epitope that is not shared with the commercial antibody. [Figure 5B] Figure 1 shows the results of a competition assay performed to evaluate the 18H3 anti-CD94 antibody. For all competition assays, 18H3 was incubated with PBMCs at a concentration of 1.3 μg / ml. Competition assays using 18H3 and other hybridoma anti-CD94 antibodies disclosed herein (1M4 and 1E4) are shown. To test the competition between 18H3 and 1M4 / 1E4, 1M4 and 1E4 were incubated with cells at concentrations of 8.5 μg / ml and 11 μg / ml, respectively. 18H3 was fluorescently tagged with AF647 and incubated with cells in parallel with 18H3-AF647. 18H3 did not compete with 1E4 and 1M4. These results suggest that the 18H3 antibody binds to an epitope that is not shared with commercial antibodies. [Figure 6A] Figure 1 shows the results of a competition assay performed to evaluate the 1M4 anti-CD94 antibody. For all competition assays, 1M4 was incubated with PBMCs at a concentration of 8.5 μg / ml. Figure 2 shows a competition assay between 1M4 and a commercial anti-CD94 antibody. The commercial anti-CD94 antibody was titrated and incubated with PBMCs in parallel with 1M4. The 1M4 antibody did not compete with the commercial antibody. [Figure 6B]Figure 1 shows the results of a competition assay performed to evaluate the 1M4 anti-CD94 antibody. For all competition assays, 1M4 was incubated with PBMCs at a concentration of 8.5 μg / ml. Competition assays using 1M4 and 1E4 antibodies are shown. To test the competition between 1M4 and 1E4, 1E4 was incubated with cells at a concentration of 11 μg / ml. 1M4 with an anti-mouse secondary antibody was incubated with cells in parallel with 1E4. The 1M4 antibody competed with 1E4. [Figure 7] Figure 1 shows the results of a competition assay performed to evaluate the 1E4 anti-CD94 antibody. For all competition assays, 1E4 was incubated with PBMCs at a concentration of 11 μg / ml. Commercially available anti-CD94 antibodies were titrated and incubated with PBMCs in parallel with 1E4. The 1E4 antibody did not compete with four of the five commercially available anti-CD94 antibodies tested, but did partially compete with 12K45. [Figure 8A] Figure 1 shows the results of an anti-CD94 antibody internalization assay. Peripheral blood mononuclear cells (PBMCs) from healthy donors were incubated with unconjugated anti-CD94 antibodies for multiple time points ranging from 30 minutes to 24 hours. Cells were maintained at either 4°C to prevent internalization or 37°C to induce internalization. Results are shown for the commercial antibodies HP-3D9 and DX22. The commercial anti-CD94 antibodies were internalized in a time-dependent manner. [Figure 8B]

[0023] Figure 1 shows the results of an anti-CD94 antibody internalization assay. Peripheral blood mononuclear cells (PBMCs) from healthy donors were incubated with unconjugated anti-CD94 antibodies for multiple time points ranging from 30 minutes to 24 hours. Cells were maintained at either 4°C to block internalization or 37°C to induce internalization. Results are shown for 18H3, 1M4, and 1E4 antibodies. 18H3, 1M4, and 1E4 antibodies did not significantly internalize upon binding to CD94. [Figure 9A]Figure 1 shows the results of an antibody-dependent cellular cytotoxicity (ADCC) assay for anti-CD94 antibodies using PBMCs from healthy donors. The 18H3 antibody is generated in Expi-CHO cells cultured in the presence of kifunensine, a potent inhibitor of the mannosidase I enzyme, and used to generate 18H3-KIF, which mimics non-fucosylated antibodies. PBMCs were plated in 96-well plates and incubated overnight in the presence of anti-CD94 antibodies ranging from 1x10-6 to 10μg / ml at 10-fold dilutions. The number of NK cells was quantified by flow cytometry. The number of NK cells in the human IgG1 and anti-CD94 antibody treatment conditions was normalized to the number of NK cells from human IgG1-treated wells. The results are shown for an ADCC assay using human IgG1 18H3 antibody. Human IgG1 18H3 depleted human primary NK cells in a concentration-dependent manner. [Figure 9B] Figure 1 shows the results of an antibody-dependent cellular cytotoxicity (ADCC) assay for anti-CD94 antibodies using PBMCs from healthy donors. Fucosylated 1E4 antibody was used in the ADCC assay. PBMCs were plated in 96-well plates and incubated overnight in the presence of anti-CD94 antibodies ranging from 1x10^-6 to 10μg / ml at 10-fold dilutions. The number of NK cells was quantified by flow cytometry. The number of NK cells in the human IgG1 and anti-CD94 antibody treatment conditions was normalized to the number of NK cells from human IgG1-treated wells. The results are shown for an ADCC assay using fucosylated 1E4 antibody. Fucosylated 1E4 depleted human primary NK cells in a concentration-dependent manner. [Figure 10]Figure 1 shows the results of an ADCC assay of anti-CD94 antibodies using PBMCs from a CLPD-NK patient. 18H3 was produced in Expi-CHO cells cultured in the presence of kifunensine, a potent inhibitor of the mannosidase I enzyme, to generate 18H3-KIF, which mimics non-fucosylated antibodies. PBMCs were plated in 96-well plates and incubated overnight in the presence of anti-CD94 antibodies ranging from 1x10-6 to 10ug / ml at 10-fold dilutions. The number of CD3-CD16+ leukemia cells was quantified by flow cytometry. The number of leukemia cells in human IgG1 and anti-CD94 antibody treatment conditions was normalized to the number of leukemia cells from human IgG1-treated wells. Partially non-fucosylated human IgG1 18H3 depleted human CLPD-NK leukemia cells in a concentration-dependent manner. [Figure 11] A summary of the properties and functional evaluation of anti-CD94 antibodies compared to commercially available anti-CD94 antibodies is shown. [Figure 12A] 1 shows the study design examining the effect of ATK-130 or isotype control on humanized IL-15 transgenic mice engrafted with PBMCs from healthy human donors. [Figure 12B] Figure 1 shows depletion of normal human NK cells and CD8 T cells in IL-15 transgenic mice. Mice were transplanted with PBMCs from healthy donors for 3 days. Mice (5 mice per group) were injected with a single dose of human IgG1 isotype control or ATX-130 (5 mg / kg) and depletion of NK cells (upper panel) and CD8 T cells (lower panel) in blood, spleen, and bone marrow was assessed by flow cytometry 48 hours after dosing. Depletion was quantified by the number of NK cells and CD8 T cells remaining in each sample. [Figure 13]Figure 1 shows depletion of LGLL cells in blood, spleen, bone marrow, and liver in IL-15 transgenic mice. Mice were transplanted with LGLL PBMCs for 28 days. Mice (5 mice per group) were injected with a single dose of human IgG1 isotype control or ATX-130 (5 mg / kg), and LGLL cell depletion in blood, spleen, bone marrow, and liver was assessed by flow cytometry 48 hours after dosing. Depletion was quantified by the number of CD94+ LGLL cells remaining in each sample. [Figure 14A] FIG. 1 shows the design of an exploratory pharmacodynamic (PD) study in non-human primates to evaluate the efficacy of ATK-130 in vivo. [Figure 14B] Figure 1 shows the depletion of cynomolgus monkey NK cells in vivo by dosing with ATX-130-KIF at 2mg / kg as assessed by flow cytometry. Two naive cynomolgus monkeys (cynomolgus monkey #1 and cynomolgus monkey #2) were administered ATX-130-KIF (2mg / kg) via intravenous (IV) infusion for 60 minutes. Cynomolgus monkey #1 was administered an additional 2mg / kg of ATX-130-KIF at week 8 and cynomolgus monkey #2 at week 16. Body weight and clinical observations were recorded periodically. PBMCs were isolated from 5mL blood draws per animal per time point. 0.3x106 cells were plated per well in technical triplicates and stained with an antibody panel. Cynomolgus monkey NK cells were identified using a CD3-CD8+ gating strategy. NK cells were quantified by calculating the percentage of this cell population among total PBMCs. Arrows indicate the time points at which doses were administered. [Figure 15A]Figure 1 shows the assessment of depletion of cynomolgus CD4 T cells, CD8 T cells, and B cells. PBMCs were isolated from 5 mL blood draws per time point for cynomolgus monkey #1. 0.3x106 cells were plated per well in technical triplicates and stained with an antibody panel. CD3+CD4+, CD3+CD8+, and CD3-CD20+ gating strategies were used to identify CD4+ T cells, CD8+ T cells, and B cells, respectively, for cynomolgus monkey #1. Cells were quantified by calculating the percentage of the cell populations among the total PBMCs. [Figure 15B] Figure 1 shows the assessment of depletion of cynomolgus CD4 T cells, CD8 T cells, and B cells. PBMCs were isolated from 5 mL blood draws per time point for cynomolgus monkey #2. 0.3x10^6 cells per well were plated in technical triplicates and stained with a panel of antibodies. CD3+CD4+, CD3+CD8+, and CD3-CD20+ gating strategies were used to identify CD4+ T cells, CD8+ T cells, and B cells, respectively, for cynomolgus monkey #2. Cells were quantified by calculating the percentage of the cell populations among total PBMCs. Figure 1 shows the results of the assessment of monocyte depletion by flow cytometry. PBMCs were isolated from 5 mL blood draws per time point for cynomolgus monkey #1 (left panel) and cynomolgus monkey #2 (right panel). 0.3x10^6 cells per well were plated in technical triplicates and stained with a panel of antibodies. A CD3-CD14+ gating strategy was used to identify monocytes, and cells were quantified by calculating the percentage of this cell population among total PBMCs. [Figure 16] Figure 16. [Figure 17] Figure 1 shows the results of assessment of CD16 expression on monocytes by flow cytometry. PBMCs were isolated from 5 mL blood draws per time point for cynomolgus monkey #1 (left panel) and cynomolgus monkey #2 (right panel). 0.3x106 cells were plated per well in technical triplicates and stained with an antibody panel. A CD3-CD14+ gating strategy was used to identify monocytes. CD16 MFI was assessed in monocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Some aspects are described below with reference to example applications for illustrative purposes. It should be understood that numerous specific details, relationships, and methods are described to provide a complete understanding of the features described herein. However, those skilled in the art will readily recognize that the features described herein can be implemented without one or more of the specific details, or by using other methods. The features described herein are not limited by the order of the illustrated acts or events, since some acts can occur in different orders and / or simultaneously with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein.

[0033] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." As used herein, the term "comprising" is synonymous with "including" or "containing," and is inclusive or open-ended.

[0034] Any reference to "or" herein is intended to include "and / or" unless otherwise specified. As used herein, the term "about" in reference to a number refers to that number plus or minus 10% of that number. The term "about" in reference to a range refers to that range and its lower limit minus 10% and its upper limit plus 10%.

[0035] I. Antibodies In some embodiments, provided herein are antibodies that bind to CD94, e.g., human CD94 expressed on the surface of NK cells or T cells. Also provided herein are antibodies that bind to CD94 and have an immunoglobulin Fc portion with modifications including reduced fucosylation, non-fucosylation, or mutations that enhance ADCC activity and / or improve affinity of the Fc region for Fc receptors such as CD16 (e.g., CD16a). Also provided herein are antibodies that bind to CD94 and have one or more of the following properties: bind to human CD94 and cynomolgus CD94, do not block HLA-E binding to CD94 / NKG2A heterodimers, have low target (e.g., CD94) internalization, are non-fucosylated or have reduced fucosylation, and / or induce or promote ADCC activity.

[0036] A. Antibody Targets and Affinity In some embodiments, the antibodies provided herein bind to CD94. In some embodiments, the antibodies provided herein bind to human CD94 (e.g., the extracellular domain of human CD94). In some embodiments, the antibodies provided herein bind to cynomolgus CD94 (e.g., the extracellular domain of cynomolgus CD94). In some embodiments, the antibodies provided herein bind to human CD94 and cynomolgus CD94. In some embodiments, the antibodies of the present disclosure bind to CD94 on the surface of NK cells and / or T cells.

[0037] In some embodiments, an antibody of the present disclosure binds to a human CD94 protein or a portion thereof, or a protein having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) homology to a human CD94 protein or a portion thereof. The amino acid sequences of representative human CD94 proteins are provided as SEQ ID NOs:64-66. [ka]

[0038] In some embodiments, the terms bind, specifically bind to, or specific to refer to a measurable and reproducible interaction, such as binding between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or with longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (K D In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0039] In some embodiments, the antibodies provided herein bind to human CD94 (Natural Killer Cell Antigen CD94; CD94 Entrez Gene ID: 3824; KLRD1 (HGNC symbol); UniProtKB Identifier: Q13241; HGNC: 6378; Ensembl: ENSG00000134539 OMIM: 602894; KP43).

[0040] In some embodiments, an antibody of the present disclosure binds to a cynomolgus monkey CD94 protein or a portion thereof, or a protein having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) homology to a cynomolgus monkey CD94 protein or a portion thereof. The amino acid sequence of the cynomolgus monkey CD94 protein is known in the art (e.g., UniProtKB identifier: Q68VD4).

[0041] In certain embodiments, the affinity of an antibody for its target (e.g., CD94) can be determined by the dissociation constant (K D Affinity can be measured using the assays described herein (e.g., in the Examples) by common methods known in the art, such as flow cytometry or Western blotting. In some embodiments, K D is measured using a radiolabeled antigen binding assay (RIA) performed with the Fab version of the antibody of the present disclosure and its target (e.g., CD94). D is measured using a surface plasmon resonance assay. A representative assay is described, for example, in Drake, A and Klakamp, ​​S.L. (2007) J. Immunol. Methods 318:147-152.

[0042] In some embodiments, the binding of an antibody of the present disclosure to CD94, e.g., human CD94 and / or cynomolgus CD94, may be assessed using any method known in the art. For example, the binding of an antibody of the present disclosure to human CD94 may be assessed in an ex vivo flow cytometry-based assay using peripheral blood mononuclear cells (PMBCs) and / or NK cells, e.g., as described in the Examples. Titration curves and EC50s may be generated and evaluated using methods known in the art, such as using Graphpad prism. In another example, the binding of an antibody of the present disclosure to cynomolgus CD94 may be assessed in an ex vivo or in vitro flow cytometry-based assay using cynomolgus CD94-expressing cells, e.g., cynomolgus CD94-expressing HEK293 cells, as described in the Examples.

[0043] In certain embodiments, an antibody of the disclosure has a K of less than about 10 μM for binding to its target (e.g., human and / or cynomolgus CD94). D In certain embodiments, an antibody of the disclosure has a K of less than about 1 μM for binding to its target (e.g., human and / or cynomolgus CD94). D In certain embodiments, an antibody of the disclosure has a binding affinity to its target (e.g., human and / or cynomolgus CD94) of less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, K of less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM DIn some embodiments, an antibody of the disclosure has a K of less than about 100 pM, less than about 75 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5 pM, less than about 1 pM, less than about 0.5 pM, or less than about 0.1 pM for binding to its target (e.g., human and / or cynomolgus CD94). D has.

[0044] In certain embodiments, an antibody of the disclosure has a K of less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM for binding to human CD94 on human NK cells or T cells. D In certain embodiments, the antibodies of the disclosure have a K of less than about 75 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM for binding to human CD94 on human NK cells or T cells. D In certain embodiments, the antibodies of the disclosure have a K of about 2 nM to about 80 nM for binding to human CD94 on human NK cells or T cells. D has.

[0045] In certain embodiments, an antibody of the disclosure has a K of less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM for binding to cyno CD94 on cells expressing cyno CD94. D In certain embodiments, an antibody of the disclosure has a K of less than about 75 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM for binding to cyno CD94 on cells expressing cyno CD94. D In certain embodiments, the antibodies of the disclosure have a K of about 2 nM to about 80 nM for binding to cynomolgus monkey CD94 on cells expressing cynomolgus monkey CD94. D has.

[0046] In some embodiments, the antibody of the present disclosure binds to its target (e.g., CD94) at the same or different epitope as an antibody known in the art for that target. In some embodiments, the antibody of the present disclosure binds to a different epitope than an antibody known in the art. In some embodiments, the antibody of the present disclosure specifically binds to human CD94, and the antibody does not bind to the same epitope on human CD94 as anti-CD94 antibody clones HP-3D9, DX22, HP-3B1, 131412, or 12K45. In some embodiments, the antibody of the present disclosure specifically binds to human CD94, and the antibody does not bind to the same epitope on human CD94 as anti-CD94 antibody clones DX22, HP-3B1, or 131412. In some embodiments, the antibody of the present disclosure specifically binds to human CD94, and the antibody binds to the same epitope on human CD94 as anti-CD94 antibody clone HP-3D9, DX22, HP-3B1, 131412, or 12K45. In some embodiments, the antibody of the present disclosure specifically binds to human CD94, and the antibody binds to the same epitope on human CD94 as anti-CD94 antibody clone HP-3D9. In some embodiments, the antibody of the present disclosure specifically binds to human CD94, and the antibody binds to the same epitope on human CD94 as anti-CD94 antibody clone 12K45.

[0047] In some embodiments, if an antibody of the disclosure does not bind to its target (e.g., CD94) at the same epitope as another antibody to that target, e.g., a commercially available antibody or an antibody known in the art for that target, the antibody of the disclosure does not block binding of the other antibody to the target in a competition assay (e.g., as described in the Examples), e.g., by 50% or more.

[0048] In some embodiments, an antibody of the present disclosure binds to its target (e.g., CD94) with higher affinity than an antibody known in the art for that target. In some embodiments, an antibody of the present disclosure binds to its target (e.g., CD94) with higher affinity than anti-CD94 antibody clones HP-3D9, DX22, HP-3B1, 131412, or 12K45. In some embodiments, an antibody of the present disclosure binds to its target (e.g., CD94) with higher affinity than anti-CD94 antibody clones HP-3B1, 131412, or 12K45.

[0049] In some embodiments, an antibody of the present disclosure specifically binds to human CD94, which antibody binds to human CD94 with greater affinity than the anti-CD94 antibody clones HP-3D9, DX22, HP-3B1, 131412, or 12K45. In some embodiments, an antibody of the present disclosure specifically binds to human CD94, which antibody binds to human CD94 with greater affinity than the anti-CD94 antibody clones HP-3B1, 131412, or 12K45.

[0050] In some embodiments, an antibody of the disclosure binds to its target (e.g., CD94) with an affinity that is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, or more, greater than another antibody known in the art for that target.

[0051] B. Representative anti-CD94 antibodies Certain aspects of the present disclosure relate to antibodies that bind (e.g., specifically bind) human CD94, the antibodies comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the antibodies are human antibodies. In some embodiments, provided herein are variants of the anti-CD94 antibody ATX-130, described in International Application No. PCT / US2021 / 052668, filed September 29, 2021.

[0052] In some embodiments, the antibody comprises a VH domain comprising the amino acid sequence EVQLX1ESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISX2SSX3X4IYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGRYYYYMDVWGKGTTVTVSS, where X1 is L or V, where X2 is T or S, where X3 is N or S, and where X4 is F or Y (SEQ ID NO: 69), and / or the amino acid sequence DIX1MTQSPSSLSASVGDRVTITCRASQSISSWLAWX2QQKPX3KX4PKX5LIYAASSLQSGVPSX6FSGSGSGTDFTLTISSLQPEDX7ATYYCQX8YNSX9PFTFGPGTKVDIK, where X1 is V or is Q, X2 is Y or F, X3 is G or E, X4 is A or V, X5 is S or L, X6 is K or R, X7 is V or F, X8 is K or Q, and X9 is A or Y (SEQ ID NO:70), wherein the antibody does not comprise a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO:2), a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3), a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:6). In some embodiments, the antibody comprises a VH domain that includes one, two, three, or four amino acid substitutions found in SEQ ID NOs:29-32 relative to the amino acid sequence of SEQ ID NO:16, and / or the antibody comprises a VL domain that includes one, two, three, four, five, six, seven, eight, or nine amino acid substitutions found in SEQ ID NOs:33-41 relative to the amino acid sequence of SEQ ID NO:17.

[0053] In some embodiments, the antibody comprises a VH domain comprising: CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1); CDR-H2 comprising an amino acid sequence selected from the group consisting of SISTSSNFIYYADSVKG (SEQ ID NO:2), SISSSSNFIYYADSVKG (SEQ ID NO:18), SISTSSSFIYYADSVKG (SEQ ID NO:21), and SISTSSNYIYYADSVKG (SEQ ID NO:24); and CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3), and / or a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4); CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5); and QKYN and a VL domain comprising a CDR-L3 comprising an amino acid sequence selected from the group consisting of SAPFT (SEQ ID NO: 6), QQYNSAPFT (SEQ ID NO: 27), and QKYNSYPFT (SEQ ID NO: 28), wherein the antibody does not comprise a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 1), a CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO: 2), a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 3), a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6).

[0054] In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO:2), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISSSSNFIYYADSVKG (SEQ ID NO:18), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISTSSSFIYYADSVKG (SEQ ID NO:21), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1), a CDR-H2 comprising the amino acid sequence SISTSSNYIYYADSVKG (SEQ ID NO:24), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3). Any of the VH domains or sets of three CDRs from a VH domain of the disclosure may be combined with any of the VL domains or sets of three CDRs from a VL domain of the disclosure.

[0055] As is known in the art, the variable domains of the heavy and light chains of an antibody (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR) (see, for example, Kindt et al. Kuby Immunology, 6 thed., W.H. Freeman and Co., page 91 (2007). Framework (or "FR" as used herein) may refer to variable domain residues other than CDR residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in a VH (or VL) in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In some embodiments, FR1, FR2, FR3, and / or FR4 of the present disclosure refer to human framework regions (i.e., of the VH or VL domain).

[0056] In some embodiments, the VH domain further comprises: (a) a FR1 comprising an amino acid sequence selected from the group consisting of EVQLLESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 48) and EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 49); (b) a FR2 comprising an amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO: 50); (c) a FR3 comprising an amino acid sequence of RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 51); and (d) a FR4 comprising the amino acid sequence of WGKGTTVTVSS (SEQ ID NO: 52). In some embodiments, the VH domain further comprises (a) an FR1 comprising the amino acid sequence of EVQLLESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 48), (b) an FR2 comprising the amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO: 50), (c) an FR3 comprising the amino acid sequence of RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 51), and (d) an FR4 comprising the amino acid sequence of WGKGTTVTVSS (SEQ ID NO: 52). In some embodiments, the VH domain further comprises (a) an FR1 comprising the amino acid sequence of EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 49), (b) an FR2 comprising the amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO: 50), (c) an FR3 comprising the amino acid sequence of RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 51), and (d) an FR4 comprising the amino acid sequence of WGKGTTVTVSS (SEQ ID NO: 52).

[0057] In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:6). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QQYNSAPFT (SEQ ID NO:27). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QKYNSYPFT (SEQ ID NO:28). Any of the VL domains or sets of three CDRs from a VL domain of the present disclosure may be combined with any of the VH domains or sets of three CDRs from a VH domain of the present disclosure.

[0058] In some embodiments, the VL domain comprises (a) a FR1 comprising an amino acid sequence selected from the group consisting of DIVMTQSPSSLSASVGDRVTITC (SEQ ID NO: 53) and DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 54); (b) a FR2 comprising an amino acid sequence selected from the group consisting of WYQQKPGKAPKSLIY (SEQ ID NO: 55), WFQQKPGKAPKSLIY (SEQ ID NO: 56), WYQQKPEKAPKSLIY (SEQ ID NO: 57), WYQQKPGKVPKSLIY (SEQ ID NO: 58), and WYQQKPGKAPKLLIY (SEQ ID NO: 59). 59), (c) an FR3 comprising an amino acid sequence selected from the group consisting of GVPSKFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 60), GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 61), and GVPSKFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 62), and (d) an FR4 comprising the amino acid sequence of FGPGTKVDIK (SEQ ID NO: 63).

[0059] In some embodiments, the VH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, and 29 to 32, and / or the VL domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, and 33 to 41. In some embodiments, the antibody does not comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a VL domain comprising the amino acid sequence of SEQ ID NO: 17.

[0060] In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 29, and the VL domain comprises the amino acid sequence of SEQ ID NO: 38.In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:29, and the VL domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:29, and the VL domain comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:29, and the VL domain comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:30, and the VL domain comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:34.In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:31, and the VL domain comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:40.In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:32, and the VL domain comprises the amino acid sequence of SEQ ID NO:41.

[0061] In some embodiments, an antibody of the disclosure comprises a VH domain comprising one, two, or three CDRs from a single VH domain listed in Table 1, and / or a VL domain comprising one, two, or three CDRs from a single VL domain listed in Table 1. In some embodiments, an antibody of the disclosure comprises a VH domain comprising one, two, or three CDRs from a single VH domain listed in Table 2, and / or a VL domain comprising one, two, or three CDRs from a single VL domain listed in Table 2. In some embodiments, an antibody of the disclosure comprises a VH and / or VL domain listed in Table 2. As exemplified below and described herein, in some embodiments, in an antibody of the disclosure, any set of three VH CDRs shown in Table 1 can be combined with any set of three VL CDRs shown in Table 1. In some embodiments, in an antibody of the disclosure, any set of three CDRs from a VH domain listed in Table 2 can be combined with any set of three CDRs from a VL domain listed in Table 2. In some embodiments, any VH domain shown in Table 2 may be combined with any VL domain shown in Table 2 in an antibody of the disclosure. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 2-1] [Table 2-2]

[0062] Many definitions of CDR sequences of antibody variable domains are known in the art and may be used to describe the antibodies of the present disclosure, for example, by their CDR sequences. In some embodiments, the CDR sequences of an antibody are defined as in Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). In some embodiments, the CDR sequences of an antibody are defined as in Chothia (see, for example, Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, the CDR sequences of an antibody are defined as in IMGT (see, for example, Lefranc, MP (1999) The Immunologist 7:132-136). In some embodiments, the CDR sequences of a single antibody are defined by a mixture of two or more definitions, for example, Kabat, Chothia, and / or IMGT.

[0063] In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7); a CDR-H2 comprising an amino acid sequence selected from the group consisting of ISTSSNFI (SEQ ID NO:8), ISSSSNFI (SEQ ID NO:19), ISTSSSFI (SEQ ID NO:22), and ISTSSNYI (SEQ ID NO:25); and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9), and / or the VL domain comprises a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO:10); a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO:11); and a CDR-H3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:12). SEQ ID NO:12), QQYNSAPFT (SEQ ID NO:27), and QKYNSYPFT (SEQ ID NO:28), and the antibody does not comprise a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISTSSNFI (SEQ ID NO:8), a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9), a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO:10), a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO:11), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:12). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISTSSNFI (SEQ ID NO:8), and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISSSSNFI (SEQ ID NO:19), and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISTSSSFI (SEQ ID NO:22), and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9).In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSYS (SEQ ID NO:7), a CDR-H2 comprising the amino acid sequence ISTSSNYI (SEQ ID NO:25), and a CDR-H3 comprising the amino acid sequence ARDLGRYYYYMDV (SEQ ID NO:9). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO:10), a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO:11), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO:12). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO:10), a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO:11), and a CDR-L3 comprising the amino acid sequence QQYNSAPFT (SEQ ID NO:27). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence QSISSW (SEQ ID NO: 10), a CDR-L2 comprising the amino acid sequence ASS (SEQ ID NO: 11), and a CDR-L3 comprising the amino acid sequence QKYNSYPFT (SEQ ID NO: 28).

[0064] In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13); a CDR-H2 comprising an amino acid sequence selected from the group consisting of STSSNF (SEQ ID NO: 14), SSSSNF (SEQ ID NO: 20), STSSSF (SEQ ID NO: 23), and STSSNY (SEQ ID NO: 26); and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15), and / or the VL domain comprises a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4); a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5); and a CDR-H3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6). No. 6), QQYNSAPFT (SEQ ID NO: 27), and QKYNSYPFT (SEQ ID NO: 28), and the antibody does not comprise a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence STSSNF (SEQ ID NO: 14), a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15), a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence STSSNF (SEQ ID NO: 14), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence SSSSNF (SEQ ID NO: 20), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence STSSSF (SEQ ID NO: 23), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15).In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTFSSY (SEQ ID NO: 13), a CDR-H2 comprising the amino acid sequence STSSNY (SEQ ID NO: 26), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 15). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QQYNSAPFT (SEQ ID NO: 27). In some embodiments, the antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5), and a CDR-L3 comprising the amino acid sequence QKYNSYPFT (SEQ ID NO:28).

[0065] In some embodiments, the antibody of the present disclosure comprises an Fc region. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the antibody comprises a non-fucosylated human Fc region. In some embodiments, the antibody binds to human cell Fc gamma receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. In some embodiments, the antibody can induce antibody-dependent cellular cytotoxicity (ADCC) against cells expressing human CD94 on the surface.

[0066] In some embodiments, the term antibody is used in its broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibodies of the present disclosure are isolated antibodies. An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with the research, diagnostic, and / or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibodies are purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody, as determined, for example, by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, for example, using Coomassie blue or silver staining. 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.

[0067] In some embodiments, a monoclonal antibody is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies, e.g., containing naturally occurring mutations or arising during the production of the monoclonal antibody preparation, which variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, in some embodiments, a monoclonal antibody is obtained from a substantially homogeneous population of antibodies. Monoclonal antibodies may be produced using any method known in the art. For example, monoclonal antibodies used in accordance with the present disclosure may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus.

[0068] C. Blockade of HLA-E binding Major histocompatibility complex class I, E (HLA-E) is a ligand for the CD94 / NKG2A heterodimer and plays an essential role in the inhibition of NK cell and CD8+ T cell activity when bound to the CD94 / NKG2A heterodimer. Thus, without wishing to be bound by theory, blocking the interaction of HLA-E with CD94 / NKG2A can result in the activation and proliferation of target cells, e.g., cells expressing CD94. Thus, for example, in subjects with NK / T cell lymphoma administered an anti-CD94 antibody, it may be beneficial for the anti-CD94 antibody not to block the interaction of HLA-E with the CD94 / NKG2A heterodimer. In some embodiments, the HLA-E is human HLA-E, also known as QA1 and HLA-6.2. For representative HLA-E genes, see, e.g., NCBI gene ID number 3133, and for representative HLA-E polypeptides, see, e.g., NP_005507.3.

[0069] In some embodiments, blocking of HLA-E binding to the CD94 / NKG2A heterodimer refers to blocking of HLA-E binding to the CD94 / NKG2A heterodimer, blocking of HLA-E binding to CD94, and / or blocking of HLA-E binding to NKG2A.

[0070] Blocking of HLA-E binding to CD94 / NKG2A heterodimers by antibodies of the present disclosure may be assessed using any method known in the art. For example, blocking of HLA-E binding to CD94 / NKG2A heterodimers by antibodies of the present disclosure may be assessed using ex vivo assays using PBMCs and / or NK cells, for example, as described in the Examples. In a representative assay, for example, PBMCs obtained from healthy donors are incubated with human Fc block (Biolegend, San Diego, CA) and cell viability dye (Thermo Fisher, Carlsbad, CA) on ice for 30 minutes protected from light. The cells are then washed once with FACS buffer (PBS with 2% low IgG FBS). Saturating concentrations of anti-CD94 antibody or isotype control antibody are incubated with the cells on ice for 30 minutes protected from light. The cells are then washed and incubated with HLA-E tetramer PE (Creative Biolabs, Shirley, NY), CD3 Pacific Blue antibody, and CD56 FITC antibody (Biolegend, San Diego, CA) on ice for 30 minutes protected from light. The cells are then subjected to a final wash in FACS buffer before quantification on a flow cytometer. Data acquisition and fluorescence compensation may be performed using methods known in the art, such as using a CytoFlex flow cytometer (Beckman Coulter, Chaska, MN). Data analysis may be performed using any method known in the art, such as using FlowJo software. NK cells are identified by gating lymphocytes based on forward and side scatter, followed by excluding doublets and dead cells, and gating on the CD3-CD56+ population. HLA-E is then quantified in the CD3-CD56+ NK cell population. The percent blockage of HLA-E binding to CD94 / NKG2A heterodimers by an anti-CD94 antibody compared to a control antibody, e.g., an isotype control antibody, is calculated as 100-((percent HLA-E positive for anti-CD94 antibody) / (percent HLA-E positive for isotype) x 100).

[0071] In some embodiments, an antibody blocks HLA-E binding to the CD94 / NKG2A heterodimer if it blocks more than about 20% of HLA-E binding to the CD94 / NKG2A heterodimer, e.g., compared to an isotype control antibody. In some embodiments, an antibody blocks HLA-E binding to the CD94 / NKG2A heterodimer if it blocks about 21%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of HLA-E binding to the CD94 / NKG2A heterodimer, e.g., compared to a control antibody, e.g., an isotype control antibody.

[0072] In some embodiments, an antibody does not block HLA-E binding to the CD94 / NKG2A heterodimer if it blocks about 20% or less of HLA-E binding to the CD94 / NKG2A heterodimer, e.g., compared to an isotype control antibody. In some embodiments, an antibody does not block HLA-E binding to the CD94 / NKG2A heterodimer if it blocks about 20%, about 18%, about 16%, about 14%, about 12%, about 10%, about 8%, about 6%, about 4%, about 2%, about 1%, about 0.5%, or 0% of HLA-E binding to the CD94 / NKG2A heterodimer, e.g., compared to a control antibody, e.g., an isotype control antibody.

[0073] In some embodiments, the antibodies provided herein do not block HLA-E binding to CD94 / NKG2A heterodimers. In some embodiments, the antibodies provided herein block about 20% or less, about 19% or less, about 18% or less, about 17% or less, about 16% or less, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, or 0% of HLA-E binding to CD94 / NKG2A heterodimers compared to a control antibody, e.g., an isotype control antibody. In some embodiments, the antibodies provided herein block 0% of HLA-E binding to CD94 / NKG2A heterodimers compared to a control antibody, e.g., an isotype control antibody.

[0074] D. Enhanced ADCC Activity In some embodiments, antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular cytotoxicity, antibody directed cytotoxicity, or ADCC refers to a cell-mediated reaction in which non-specific cytotoxic cells bearing Fc receptors, such as natural killer cells (NK cells), neutrophils, and macrophages, recognize antibodies bound to target cells and then cause lysis of the target cells. The main mediator cells are natural killer (NK) cells. NK cells express FcγRIII (Ravetch et al. (1991) Annu. Rev. Immunol., 9:457-92). In some embodiments, ADCC activity refers to the ability of an antibody or Fc fusion protein to induce an ADCC response.

[0075] In some embodiments, the antibodies provided herein have enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. In some embodiments, enhanced ADCC activity refers to an antibody or an Fc region of an antibody mediating or inducing ADCC more efficiently and / or more effectively in vitro or in vivo in the presence of effector cells than a native or wild-type antibody and / or a native or wild-type Fc region of an antibody, which can be determined, for example, using an ADCC assay as described herein or known in the art. In some embodiments, the effector cells are white blood cells that bear one or more Fc receptors and perform effector functions. In some embodiments, such cells bear at least FcγRIII and perform ADCC effector functions. Examples of human white blood cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer cells (NKs), monocytes, cytotoxic T cells, and neutrophils.

[0076] In some embodiments, ADCC activity was measured using peripheral blood mononuclear cells (PBMCs) and / or NK effector cells. 51ADCC activity can be assessed directly using in vitro assays, using a Cr release assay (see, e.g., Shields et al. (2001) J. Biol. Chem., 276:6591-6604), or another suitable method. ADCC activity can be expressed as the number of cells remaining following an ADCC assay, or the concentration of antibody or Fc fusion protein at which half-maximal lysis of target cells occurs (e.g., EC50 or IC50). In some embodiments, ADCC activity is determined using an ex vivo assay using PBMCs, LGL cells, and / or NK cells, e.g., as described in the Examples, and the ADCC activity of an antibody of the present disclosure is described as the percentage of target cells remaining following an ADCC assay and / or the IC50 or EC50 of the antibody (i.e., the concentration of an antibody of the present disclosure at which half-maximal target cell depletion or cell lysis is achieved). The IC50 or EC50 of the antibody may be determined using any method known in the art, e.g., using dose-response curves and GraphPad Prism.

[0077] In some embodiments, the antibodies provided herein induce ADCC activity with an IC50 or EC50 of about 1 ng / ml to about 100 ng / ml (e.g., any of about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about 100 ng / ml) measured using an ex vivo assay. In some embodiments, the antibodies provided herein induce ADCC activity with an IC50 or EC50 of about 20 ng / ml or less, as measured using an ex vivo assay. In some embodiments, the antibodies provided herein induce ADCC activity with an IC50 or EC50 of about 60 ng / ml or less, as measured using an ex vivo assay. In some embodiments, the antibodies of the present disclosure exhibit an IC50 or EC50 that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than the IC50 or EC50 of a control antibody (e.g., a wild-type control antibody, or an antibody known in the art or commercially available against the same target).

[0078] In some embodiments, IC50 or EC50 refers to the concentration of a compound (e.g., an antibody) that induces a response halfway between baseline and maximum after a specified exposure time. For example, IC50 or EC50 may be used to measure the potency of an antibody in mediating and / or inducing effector function, e.g., ADCC activity. In some embodiments, IC50 or EC50 of a dose-response curve represents the concentration of a compound (e.g., an antibody) at which 50% of its maximum effect is observed.

[0079] In some embodiments, the antibodies of the present disclosure have a higher target cell lysis maximum compared to a control antibody (e.g., a wild-type control antibody, or an antibody known in the art or commercially available against the same target). For example, the antibodies of the present disclosure may exhibit a target cell lysis maximum that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the target cell lysis maximum of a control antibody (e.g., a wild-type control antibody, or an antibody known in the art or commercially available against the same target).

[0080] (i) Enhanced binding to Fc receptors In some embodiments, the antibodies provided herein comprise a human immunoglobulin Fc region that has enhanced ADCC activity compared to a wild-type Fc region. In some embodiments, the antibodies provided herein bind to human cell Fc receptors to a greater extent than antibodies comprising a wild-type Fc region. In some embodiments, an Fc receptor (FcR) is a receptor that can bind to the Fc region of an antibody. Certain Fc receptors can bind to IgG (i.e., gamma-receptors), including FcyRI, FcyRII, and FcyRIII subclasses, as well as allelic variants and alternative splicing events thereof. For a general overview of Fc receptors, see Ravetch and China: Annu. Port. Immunol. 9, 457 (1991); Capel et al. Immunomethods, 4, 25 (1994); and de Haas et al., J. Leg. Clin. Med. 126, 330 (1995).

[0081] In some embodiments, the antibody provided herein binds to human cell Fc gamma receptor IIIA to a greater extent than an antibody comprising wild-type Fc region.In some embodiments, human cell Fc gamma receptor IIIA comprises a valine or phenylalanine residue at amino acid residue 158.See, for example, UniProt accession P08637 or VAR_003960.In some embodiments, human cell Fc gamma receptor IIIA comprises the sequence of SEQ ID NO:67 or 68. Human Cellular Fc Gamma Receptor IIIA 158F [ka] Human Cell Fc Gamma Receptor IIIA 158V [ka]

[0082] In some embodiments, the antibodies provided herein are of the IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or IgA2), IgD, IgM, or IgE isotype. In some embodiments, the antibodies provided herein are of the IgG isotype. In some embodiments, the antibodies provided herein are of the IgG1 isotype. In some embodiments, the antibodies provided herein bind to human cell Fc gamma receptor IIIA (FcγRIIIA) to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. In some embodiments, the human cell Fc gamma receptor IIIA comprises a valine or phenylalanine residue at amino acid residue 158. Representative assays for determining binding to human cell Fc gamma receptor IIIA are known in the art, see, e.g., Lazar, GA et al. (2006) Proc. Natl. Acad. Sci. 103:4005-1010, and Ferrara, C. et al. (2011) Proc. Natl. Acad. Sci. 108:12669-12674.

[0083] In some embodiments, the Fc region is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. In some embodiments, the Fc region includes a native Fc region or a variant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In some embodiments, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. In some embodiments, a wild-type or native Fc region is an Fc region that includes an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. In some embodiments, a variant Fc region is an Fc region that comprises an amino acid sequence in which at least one amino acid differs from the native or wild-type sequence of the Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution, e.g., about 1-10 or 1-5 amino acid substitutions. In some embodiments, the Fc region variant is at least about 80% (e.g., at least about 90%, or at least about 95%) homologous to the native or wild-type sequence Fc region and / or the Fc region of the original polypeptide. In some embodiments, the at least one amino acid substitution in the variant Fc region enhances an effector function of the variant Fc region compared to the native or wild-type Fc region. In some embodiments, an effector function is a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype.Examples of antibody effector functions include Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0084] The binding affinity of an antibody to an Fc receptor may be assessed using any method known in the art, such as, for example, using surface plasmon resonance and / or ELISA, as described in Shields et al. (2001) J. Biol. Chem., 276:6591-6604. In some embodiments, the affinity of an antibody of the present disclosure to FcγRIIIA may exceed that of a wild-type control by any of at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or more.

[0085] In some embodiments, affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or target). For example, the affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein.

[0086] In some embodiments, a statement that a molecule (e.g., an antibody and / or Fc region) binds to a greater extent than another molecule (e.g., an antibody and / or Fc region) or that a molecule (e.g., an antibody and / or Fc region) binds with greater affinity than another molecule (e.g., an antibody and / or Fc region), or other grammatical equivalents, refers to a molecule (e.g., an antibody and / or Fc region) that binds more tightly (e.g., has a lower dissociation constant) to a target (e.g., an Fc receptor, a cell surface protein) than another molecule (e.g., an antibody and / or Fc region) under substantially the same conditions in a binding assay (e.g., as described herein and / or known in the art). For example, a statement that antibody "X" binds to an Fc receptor to a greater extent than antibody "Y" indicates that antibody "X" binds more tightly (e.g., has a lower dissociation constant) to an Fc receptor than antibody "Y" under substantially the same conditions in a binding assay (e.g., as described herein and / or known in the art). In another example, a statement that antibody "X" binds to a target (e.g., a cell surface protein, such as CD94) with higher affinity than antibody "Y" indicates that antibody "X" binds more tightly (e.g., has a lower dissociation constant) to a target (e.g., a cell surface protein, such as CD94) than antibody "Y" under substantially the same conditions in a binding assay (e.g., as described herein and / or known in the art).

[0087] (ii) Reduced fucosylation In some embodiments, the antibody of the present disclosure is non-fucosylated or fucose-deficient, e.g., a glycosylated antibody variant comprising an Fc region in which the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, the antibody with reduced fucose or lacking fucose has improved ADCC function. A non-fucosylated or fucose-deficient antibody has reduced fucose compared to the amount of fucose on the same antibody produced in a cell line. In some embodiments, a non-fucosylated or fucose-deficient antibody composition of the present disclosure is a composition in which less than about 50% of the N-linked glycans attached to the Fc region of the antibody in the composition contain fucose.

[0088] In some embodiments, fucosylation or fucosylated refers to fucose residues in the oligosaccharides attached to the peptide backbone of the antibodies of the present disclosure. Specifically, a fucosylated antibody contains α(1,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or both of the N-linked oligosaccharides attached to the antibody Fc region, e.g., at position Asn297 (EU numbering of Fc region residues) of the human IgG1 Fc domain. Asn297 may also be located about +3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence diversity in immunoglobulins.

[0089] In some embodiments, the degree of fucosylation is the percentage of fucosylated oligosaccharides relative to all oligosaccharides as determined by methods known in the art, such as, for example, in an antibody composition treated with N-glycosidase F as assessed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). In a composition of fully fucosylated antibodies, at least 90% or essentially all of the oligosaccharides contain a fucose residue, i.e., are fucosylated. Thus, each individual antibody in such a composition typically contains a fucose residue in each of the two N-linked oligosaccharides in the Fc region. In some embodiments, in a composition of fully non-fucosylated antibodies, less than about 10% of the oligosaccharides are fucosylated or essentially none of the oligosaccharides are fucosylated, and each individual antibody in such a composition does not contain a fucose residue in either of the two N-linked oligosaccharides in the Fc region. In a composition of partially fucosylated antibodies, only a portion of the oligosaccharides contain fucose. The individual antibodies in such compositions can contain no fucose residues in any of the N-linked oligosaccharides in the Fc region, or can contain a fucose residue in one or both of the N-linked oligosaccharides in the Fc region, so long as essentially all of the individual antibodies in the composition lack fucose residues in the N-linked oligosaccharides in the Fc region or contain a fucose residue in both of the N-linked oligosaccharides in the Fc region. In one embodiment, a composition of partially fucosylated antibodies has a degree of fucosylation of about 10% to about 80% (e.g., about 50% to about 80%, about 60% to about 80%, or about 70% to about 80%).

[0090] In some embodiments, the glycosylated antibody variant comprises an Fc region in which the carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function. Examples of defucosylated or fucose-deficient antibodies are described in US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, Okazaki et al. al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

[0091] Antibodies with reduced fucosylation or without fucosylation may be produced using any method known in the art. In some embodiments of the antibodies of the present disclosure, at least one or two of the heavy chains of the antibody may be without fucosylation. For example, antibodies of the present disclosure with reduced fucosylation or without fucosylation may be produced in a cell line with alpha 1,6-fucosyltransferase (Fut8) knockout and / or overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III) and / or overexpressing Golgi μ-mannosidase II (ManII). Antibodies with reduced fucosylation, or no fucosylated antibodies, may also be produced using cell lines deficient in "FUT8," i.e., alpha-1,6 fucosyltransferase, which catalyzes the transfer of fucose; for example, using Chinese hamster ovary (CHO) cells deficient in FUT8 (Yamane-Ohnuki et al., 2004); or using small interfering RNA (siRNA) to block expression of the FUT8 gene (Mori et al., 2004). Other cell lines that can be used to generate non-fucosylated or defucosylated antibodies or antibodies with reduced fucosylation are known in the art, such as Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US2003 / 0157108A1 (Presta, L); and WO2004 / 056312A1 (Adams et al., especially at Example 11)), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (Yamane-Ohnuki et al. al. Biotech. Bioeng. 87:614 (2004)), as well as cells overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi μ-mannosidase II (ManII).

[0092] In some embodiments, the antibody of the present disclosure has a reduced amount of fucose compared to the amount of fucose on the same antibody produced in wild-type CHO cells. For example, the antibody may have a lower amount of fucose than it would otherwise have if produced by a native CHO cell (e.g., a CHO cell that produces a native glycosylation pattern, such as a CHO cell that contains a native FUT8 gene). In some embodiments, the antibody provided herein is an antibody in which less than about 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the N-linked glycans thereon contain fucose. In certain embodiments, the antibody provided herein is an antibody in which none of the N-linked glycans thereon contain fucose, i.e., the antibody does not contain any fucose, does not have fucose, or is not fucosylated or defucosylated. The amount of fucose can be determined by one of skill in the art, for example, by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (Eu numbering of Fc region residues) in the Fc region, although Asn297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variability in antibodies. In some embodiments, at least one or two of the heavy chains of the antibody are non-fucosylated.

[0093] Antibodies lacking 1,6-fucose in their heavy chain glycosylation may have enhanced binding affinity to FcγRIII receptors and increased ADCC activity (see, e.g., Shields et al., 2002; Shinkawa et al, 2002; Okazaki, 2004; Dall'Ozzo, 2004). In some embodiments, the antibodies provided herein comprise an Fc region having modifications including reduced fucosylation, non-fucosylation, and / or mutations that enhance ADCC activity and / or improve affinity of the Fc region for Fc receptors such as FcγRIII and CD16 (e.g., CD16a). In some embodiments, the molecules (e.g., antibodies provided herein) induce antibody-directed cytotoxicity (ADCC) and deplete or reduce the number of CD94-expressing NK cells and / or T cells to a greater extent than fucosylated or wild-type antibodies.

[0094] In some embodiments, the antibodies of the present disclosure are engineered to improve ADCC activity by reducing fucosylation. In some embodiments, the molecules provided herein (e.g., the antibodies provided herein) can induce antibody-directed cytotoxicity (ADCC) to a greater extent than fucosylated or wild-type antibodies and can deplete or reduce the number of NK cells and / or T cells expressing CD94. In some embodiments, at least one or two of the heavy chains of the antibodies of the present disclosure are afucosylated. In some embodiments, the antibodies of the present disclosure are modified such that the carbohydrate of the antibody is afucosylated. In some embodiments, the antibodies of the present disclosure are modified such that less than about 90% of the carbohydrate of the antibody contains fucose, e.g., less than any of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1%. In some embodiments, the antibodies of the disclosure are modified such that less than about 40% of the carbohydrates of the antibody contain fucose. In some embodiments, the antibodies provided herein are non-fucosylated.

[0095] In some embodiments, the molecules (e.g., antibodies) provided herein induce antibody-directed cytotoxicity (ADCC) and deplete or reduce the numbers of CD94-expressing NK cells and / or T cells to a greater extent than fucosylated or wild-type antibodies.

[0096] (iii) Mutations that enhance ADCC activity The antibody of the present disclosure may comprise a variant Fc region. In some embodiments, the variant Fc region comprises at least one amino acid substitution in the Fc region that improves ADCC activity. For example, the antibody of the present disclosure may have a variant IgG1 Fc region that comprises one or more of the Fc mutations selected from S239D, A330L, I332E, F243L, and G236A. In another example, the antibody of the present disclosure may have a human IgG1 Fc variant region that comprises one or more of the Fc mutations selected from S239D, A330L, I332E, F243L, and G236A. Other amino acid substitutions known to enhance ADCC activity may also be used, for example, as described in Lazar et al., PNAS 103, 4005-4010 (2006); Shields et al., J. Biol. Chem. 276, 6591-6604 (2001); Stewart et al., Protein Engineering, Design and Selection 24, 671-678 (2011); and Richards et al., Mol Cancer Ther 7, 2517-2527 (2008).

[0097] (iv) Decreased internalization In some embodiments, the antibodies of the present disclosure have a lower degree of internalization, e.g., receptor-induced internalization or target internalization (i.e., internalization of surface-expressed CD94), e.g., compared to a wild-type control antibody or an antibody known in the art or commercially available against the same target. An antibody with lower internalization has a higher receptor (e.g., CD94) occupancy on the cell surface and a higher level of receptor-antibody complexes on the cell surface, which may enhance ADCC activity. The antibodies of the present disclosure may be tested for their internalization ability in vitro. The antibodies of the present disclosure may be tested for their internalization ability in an ex vivo assay, e.g., using PBMCs and / or NK cells, e.g., as described in the Examples. Target (e.g., CD94) internalization may be expressed as a percentage decrease in mean fluorescence intensity (MFI) over a period of time, e.g., using a flow cytometry-based assay, e.g., as described in the Examples. For example, target internalization (i.e., the internalization ability of an antibody of the present disclosure) can be expressed as a percentage reduction in MFI, calculated by calculating the difference in MFI over a 24 hour period (e.g., between 0.5 hours and 24 hours) in cells incubated with an antibody at 37°C, e.g., as described in the Examples, and multiplying by 100.

[0098] In some embodiments, an antibody has a high degree of internalization if the antibody results in a greater than 50% reduction in MFI, as measured, for example, by ex vivo assays using PBMCs and / or NK cells as described in the Examples, calculated by calculating the difference in MFI over a 24 hour period (e.g., between 0.5 hours and 24 hours) in cells incubated with the antibody at 37° C. and multiplied by 100. In some embodiments, an antibody has a high degree of internalization if incubation of the antibody with cells expressing human CD94 on their surface at 37° C. for 24 hours results in a greater than 50% reduction in surface antibody staining due to internalization, as assessed using methods known in the art and / or as described above.

[0099] In some embodiments, an antibody has a low degree of internalization if the antibody causes less than 50% (e.g., any of about 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, or 0%) reduction in MFI, calculated by calculating the difference in MFI over a 24 hour period (e.g., between 0.5 hours and 24 hours) in cells incubated with the antibody at 37° C. and multiplying by 100, when measured by an ex vivo assay using PBMCs and / or NK cells, e.g., as described in the Examples. In some embodiments, an antibody of the disclosure has a low degree of internalization, resulting in about 50% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2.5% or less, about 1% or less, or about 0% reduction in MFI, calculated as described above. In some embodiments, an antibody has a low degree of internalization if incubation of the antibody with cells expressing human CD94 on their surface at 37° C. for 24 hours results in less than a 50% reduction in surface antibody staining due to internalization, as assessed using methods known in the art and / or as described above.

[0100] In some embodiments, antibodies of the disclosure have or result in an internalization activity that is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or about 100% less than the internalization of a control antibody, e.g., a commercial or wild-type control antibody, or an isotype control antibody, where internalization is assessed as described above and / or using any suitable method known in the art.

[0101] Antibody candidates with no or low internalization activity may be further tested for binding to cynomolgus and / or human targets (e.g., cynomolgus and / or human CD94). Antibodies that bind to cynomolgus and / or human targets may be used in in vitro and in vivo cell killing assays (e.g., ADCC assays). The cell killing activity (e.g., ADCC activity) of selected antibodies may be compared to commercially available antibodies or antibodies known in the art.

[0102] E. Antibody Generation The antibodies of the present disclosure may be generated using any technique and / or method known in the art. Techniques for preparing antibodies, e.g., monoclonal antibodies (mAbs), against virtually any target antigen are well known in the art. See, for example, Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL.1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies can be obtained by injecting a composition containing an antigen (e.g., CD94, or a portion thereof) into a mouse, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to generate hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies against the antigen, culturing the clones that produce antibodies against the antigen, and isolating the antibodies from the hybridoma culture. One of skill in the art will recognize that if the antibody is to be administered to a human subject, the antibody will bind to a human antigen (eg, human CD94, or a portion thereof).

[0103] MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques, including affinity chromatography with Protein-A or Protein-G Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. See also, Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

[0104] After initial generation of antibodies against an immunogen (e.g., CD94, or a portion thereof), the antibodies can be sequenced and then prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those of skill in the art, as discussed below.

[0105] In an exemplary method for generating antibodies of the present disclosure, a recombinant target (e.g., CD94) may be used to immunize mice. Following immunization of mice, for example, as described above, the antibodies generated may be analyzed for specific or selective binding to their target (e.g., CD94) by ELISA and flow cytometry. Antibodies may be selected based on their ability to bind to the target (e.g., CD94).

[0106] In some embodiments, non-human primate antibodies may be generated. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., WO91 / 11465 (1991), and Losman et al., Int. J. Cancer 46:310 (1990).

[0107] In some embodiments, the antibody may be a human antibody. In some embodiments, the antibody may be a monoclonal human antibody. In some embodiments, the human antibody has an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. Such an antibody may be obtained from a transgenic mouse that has been engineered to produce a specific human antibody in response to antigenic stimulation, e.g., CD94, or a portion thereof. Methods for making fully human antibodies using either combinatorial techniques or transgenic animals transformed with human immunoglobulin loci are known in the art (e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol. 3:544-50). In certain embodiments, the claimed methods and procedures may utilize human antibodies generated by such techniques. Other methods of producing fully human antibodies include phage display, e.g., as described in Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40, generation of antibodies in normal humans or from humans exhibiting a particular disease state, e.g., as described in Dantas-Barbosa et al., 2005, or the use of transgenic animals (e.g., mice) that have been genetically engineered to produce human antibodies using standard immunization protocols discussed above, such as those described in Green et al., 1999, J. Immunol. Methods 231:11-23; Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994); and Taylor et al., Int. Immun. 6:579 (1994).

[0108] (i) In vitro cell killing assay The generation of the antibodies of the present disclosure may involve testing the in vitro ADCC activity of the antibodies. The improved cell killing or ADCC activity of the antibodies of the present disclosure may be tested using methods as described above, as described in the Examples, and / or known in the art. The improved cell killing or ADCC activity of the antibodies of the present disclosure may be tested with respect to depletion of NK cells and / or T cells expressing CD94. Depletion of NK cells and / or T cells expressing CD94 may be tested using representative in vitro models that recapitulate activity in humans (Tomasevic, et al, Growth Factors, 2014, 32(6):223-235; Huang, et al, JCI insight, 2016;1(7):e86689). Peripheral blood lymphocytes (PBLs) isolated from the blood of normal (i.e., healthy) donors are incubated with antibodies with human Fc regions with and without fucose and / or with and without Fc region mutations. The level of killing of CD94-expressing NK cells and / or T cells in the PBLs (e.g., in a PBL sample) is measured using any method known in the art, such as flow cytometry (e.g., as described in the Examples). The cell killing activity (e.g., ADCC activity) of the antibodies may be tested using the assays described above using a variety of biological samples, such as blood, synovial fluid, bone marrow, and intact spleen cell homogenates from patients with diseases such as NK / T cell lymphoma, e.g., extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (unspecified type), angioimmunoblastic TCL, adult TCL, chronic NK cell lymphoproliferative disorder (CLPD-NK), LGL leukemia, Felty syndrome, CLPD-NK, IBM and RA associated with LGL and / or aggressive NK leukemia.

[0109] In addition to the cell killing assays described above, in vitro ADCC and antibody-dependent cellular phagocytosis (ADCP) assays using antibodies of the disclosure, purified target cells (e.g., NK cells and / or T cells expressing CD94), and / or effector cells such as NK cells or monocytes / macrophages may be performed to assay the cell killing, ADCC and / or ADCP activity of antibodies of the disclosure. For example, cell killing, ADCC and / or ADCP assays as described in Kolbeck et al., J Allergy Clin Immunol. 2010;125(6):1344-1353.e2, Gomez-Roman et al., J.Immunol.Methods, 2006,308,pp.53-67, and Ackerman et al., J.Immunol.Methods, 2011,366,pp.8-19, as well as other assay methods known in the art, may be used. The in vitro activity of antibodies of the present disclosure may be compared to commercially available antibodies or antibodies known in the art against the same target.

[0110] (ii) In vivo cell killing assay The generation of antibodies of the present disclosure may involve testing the in vivo ADCC activity of the antibodies, e.g., to demonstrate the in vivo activity of selected antibody candidates with respect to depleting or reducing the levels of NK cells and / or T cells expressing CD94. The in vivo cell killing activity (e.g., ADCC and / or ADCP activity) of antibodies of the present disclosure may be determined using any method known in the art. For example, the ability of antibodies of the present disclosure to deplete or reduce NK cells and / or T cells expressing CD94 in vivo may be tested in cynomolgus monkeys using methods known in the art. For example, in a representative method for testing the in vivo cell killing activity (e.g., ADCC and / or ADCP activity) of antibodies of the present disclosure, blood is drawn from a cohort of cynomolgus monkeys one day prior to administration of a single dose of an antibody of the present disclosure, e.g., antibody treatment, to determine pre-administration levels of NK cells and / or T cells expressing CD94 by flow cytometry. After administration of the antibody of the present disclosure, for example, when treated with the antibody of the present disclosure, blood is taken from the monkey at the following time points: 1 hour, 1 day, 7 days, 14 days, and 30 days. The levels of NK cells and / or T cells expressing CD94 in blood and other biological samples such as synovial fluid, bone marrow, and spleen are determined by flow cytometry at each of these time points. The in vivo activity of the antibody of the present disclosure may be compared with commercially available antibodies against the same target or antibodies known in the art. The anti-CD94 antibody of the present disclosure may be compared with anti-CD94 antibody clones DX22, HP-3D9, HP-3B1, 131412, or 12K45.

[0111] One of ordinary skill in the art will readily appreciate that other methods known in the art for testing in vivo ADCC activity can also be used to assay the in vivo ADCC activity of the antibodies of the disclosure (e.g., transgenic animals such as transgenic mice).

[0112] Other known antibodies against targets (eg, CD94) may also be used in the methods provided herein. For example, the anti-CD94 antibodies of the present disclosure include the following anti-CD94 antibodies: HP-3D9 (LSBio Catalog No. LS-C134679-100; Abnova Catalog No.: MAB6947); 2I2; 131412 (R&D Systems Catalog No.: MAB1058); 13B146 (USBiological Catalog No.: 030068); 13B147 (USBiological Catalog No.: 030069); 1H1 (Abnova Catalog No.: MAB10543); 3G2 (Biorbyt Catalog No.: orb69389); DX22 (Biolegend Catalog No. 305502); REA113 (Miltenyi Biotec Catalog No.: 130-098-967); KP43; EPR21003; AT13E3 (ATGen Catalog: ATGA0487); HP-3B1; 12K45; and B-D49 (e.g., for in vitro or in vivo ADCC activity, or for any other property described herein).

[0113] (iii) Humanization The antibodies of the present disclosure may be humanized according to any method known in the art. In some embodiments, a humanized antibody is a chimeric antibody comprising amino acid residues from a non-human hypervariable region (HVR) and amino acid residues from a human framework region (FR). In certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, a humanized form of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0114] For example, a monoclonal antibody may be humanized by transferring mouse CDRs from the heavy and light chain variable regions of a mouse immunoglobulin into the corresponding variable domains of a human antibody. The mouse framework regions (FR) in the chimeric monoclonal antibody may also be replaced with human FR sequences. To preserve the stability and antigen specificity of the humanized monoclonal antibody, one or more human FR residues may be replaced with the mouse counterpart. The humanized monoclonal antibody may be used for therapeutic treatment of a subject. Techniques for producing humanized monoclonal antibodies are well known in the art, as described, for example, in Jones et al., 1986, Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen et al., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad. Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest et al., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993, 150:2844.

[0115] An antibody of the disclosure may have one or more of the above-mentioned properties, e.g., enhanced in vitro and / or in vivo cell killing activity (e.g., ADCC and / or ADCP activity), enhanced binding to one or more Fc receptors, reduced fucosylation or non-fucosylation, cross-reactivity (e.g., binding to human CD94 and cynomolgus CD94), lack of blocking of HLA-E binding to the CD94 / NKG2A heterodimer, competition with commercially available antibodies (e.g., commercially available anti-CD94 antibodies), low internalization, and / or a desirable affinity for its target protein (e.g., CD94, human CD94, and / or cynomolgus CD94).

[0116] In some embodiments, the antibody of the present disclosure is soluble at a concentration of about 10 mg / mL or more. In some embodiments, the antibody of the present disclosure forms low levels of soluble aggregates (e.g., less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% soluble aggregates). In some embodiments, the antibody of the present disclosure has the ability to maintain binding to its target (e.g., CD94) during storage, for example, at about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, or longer. In some embodiments, the antibodies of the disclosure have stability (e.g., lack of degradation products, e.g., as measured by SDS-PAGE) during storage, e.g., at any of about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, or about 8°C, for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, or longer.

[0117] In some embodiments, the antibody of the present disclosure is tested for toxicology. The toxicity analysis of the antibody of the present disclosure may be carried out using any method known in the art. In a typical toxicity analysis, the antibody of the present disclosure is tested for toxicity in cynomolgus monkeys at a dose that is more than 5 times higher than the dose that is expected to be used in human subjects (e.g., about 5 times higher, about 10 times higher, about 15 times higher, about 20 times higher, about 25 times higher, about 30 times higher, about 35 times higher, about 40 times higher, about 45 times higher, about 50 times higher, about 55 times higher, about 60 times higher, about 65 times higher, about 70 times higher, about 75 times higher, about 80 times higher, about 85 times higher, about 90 times higher, about 95 times higher, about 100 times higher, or more).

[0118] In some embodiments, the antibodies of the present disclosure can deplete or reduce the levels of NK cells and / or T cells expressing CD94 in vitro and / or in vivo. The depletion or reduction in the levels of NK cells and / or T cells expressing CD94 may be measured using any method known in the art. For example, the depletion of NK cells and / or T cells expressing CD94 may be measured using cell killing, ADCC, and / or ADCP assays, for example, as described above and / or in the Examples.

[0119] In some embodiments, antibodies of the disclosure are soluble at concentrations of 10 mg / mL or greater, have low levels of soluble aggregates (<5%), maintain binding to the target (>90% efficacy) as measured by ELISA, and have no degradation products as measured by SDS PAGE when incubated for 3 months at 2-8° C. In some embodiments, toxicity analysis of antibodies of the disclosure can be performed in cynomolgus monkeys at doses more than 5-fold higher than the doses expected to be used in human subjects.

[0120] In some embodiments, antibodies that bind CD94 may deplete or reduce levels of NK cells and / or T cells expressing CD94 and may have clear benefit to patients (e.g., human patients) with diseases or disorders such as LGL leukemia, rheumatoid arthritis, Felty's syndrome, CLPD-NK, aggressive NK leukemia, IBM, IBD, or NK / T cell lymphoma, e.g., extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (unspecified type), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL. Furthermore, the antibody treatment may be better tolerated and have fewer side effects than first-line and second-line therapies, including chemotherapy such as cyclophosphamide, doxorubicin, vincristine, and prednisone; stem cell transplantation; splenectomy; alemtuzumab; bevacizumab; pralatrexate; avelumab; proteasome inhibitors such as carfilzomib and bortezomib; HDAC inhibitors such as romidepsin, belinostat, and vorinostat; and antibody-drug conjugates such as brentuximab vedotin. The antibody treatment may show more selective depletion of disease-inducing cells compared to current therapies, which are non-selective. Non-limiting examples of diseases and disorders in which NK cells and / or T cells expressing CD94 may play a role are LGL leukemia, rheumatoid arthritis, Felty's syndrome, CLPD-NK, aggressive NK leukemia, IBM, IBD, or NK / T cell lymphoma, such as extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL.Thus, the invention provides a method of reducing or depleting the number of IELs that express CD94 in a human subject upon administration of a molecule that binds to CD94 and comprises (a) a region that specifically binds to a target (e.g., CD94) and (b) an immunoglobulin Fc region.

[0121] II. Methods of Use and Treatment As discussed above, NK cells, CD4+ T cells, CD8+ T cells, and / or CD8+ and CD4+ T cells have been implicated in the pathogenesis of a number of diseases and disorders, including NK / T cell lymphomas. Many of these disorders or diseases are characterized by the clonal expansion of NK cells and / or CD8+ and CD4+ T cells.

[0122] In some embodiments, provided herein are molecules (e.g., antibodies) that bind to CD94 expressed, for example, on the surface of NK cells and / or T cells. Also provided herein are molecules (e.g., antibodies) that bind to CD94 and have reduced fucosylation, afucosylation (e.g., have an immunoglobulin Fc portion with a modification or mutation that reduces or eliminates fucosylation). Also provided herein are molecules (e.g., antibodies) that bind to CD94 and have a modification or mutation that enhances ADCC activity and / or improves affinity of the Fc region for an Fc receptor, such as CD16 (e.g., CD16a). Also provided herein are molecules (e.g., antibodies) that bind to CD94 and have one or more of the following properties: bind to human CD94 and cynomolgus CD94, do not block HLA-E binding to the CD94 / NKG2A heterodimer, have a low degree of internalization, are non-fucosylated or have reduced fucosylation, and / or induce, promote, or enhance ADCC activity.

[0123] In some embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an antibody described herein that specifically binds human CD94. In some embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an antibody that specifically binds human CD94, the antibody having one or more of the following properties: does not block binding of HLA-E to the CD94 / NKG2A heterodimer, has a low degree of internalization, is non-fucosylated or has reduced fucosylation, and / or induces, promotes, or enhances ADCC activity. In some embodiments, the disease or disorder is selected from chronic NK cell lymphoproliferative disorder (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, inflammatory bowel disease, or NK / T cell lymphoma, e.g., extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (unspecified type), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, subcutaneous panniculitis TCL, or microscopic colitis. In some embodiments, administration of the antibody results in a reduction in the number of NK cells and / or T cells expressing CD94. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood NK cells, CD8+, CD4+, or CD8+ / CD4+ T cells, and / or LGL cells in the subject, e.g., expressing CD94. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood LGL cells in the subject, e.g., expressing CD94. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood NK cells in the subject, e.g., expressing CD94. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood CD8+ / CD4+ T cells in the subject, e.g., expressing CD94. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood CD8+ T cells in the subject, e.g., expressing CD94.In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood CD4+ T cells in the subject that express, for example, CD94.

[0124] Also provided herein are methods for reducing the number of peripheral blood NK and / or T cells expressing CD94, comprising administering to a subject an effective amount of an antibody of the present disclosure that specifically binds human CD94. In some embodiments, the antibody has one or more of the following properties: does not block HLA-E binding to the CD94 / NKG2A heterodimer, has a low degree of internalization, is non-fucosylated or has reduced fucosylation, and / or induces, promotes, or enhances ADCC activity. In some embodiments, the subject has a disease or disorder selected from chronic NK cell lymphoproliferative disorder (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, inflammatory bowel disease, or NK / T cell lymphoma, e.g., extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (unspecified type), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, subcutaneous panniculitis TCL, or microscopic colitis. In some embodiments, administration of the antibody to a subject results in a reduction in the number of peripheral blood NK cells and / or T cells expressing CD94 compared to before administration of the antibody.

[0125] Also provided herein are methods for inducing ADCC activity in a subject, the methods comprising administering to the subject an effective amount of an antibody of the present disclosure that specifically binds human CD94. In some embodiments, the antibody has one or more of the following properties: does not block binding of HLA-E to the CD94 / NKG2A heterodimer, has a low degree of internalization, is non-fucosylated or has reduced fucosylation, and / or induces, enhances, or promotes ADCC activity. In some embodiments, the subject has a disease or disorder selected from chronic NK cell lymphoproliferative disorder (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, inflammatory bowel disease, or NK / T cell lymphoma, e.g., extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (unspecified type), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, subcutaneous panniculitis TCL, or microscopic colitis. In some embodiments, administration of the antibody to a subject results in a reduction in the number of peripheral blood NK cells and / or T cells expressing CD94 compared to before administration of the antibody.

[0126] In some embodiments, the disease or disorder is CLPD-NK. In some embodiments, the disease or disorder is LGL leukemia. In some embodiments, the disease or disorder is Felty syndrome. In some embodiments, the disease or disorder is rheumatoid arthritis. In some embodiments, the disease or disorder is aggressive NK leukemia. In some embodiments, the disease or disorder is inclusion body myositis. In some embodiments, the disease or disorder is inflammatory bowel disease. In some embodiments, the disease or disorder is T-cell large granular lymphocytic leukemia (T-LGLL). In some embodiments, the disease or disorder is natural killer cell large granular lymphocytic leukemia (NK-LGLL). In some embodiments, the disease or disorder is NK / T cell lymphoma. In some embodiments, the disease or disorder is extranodal NK / T cell lymphoma. In some embodiments, the disease or disorder is hepatosplenic T cell lymphoma (TCL). In some embodiments, the disease or disorder is enteropathy-associated TCL. In some embodiments, the disease or disorder is cutaneous TCL. In some embodiments, the disease or disorder is anaplastic large cell lymphoma (ALK+). In some embodiments, the disease or disorder is anaplastic large cell lymphoma (ALK-). In some embodiments, the disease or disorder is peripheral TCL (non-specific). In some embodiments, the disease or disorder is angioimmunoblastic TCL. In some embodiments, the disease or disorder is adult TCL. In some embodiments, the disease or disorder is monomorphic epitheliotropic enteric TCL. In some embodiments, the disease or disorder is epidermotropic CD8+ cutaneous TCL. In some embodiments, the disease or disorder is primary cutaneous gamma / delta TCL. In some embodiments, the disease or disorder is subcutaneous panniculitis TCL. In some embodiments, the disease or disorder is microscopic colitis.

[0127] Also provided herein is a method for treating CLPD-NK in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an antibody of the present disclosure, wherein the antibody specifically binds to human CD94. In some embodiments, administration of the antibody to the human subject results in amelioration of symptoms of CLPD-NK in the human.

[0128] Also provided herein is a method for treating NK / T cell lymphoma in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an antibody of the present disclosure, the antibody specifically binding to human CD94. In some embodiments, administering the antibody to the human subject results in amelioration of symptoms of NK / T cell lymphoma in the human. In some embodiments, the NK / T cell lymphoma is selected from extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (unspecified type), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic enteric TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL.

[0129] Also provided herein is a method for treating CLPD-NK in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an antibody of the present disclosure, which specifically binds to human CD94, and which has one or more of the following properties: does not block binding of HLA-E to the CD94 / NKG2A heterodimer, has a low degree of internalization, is non-fucosylated or has reduced fucosylation, and / or induces, enhances, or promotes ADCC activity. In some embodiments, administration of the antibody to a human subject results in amelioration of CLPD-NK symptoms in the human.

[0130] Also provided herein is a method for treating NK / T cell lymphoma in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an antibody of the present disclosure, which specifically binds to human CD94, and which has one or more of the following properties: does not block binding of HLA-E to the CD94 / NKG2A heterodimer, has a low degree of internalization, is non-fucosylated or has reduced fucosylation, and / or induces, enhances, or promotes ADCC activity. In some embodiments, administration of the antibody to a human subject results in amelioration of symptoms of NK / T cell lymphoma in the human. In some embodiments, the NK / T cell lymphoma is selected from extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic enteric TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL. In some embodiments, the NK cell lymphoma or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL. In some embodiments, the NK cell lymphoma or T cell lymphoma is characterized by NK cells and / or T cells expressing CD94 (e.g., human CD94).

[0131] Also provided herein is a method for treating microscopic colitis in a human subject in need thereof, comprising administering to the human subject an effective amount of an antibody of the present disclosure, which specifically binds to human CD94, and which has one or more of the following properties: does not block HLA-E binding to CD94 / NKG2A heterodimer, has low internalization, is non-fucosylated or has reduced fucosylation, and / or induces, enhances, or promotes ADCC activity. In some embodiments, administering the antibody to a human subject results in the amelioration of symptoms of microscopic colitis in the human. Microscopic colitis is a gastrointestinal disease characterized by inflammation of the colon that leads to persistent non-bloody watery diarrhea. It is called microscopic because tissue destruction can only be seen under a microscope, not by macroscopic examination. The colon in these patients appears macroscopically normal or has a near-normal colonic mucosa. There are currently two subtypes of microscopic colitis: collagenous colitis, characterized by the accumulation of a collagen layer in the intestinal lining, and lymphocytic colitis, characterized by an increase in lymphocytes in colonic tissue. It currently affects 100 / 100,000 individuals worldwide, of which 39.3% of cases are of the lymphocytic subtype (Hisamatsu et al. (2016) Inflamm. Intest. Dis. 2:52-62). The current cause of microscopic colitis is unknown, but common factors include medications, bacterial and viral infections, autoimmune diseases such as rheumatoid arthritis, celiac disease, or psoriasis, and accumulation of bile acids. The current method for diagnosis is histological examination of intestinal tissue, as the disease cannot be diagnosed without histopathological examination of biopsy specimens. Common symptoms of microscopic colitis include persistent watery diarrhea resulting in weight loss, bloating, anemia, malnutrition, etc. There is no cure or adequate treatment for microscopic colitis, except for antidiarrheal medications, a low-fat, low-fiber, and low-dairy diet, steroids, bile acid blockers, anti-inflammatory drugs, TNF inhibitors, or, in the rarest of circumstances, surgery to remove part or all of the colon.Although it is not clear what the primary pathogenic cells are in microscopic colitis, studies have shown that there is an increase in CD8+ intraepithelial lymphocytes (IELs) in the colon of patients with microscopic colitis (Goranzon et al. (2013) J. Crohns Colitis 7:e434-442). There is evidence in the literature suggesting that CD94 is highly expressed on IELs in microscopic colitis (Barmeyer et al. (2016) Inflamm. Bowel Dis. 22:539-547). Without wishing to be bound by theory, it is believed that anti-CD94 (e.g., non-fucosylated IgG1 antibodies) can perform ADCC against IEL cells via sibling killing. The literature also suggests that CD16 is highly elevated in biopsies of microscopic colitis versus normal controls, suggesting that IELs may bind anti-CD94 and induce ADCC against other IEL cells (Barmeyer et al. (2016) Inflamm. Bowel Dis. 22:539-547). Without wishing to be bound by theory, it is believed that IEL cells are cytotoxic and can act as effector cells as well as target cells.

[0132] Also provided herein is a method for enhancing chimeric antigen receptor T cell (CAR-T) therapy in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an antibody of the present disclosure, the antibody specifically binds to human CD94, the antibody having one or more of the following properties: does not block HLA-E binding to CD94 / NKG2A heterodimer, has a low degree of internalization, is non-fucosylated or has reduced fucosylation, and / or induces, enhances, or promotes ADCC activity. In some embodiments, administration of the antibody to the human subject results in depletion of NK cells in the subject prior to administration of CAR-T treatment. In some embodiments, the CAR-T therapy is for the treatment of cancer, i.e., the subject has / is being treated for cancer. The short half-life of CAR-T cells for the treatment of various hematological and solid cancers significantly impedes the development of cell therapy. Lymphocyte-depleting chemotherapy such as cyclophosphamide with or without fludarabine (Flu) is commonly used prior to CAR-T treatment. However, these standard chemotherapies do not adequately deplete NK cells, resulting in the elimination of off-the-shelf CAR-T cells shortly after infusion. Without wishing to be bound by theory, anti-CD94 antibody treatment could potentially transiently deplete NK cells, thereby allowing engraftment of HLA class I-deficient cells ("universal CAR T cells") or organs and preventing NK cell-mediated rejection. Transient depletion of NK cells would provide a window for engraftment of HLA class I-negative cells and should not pose a safety risk. After the CD94-depleting antibody disappears, NK cell numbers can return to normal. Newly generated NK cells should tolerate the transferred HLA class I-negative cells (based on mixed bone marrow chimera experiments). Thus, anti-CD94 antibody treatment may provide an opportunity to enhance CAR-T therapy, for example, in cancer patients.

[0133] In some embodiments, the terms treat, treating, treating, ameliorate, ameliorating, reducing one or more symptoms, reducing symptoms, reducing one or more symptoms, reducing symptoms, and other grammatical equivalents refer to alleviating, relieving, or improving one or more symptoms of a disease or disorder, preventing further symptoms, improving or preventing the underlying cause of a symptom, inhibiting a disease or disorder, e.g., preventing the onset of a disease or disorder, relieving a disease or disorder, causing regression of a disease or disorder, reducing pathology caused by a disease or disorder, or arresting a symptom of a disease or disorder, and are intended to include prophylaxis. In some embodiments, these terms further include achieving a therapeutic benefit and / or a prophylactic benefit. In some embodiments, a therapeutic benefit refers to eradication or improvement of the underlying disease or disorder being treated. Therapeutic benefit is also achieved by eradicating or improving one or more of the physiological symptoms associated with the underlying disease or disorder, such that in some embodiments, the patient is still suffering from the underlying disease or disorder, but the patient is observed to improve. With regard to prophylactic benefit, the pharmaceutical composition is administered to a patient who is at risk of developing a particular disease or disorder, or who reports one or more of the physiological symptoms of the disease or disorder, even if the disease or disorder has not been diagnosed.

[0134] In some embodiments, an effective amount, therapeutically effective amount, or pharma- ceutical effective amount may be a sufficient quantity of at least one pharmaceutical composition or compound (e.g., an antibody of the present disclosure) being administered that will relieve to some extent one or more of the symptoms of the disease or condition being treated.

[0135] In some embodiments, the reduction in the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject occurs within the first 24 hours, e.g., within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours, after administration of the antibody to the subject.

[0136] In some embodiments, the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject (e.g., in a peripheral blood sample obtained from the subject) is reduced to below the lower limit for clinical diagnosis of a disease or disorder. In some embodiments, the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject (e.g., in a peripheral blood sample obtained from the subject) is reduced to below 2×10 9 cells / L or less. See, e.g., Lamy, T. et al. (2017) Blood 129:1082-1094. In some embodiments, the reduction in the number of peripheral blood NK cells and / or T cells expressing CD94 in the subject to below the lower limit of clinical diagnosis of the disease or disorder is present in the subject for at least about 1 week, e.g., at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or longer, after administration of the antibody to the subject. In some embodiments, the number of peripheral blood NK cells and / or T cells expressing CD94 in the subject is 2×10 in the subject (e.g., in a peripheral blood sample obtained from the subject). 9The reduction to cells / L or less is present in the subject for at least about 1 week, e.g., at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or longer, after administration of the antibody to the subject.

[0137] In some embodiments, the number of peripheral blood NK cells and / or T cells expressing CD94 in the subject is reduced to below the limit of detection of peripheral blood NK cells and / or T cells expressing CD94 in the subject. In some embodiments, the reduction in the number of peripheral blood NK cells and / or T cells expressing CD94 in the subject to below the limit of detection of peripheral blood NK cells and / or T cells expressing CD94 exists in the subject for at least about one week, e.g., at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, or longer, following administration of the antibody to the subject. In some embodiments, peripheral blood NK cells and / or T cells expressing CD94 are detected by flow cytometry (e.g., performed on a peripheral blood sample from the subject).

[0138] In some embodiments, peripheral blood NK cells that express CD94 are CD3+ CD56 bright. In some embodiments, peripheral blood T cells that express CD94 are CD4+, CD8+, or CD8+ and CD4+.

[0139] In some embodiments, the reduction in the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject is reversible, hi some embodiments, the reduction in the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject is reversible within any of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or longer, following administration of the antibody to the subject.

[0140] In some embodiments, a statement that a cell or cell population is positive (+) for or expresses a particular marker (e.g., CD3, CD4, CD8, CD16, CD94, NKG2A, etc.) refers to the detectable presence of the particular marker on or within the cell. In some embodiments, a statement that a cell or cell population is positive (+) for or expresses a surface marker (e.g., a cell surface protein) refers to the presence of cell surface expression of the particular marker, as detected, for example, by flow cytometry, for example, by staining with and detection of an antibody that specifically binds to the marker, where the staining is detectable by flow cytometry at a level substantially greater than that detected using isotype-matched controls and / or fluorescence minus one (FMO) gating controls, performing the same procedure under otherwise identical conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially greater than that of cells known to be negative for the marker.

[0141] In some embodiments, a statement that a cell or cell population is negative (-) for or does not express a particular marker (e.g., CD3, CD4, CD8, CD16, CD94, NKG2A, etc.) refers to the absence of detectable presence of the particular marker on or within the cell. In some embodiments, a statement that a cell or cell population is negative (-) for or does not express a surface marker (e.g., a cell surface protein) refers to the absence of cell surface expression of the particular marker, as detected, for example, by flow cytometry, for example, by staining with and detection of an antibody that specifically binds to the marker, where the staining is detectable by flow cytometry at a level substantially similar to or below that detected using isotype-matched controls and / or fluorescence minus one (FMO) gating controls, performing the same procedure under otherwise identical conditions, and / or at a level substantially similar to or below that of cells known to be positive for the marker, and / or at a level substantially similar to or below that of cells known to be negative for the marker.

[0142] In some embodiments, the antibody has an EC50 or IC50 of about 1 ng / ml to about 100 ng / ml, e.g., about 1 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about 100 ng / ml, for reducing peripheral blood NK cells and / or T cells expressing CD94 in a subject. In some embodiments, the antibody has an IC50 or EC50 of about 10 ng / ml to about 80 ng / ml for reducing peripheral blood NK cells and / or T cells expressing CD94 in a subject. In some embodiments, the antibody has an IC50 or EC50 of about 20 ng / ml for reducing peripheral blood NK cells and / or T cells expressing CD94 in a subject. In some embodiments, the antibody has an IC50 or EC50 of about 60 ng / ml for reducing peripheral blood NK cells and / or T cells expressing CD94 in a subject. The EC50 or IC50 may be measured using any method known in the art, for example, as described in the Examples.

[0143] In some embodiments, the subject is a human, a primate, a non-human primate (e.g., African green monkeys, cynomolgus monkeys, rhesus monkeys, etc.), a domestic mammal, a game mammal, or a captive mammal. In some embodiments, the subject is a human. In some embodiments, the human subject is an infant, a toddler, a child, a young adult, an adult, or a geriatric individual. In some embodiments, the subject has a disease involving T cells and / or NK cells expressing CD94, e.g., CLPD-NK, LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, inflammatory bowel disease, or NK / T cell lymphoma, e.g., extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic enteric TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL.

[0144] In some embodiments, administration of the antibody to a subject does not result in tumor lysis syndrome in the subject. Tumor lysis syndrome may be measured or diagnosed according to any method known in the art, such as the Cairo-Bishop classification system for tumor lysis syndrome (see, e.g., Cairo and Bishop (2004) Br J Haematol, 127(1):3-11).

[0145] In some embodiments, the antibodies of the disclosure bind to CD94. In some embodiments, the antibodies of the disclosure deplete and / or reduce the levels of NK cells and / or T cells that express CD94. In some embodiments, the antibodies of the disclosure have a clear benefit to patients (e.g., human patients) with a disease or disorder such as CLPD-NK, LGL leukemia, rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, IBD, or NK / T cell lymphoma, e.g., extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (unspecified type), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, subcutaneous panniculitis TCL, or other diseases associated with LGL, T cells, and / or NK cells. In some embodiments, the antibodies of the disclosure are used in the treatment of diseases or disorders (e.g., CLPD-NK, CLPD-NK+ ... It is better tolerated and has fewer side effects than first- and second-line therapy for LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, inflammatory bowel disease, or NK / T-cell lymphoma, e.g., extranodal NK / T-cell lymphoma, hepatosplenic T-cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, or adult TCL).In some embodiments, the antibodies of the disclosure exhibit more selective depletion of disease-inducing cells, e.g., NK cells and / or T cells expressing CD94, compared to current therapies that are non-selective, such as chemotherapy, e.g., cyclophosphamide, doxorubicin, vincristine, prednisone; stem cell transplantation; splenectomy; alemtuzumab; bevacizumab; pralatrexate; avelumab; proteasome inhibitors such as carfilzomib and bortezomib; HDAC inhibitors such as romidepsin, belinostat, and vorinostat; and antibody-drug conjugates such as brentuximab vedotin. Thus, in some embodiments, the present disclosure provides methods of reducing the number of or depleting NK cells and / or T cells that express CD94 in a human subject upon administration of a molecule (e.g., an antibody of the present disclosure) that binds to a cell surface protein on NK cells and / or T cells, such as CD94, and comprises (a) a region that specifically binds to a target and (b) an immunoglobulin Fc region.

[0146] In some embodiments, the methods provided herein further include administering to the subject an IL-2 polypeptide, e.g., a therapeutic IL-2 polypeptide. Suitable IL-2 polypeptides, e.g., therapeutic IL-2 polypeptides, for administration to a subject (e.g., a subject having a disease or disorder described herein) according to the methods provided herein are known in the art. Exemplary IL-2 polypeptides include, but are not limited to, Aldesleukin, Interking, and Neoleukin 2 / 15.

[0147] A. Administration and Dosing Regimens (i) Route of Administration In some embodiments, administer, administering, administration, and the like refer to methods used to enable delivery of a therapeutic or pharmaceutical composition to a desired site of biological action. In some embodiments, the antibodies of the present disclosure (and any additional therapeutic agents) for use in any of the methods provided herein may be administered to a subject (e.g., a human) by any suitable means, including parenteral, intrapulmonary, intranasal, and intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibodies of the present disclosure are administered by intravenous infusion. Dosing of the antibodies of the present disclosure can be by any suitable route, for example, injections, such as intravenous or subcutaneous injections, depending in part on whether administration is acute or chronic.

[0148] (ii) Medication regimen The antibodies of the present disclosure for use in any of the methods provided herein may be administered to a subject using a variety of dosing schedules or regimens, including, but not limited to, single or multiple doses over various time points, bolus doses, and pulse infusions. The specific dosage of the antibodies of the present disclosure to be administered will vary according to the particular target specificity, the type of disease or disorder, the subject, and the nature and severity of the disease, the physical condition of the subject, the treatment regimen (e.g., whether a concomitant therapeutic agent is used), and the route of administration selected. In some embodiments, the dose of the antibodies of the present disclosure may range from about 0.0001 mg / kg to 100 mg / kg based on the subject's body weight. A representative dosing regimen of the antibodies of the present disclosure involves administration of the antibodies in multiple doses over an extended period of time, for example, at least six months.

[0149] Other known antibodies against CD94 may also be used in the methods provided herein. For example, the following anti-CD94 antibodies: HP-3D9 (LSBio Catalog No. LS-C134679-100; Abnova Catalog No.: MAB6947); 2I2; 131412 (R&D Systems Catalog No.: MAB1058); 13B146 (USBiological Catalog No.: 030068); 13B147 (USBiological Catalog No.: 030069); 1H1 (Abnova Catalog No.: MAB10543); 3G2 (Biorbyt Catalog No.: orb69389); DX22 (Biolegend Catalog No.: 305502); REA113 (Miltenyi Biotec catalog number: 130-098-967); KP43; EPR21003; AT13E3 (ATGen catalog: ATGA0487); HP-3B1; 12K45; and B-D49 may be used.

[0150] B. Disease There are nine distinct diseases involving NK / T cell lymphoma: extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (non-specified type), angioimmunoblastic TCL, and adult TCL. See, e.g., Bajaj, 2019. NK / T cell lymphoma affects various organs, such as the skin, GI, liver, spleen, and bone marrow. Symptoms of NK / T cell lymphoma include lymphadenopathy in the neck. Advantageously, the methods described herein may be used to reduce the number of abnormal or pathological NK cells and / or T cells (e.g., CD4+ T cells, CD8+ T cells, CD4+ and CD8+ T cells) expressing CD94, for example, via mechanisms such as ADCC using NK cells, which essentially uses the pathological cells to eliminate each other.

[0151] The methods described herein may also be used to reduce the number of abnormal or pathological NK and / or T cells, for example, that express CD94 and are associated with NK / T cell lymphoma, e.g., extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (unspecified type), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL, through mechanisms such as ADCC using NK cells, which essentially uses the pathological cells to eliminate each other.

[0152] There are three distinct diseases in which LGL is involved: T-cell LGL (T-LGL) leukemia, chronic NK cell lymphoproliferative disorder (CLPD-NK, formerly known as NK-LGL), and aggressive NK cell leukemias such as aggressive natural killer leukemia (ANKL) and extranodal NKL, nasal type (ENKL).

[0153] In addition to NK leukemia or T LGL leukemia, NK cells or LGL cells play a major role in rheumatoid arthritis (RA), Felty's syndrome, aggressive NK leukemia, inclusion body myositis (IBM), inflammatory bowel disease (IBD), and other diseases. Non-limiting examples of diseases and disorders in which LGL cells and NK cells play a role include LGL leukemia, rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, and IBD. Advantageously, the methods described herein may be used to reduce the number of abnormal or pathological NK cells (e.g., CLPD-NK, ANKL, or ENKL cells), for example, through mechanisms such as ADCC using NK cells, which essentially uses pathological cells to eliminate each other. For representative descriptions of the symptoms of these diseases, see, e.g., Lamy, et al, Blood, 2017xVol. 129, No. 9; Loughran Blood, VOI 82, NO 1 (July 1), 1993: pp 1-14; Semenzato G, et al, Blood. 1997; 89(1): 256-260; and Bourgault-Rouxel, et al, Leuk Res. 2008; 32(1): 45-48.

[0154] (i)CLPD-NK Chronic NK-cell lymphoproliferative disorder (CLPD-NK), also known as NK-LGL leukemia, chronic NK-cell lymphocytosis, chronic NK-LGL lymphoproliferative disorder (LPD), NK-cell lineage granular lymphoproliferative disorder, NK-cell LGL lymphocytosis, or indolent granular NK-cell LPD, is generally characterized by a sustained (e.g., 6 months or more) increase in peripheral blood NK cells (e.g., ≥ 2 × 10 9 / L).

[0155] Symptoms of CLPD-NK include variable cytopenias such as neutropenia and anemia, fatigue, fever, night sweats, recurrent infections, rheumatoid arthritis, lymphadenopathy, hepatosplenomegaly, skin lesions, hematologic neoplasms, vasculitis, neuropathy, and autoimmune disorders.

[0156] In some embodiments of the methods provided herein, the disease or disorder is CLPD-NK, and administration of the antibody results in a reduction in one or more symptoms of CLPD-NK in the subject. In some embodiments, a reduction in the number of peripheral blood LGL cells and / or NK cells in the subject after administration of the antibody results in a reduction in one or more symptoms of CLPD-NK in the subject.

[0157] CLPD-NK symptoms may be measured by any method known in the art, such as using laboratory tests to measure anemia, neutropenia, complete blood count, and / or magnetic resonance imaging (MRI), CT scan, palpation, or ultrasound examination (e.g., to determine hepatosplenomegaly), bone marrow examination, and flow cytometry. Methods for measuring CLPD-NK symptoms are described, for example, in Swerdlow, SH et al. (2016) Blood 127:2375-2390.

[0158] (ii) LGL leukemia Large granular lymphocytic (LGL) leukemia is a chronic lymphoproliferative disorder that shows a chronic increase in large granular lymphocytes (LGL) in the peripheral blood, and is referred to as T-cell LGL leukemia.

[0159] Symptoms of LGL leukemia include splenomegaly, B symptoms (e.g., generalized symptoms such as fever, night sweats, and weight loss), anemia, neutropenia, and recurrent infections. Rheumatoid arthritis is also often seen in people with T-cell LGL leukemia.

[0160] In some embodiments of the methods provided herein, the disease or disorder is LGL leukemia, and administration of the antibody results in a reduction in one or more symptoms of LGL leukemia in the subject. In some embodiments, a reduction in the number of peripheral blood LGL cells and / or NK cells in the subject after administration of the antibody results in a reduction in one or more symptoms of LGL leukemia in the subject.

[0161] Symptoms of LGL leukemia may be measured by any method known in the art, such as by laboratory tests to measure anemia, neutropenia, and other cytopenias, complete blood counts, magnetic resonance imaging (MRI), CT scans, palpation, or ultrasound examination (e.g., to determine splenomegaly), bone marrow examination, and flow cytometry. Methods for measuring symptoms of LGL leukemia are described, for example, in Swerdlow, SH et al. (2016) Blood 127:2375-2390.

[0162] (iii) Felty syndrome Felty's syndrome is an autoimmune disease characterized by rheumatoid arthritis, splenomegaly (e.g., inflammatory splenomegaly), and reduced numbers of neutrophils in the blood. Symptoms of Felty's syndrome include painful, stiff, and / or swollen joints, physical findings associated with rheumatoid arthritis, splenomegaly, neutropenia, infections, keratoconjunctivitis sicca, fever, weight loss, fatigue, skin discoloration, erosions (e.g., ulcers), hepatomegaly, anemia, thrombocytopenia, liver dysfunction, lymphadenopathy, and vasculitis.

[0163] In some embodiments of the methods provided herein, the disease or disorder is Felty syndrome, and administration of the antibody results in a reduction in one or more symptoms of Felty syndrome in the subject. In some embodiments, a reduction in the number of peripheral blood LGL cells and / or NK cells in the subject after administration of the antibody results in a reduction in one or more symptoms of Felty syndrome in the subject. Symptoms of Felty syndrome include, but are not limited to, joint inflammation, joint pain, and splenomegaly.

[0164] Symptoms of Felty's syndrome may be measured by any method known in the art, such as laboratory tests to measure anemia, neutropenia, thrombocytopenia, and other cytopenias, complete blood count, magnetic resonance imaging (MRI), CT scan, or ultrasound (e.g., to determine splenomegaly and / or hepatomegaly), laboratory tests for liver function abnormalities, palpation to determine splenomegaly and / or hepatomegaly, flow cytometry, Disease Activity Score-28 (DAS-28, e.g., used to monitor symptoms of rheumatoid arthritis), and using the DAS-28 with erythrocyte sedimentation rate (ESR).

[0165] (iv) Rheumatoid arthritis Rheumatoid arthritis is an autoimmune disorder that primarily affects the joints, but can also affect other organs, and may be associated with cardiovascular disease, osteoporosis, interstitial lung disease, infections, cancer, fatigue, and depression. Symptoms of rheumatoid arthritis include swollen, tender, and hot joints, joint inflammation, joint pain, joint stiffness, splenomegaly, rheumatoid nodules (e.g., in the skin), necrotizing granulomas, vasculitis, pyoderma gangrenosum, Sweet's syndrome, drug reactions, erythema nodosum, lobular panniculitis, atrophic skin of the fingers, palmar erythema, fragile skin, diffuse alopecia areata, pulmonary fibrosis, Kaplan's syndrome, exudative pleural effusion, atherosclerosis, myocardial infarction, stroke, pericarditis, endocarditis, left ventricle, and pulmonary fibrosis. These include ventricular heart failure, valvular inflammation, cardiac and / or vascular fibrosis, anemia, elevated platelet count, decreased white blood cell count, renal amyloidosis, episcleritis, scleritis, keratoconjunctivitis sicca, keratitis, decreased vision, liver damage, peripheral neuropathy, mononeuritis multiplex, carpal tunnel syndrome, myelopathy, atlantoaxial subluxation, spondylolisthesis, fatigue, low-grade fever, malaise, morning stiffness, loss of appetite, weight loss, osteoporosis, cancer (e.g., lymphoma, skin cancer), and periodontitis.

[0166] In some embodiments of the methods provided herein, the disease or disorder is rheumatoid arthritis, and administration of the antibody results in a reduction in one or more symptoms of rheumatoid arthritis in the subject. In some embodiments, a reduction in the number of peripheral blood LGL cells and / or NK cells in the subject after administration of the antibody results in a reduction in one or more symptoms of rheumatoid arthritis in the subject.

[0167] In some embodiments, rheumatoid arthritis symptoms and disease state / progression are measured according to the 2010 ACR / EULAR rheumatoid arthritis classification criteria (see, e.g., Aletaha et al., (2010) Annals of Rheumatic Diseases, 69(9):1580-8). Rheumatoid arthritis symptoms may also be measured by any method known in the art, such as using erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, anti-citrullinated protein antibodies, anemia and other cytopenias, elevated platelet count, decreased white blood cell count, complete blood count, laboratory tests to measure renal amyloidosis, medical imaging such as x-ray, MRI, CT scan, ultrasound (e.g., ultrasound using high frequency transducers; Doppler ultrasound), flow cytometry, Disease Activity Score-28 (DAS-28), and DAS-28 using erythrocyte sedimentation rate (ESR).

[0168] (v) Aggressive NK leukemia Aggressive NK cell leukemia is an aggressive disease with a systemic proliferation of NK cells and a rapidly debilitating clinical course. Aggressive NK cell leukemia may also be referred to as aggressive NK cell lymphoma. Symptoms of aggressive NK cell leukemia include systemic symptoms (e.g., malaise, weight loss, fatigue), hepatosplenomegaly, lymphadenopathy, coagulopathy, hemophagocytic syndrome, multiple organ failure, infections such as Epstein-Barr virus, necrosis, and allergic reactions (e.g., to insect bites such as mosquito bites) that may result in systemic symptoms such as fever, lymphadenopathy, abdominal pain, diarrhea, and anaphylaxis.

[0169] In some embodiments of the methods provided herein, the disease or disorder is aggressive NK cell leukemia, and administration of the antibody results in a reduction in one or more symptoms of aggressive NK cell leukemia in the subject. In some embodiments, a reduction in the number of peripheral blood LGL cells and / or NK cells in the subject after administration of the antibody results in a reduction in one or more symptoms of aggressive NK cell leukemia in the subject.

[0170] Symptoms of aggressive NK leukemia may be measured by any method known in the art, such as, for example, using laboratory tests to measure anemia, neutropenia, and other cytopenias, complete blood counts, and / or magnetic resonance imaging (MRI), CT scans, palpation, or ultrasound examinations (e.g., to determine splenomegaly), bone marrow examinations, and flow cytometry. Methods for measuring symptoms of aggressive NK leukemia are described, for example, in Swerdlow, SH et al. (2016) Blood 127:2375-2390.

[0171] (vi) Inclusion body myositis Inclusion body myositis (IBM), also known as sporadic inclusion body myositis, is an inflammatory muscle disease characterized by autoimmune and degenerative processes that result in progressive weakness and wasting of distal and / or proximal muscles. Generally, IBM is characterized by infiltration of immune cells into muscle tissue. In some cases, patients with IBM have elevated blood creatine kinase levels. Symptoms of IBM include progressive muscle weakness, muscle wasting / atrophy, frequent tripping and falling, difficulty manipulating fingers, foot drop, limited movement, impaired balance, muscle pain, difficulty swallowing, and fatigue.

[0172] In some embodiments of the methods provided herein, the disease or disorder is IBM, and administration of the antibody results in a reduction in one or more symptoms of IBM in the subject. In some embodiments, a reduction in the number of peripheral blood LGL cells and / or NK cells in the subject after administration of the antibody results in a reduction in one or more symptoms of IBM in the subject.

[0173] Symptoms of IBM may be measured by any method known in the art, such as muscle biopsy, blood tests (e.g., to measure creatine kinase), electromyography (EMG) tests, blood tests to measure antibodies to NT5C1A, flow cytometry, and myositis disease activity assessment tools, including but not limited to the Myositis Intention to Treat Activity Index (MITAX) and Myositis Disease Activity Assessment Visual Analogue Scales (MYOACT).

[0174] (vii) Inflammatory bowel disease Inflammatory bowel disease (IBD) refers to a class of inflammatory conditions of the colon and small intestine. Types of IBD include ulcerative colitis and Crohn's disease. Symptoms of IBD include diarrhea, fever, fatigue, abdominal pain, abdominal cramps, bloody stools, loss of appetite, and weight loss.

[0175] In some embodiments of the methods provided herein, the disease or disorder is IBD, and administration of the antibody results in a reduction in one or more symptoms of IBD in the subject. In some embodiments, a reduction in the number of peripheral blood LGL cells and / or NK cells in the subject after administration of the antibody results in a reduction in one or more symptoms of IBD in the subject.

[0176] Symptoms of IBD may be measured by any method known in the art, such as clinical blood tests for anemia, other cytopenias, or infections, fecal occult blood tests, colonoscopy, flexible sigmoidoscopy, upper endoscopy, capsule endoscopy, balloon enteroscopy, X-ray, CT scan, MRI scan, ultrasound, and flow cytometry.

[0177] III. Pharmaceutical Formulations In some embodiments, a pharmaceutical composition, composition, or pharmaceutical formulation refers to a biologically active compound (e.g., an antibody of the present disclosure) optionally mixed with at least one pharma- ceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, and the like.

[0178] Pharmaceutical compositions, pharmaceutical formulations, and / or compositions of any of the antibodies of the present disclosure for use in any of the methods described herein may be prepared in the form of a lyophilized formulation or aqueous solution by mixing such antibodies having the desired purity with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0179] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmacopoeitic carriers include, but are not limited to, peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoeitic carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain representative sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0180] The formulations herein may also contain more than one active ingredients as necessary for the particular indication (e.g., disease or disorder) being treated, preferably active ingredients with complementary activities that do not adversely affect each other.

[0181] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0182] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0183] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0184] IV. KITS AND ARTICLES In another aspect of the disclosure, a kit or article of manufacture is provided that contains materials useful for the methods provided herein, such as treating the above-mentioned diseases or disorders, reducing the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject, or inducing ADCC activity in a subject. The kit or article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective, by itself or in combination with another composition, for the methods provided herein, such as treating the above-mentioned diseases or disorders, reducing the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject, or inducing ADCC activity in a subject, and the container may have a sterile access port (e.g., the container may be an intravenous infusion bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the disclosure. The label or package insert indicates that the composition is used for the methods provided herein, e.g., treating a disease or disorder as described above, reducing the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject, or inducing ADCC activity in a subject. Additionally, the kit or article of manufacture may include (a) a first container in which the composition is housed, the composition comprising an antibody of the present disclosure, and (b) a second container in which the composition is housed, the composition comprising an additional therapeutic agent. The kit or article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition may be used, e.g., to treat a particular disease or disorder as described herein, to reduce the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject, or to induce ADCC activity in a subject. Alternatively or additionally, the kit or article of manufacture may further include a second (or third) container comprising a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, or dextrose solution.It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0185] The following description is presented to enable those skilled in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described and shown herein, but should be accorded the scope consistent with the claims. EXAMPLES

[0186] Example 1: Generation and evaluation of anti-CD94 antibodies This example describes the generation and characterization of antibodies specific for human CD94.

[0187] Materials and Methods Generation and screening of anti-CD94 antibodies Four-week-old ATX-Gx Alloy transgenic mice (human antibody producing mice) were subcutaneously immunized with C-terminal His-tagged CD94 for 5 weeks with weekly antigen boosts. Antibody titers in mouse serum were assessed via ELISA and flow cytometry before and after boosts. Mice with the highest serum antibody titers were selected to provide B cells for hybridoma generation.

[0188] Prior to cell fusion, mice were given one additional boost of CD94-His antigen. Mice were then sacrificed and their spleens harvested. Spleen cells and SP2 / 0-Ag14 myeloma cells were mixed, and then fusion was induced by incubation at 37°C in the presence of polyethylene glycol (PEG) or by electroporation. Cells were then harvested and plated in 96-well plates using limiting dilution to achieve one cell per well. Cells were then treated with hypoxanthine, aminopterin, and thymidine (HAT) medium and selected in culture for 2 weeks.

[0189] To identify candidates specific for CD94, hybridoma supernatants were screened using ELISA and flow cytometry. For ELISA, CD94-His antigen was immobilized on a plate and 100 μl of each supernatant was incubated with the antigen. A fluorescently labeled secondary antibody was used to detect the antibodies captured on the ELISA plate, and positive hits were verified by analysis of antibody binding on human primary NK cells using flow cytometry. Cynomolgus CD94 cross-reactivity was assessed by antibody binding to cynomolgus-CD94 expressing BaF3 cells using flow cytometry.

[0190] Verification of the VH and VL sequences was performed using standard RNA extraction of the hybridomas followed by reverse transcription of the RNA to cDNA and PCR using Alloy ATC-Gx specific primers.

[0191] Healthy donor and patient samples Fresh healthy donor buffy coats were obtained from the Stanford Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated via separation with ficoll-paque (GE Healthcare, Chicago, IL) and cryopreserved in Bambanker cell freezing medium (Bulldog-Bio, Portsmouth, NH). Briefly, buffy coats were diluted 1:1 in phosphate-buffered saline (PBS), followed by layering the diluted buffy coats and centrifuging at 760 g in ficoll. The PBMC layer was isolated and washed in PBS prior to downstream analysis. Peripheral blood leukocytes (PBLs) were isolated by red blood cell lysis.

[0192] Antibody affinity assays PBMCs from healthy donors were seeded in 96-well plates at a density of 100,000 cells per well and incubated with human Fc block (Biolegend) and cell viability dye (Thermo Fisher) for 30 minutes on ice protected from light. Cells were washed once with FACS buffer (PBS with 2% low IgG FBS). Anti-CD94 antibodies at concentrations of 100 nM to 0.046 nM in a 1:3 dilution were incubated with cells for 30 minutes on ice protected from light. Cells were then washed and incubated with goat IgG anti-mouse Fcy-specific AF647 secondary antibody or goat (Fab)2 fragment anti-human Fcy-specific AF647 secondary antibody (Jackson immuno research), anti-CD3 Pacific Blue-labeled antibody, and anti-CD56 FITC-labeled antibody (Biolegend) for 30 minutes on ice protected from light. After incubation, cells were given a final wash in FACS buffer before quantification on a Cytoflex (Beckman Coulter). All data acquisition and fluorescence compensation was performed using CytoFlex (Beckman Coulter, Atlanta, GA). Data analysis was performed using FlowJo software. NK cells were identified by gating on lymphocytes based on forward and side scatter, followed by exclusion of doublets and dead cells, and gating on the CD3-CD56+ population. CD94 expression was then quantified in the CD3-CD56+ NK cell population. Antibody titration curves and EC50s were generated using Graphpad Prism.

[0193] Cynomolgus CD94 cross-reactivity assay of antibodies Cynomolgus CD94-expressing HEK293 cells were seeded in 96-well plates at a density of 100,000 cells per well and incubated with cell viability dye (Thermo Fisher) on ice for 30 min protected from light. Cells were washed once with FACS buffer (PBS with 2% low IgG FBS). Hybridoma supernatants, anti-CD94 antibodies, and isotype controls were incubated with cells on ice for 30 min protected from light. Cells were then washed and incubated with goat IgG anti-mouse Fcy-specific AF647 secondary antibody or goat (Fab)2 fragment anti-human Fcy-specific AF647 secondary antibody (Jackson ImmunoResearch) on ice for 30 min protected from light. After incubation, cells were subjected to a final wash in FACS buffer before quantification with a Cytoflex (Beckman Coulter). All data acquisition and fluorescence compensation were performed using a CytoFlex (Beckman Coulter, Atlanta, GA). Data analysis was performed using FlowJo software. HEK293 cells were identified by gating on the major cell populations using forward and side scatter, followed by exclusion of doublets and dead cells. CD94 expression was then quantified in this population. MFI bar graphs were generated using Graphpad Prism.

[0194] HLA-E blocking assay PBMCs from healthy donors were seeded in 96-well plates at a density of 100,000 cells per well and incubated with human Fc block (Biolegend) and cell viability dye (Thermo Fisher) for 30 min on ice protected from light. Cells were washed once with FACS buffer (PBS with 2% low IgG FBS). Anti-CD94 antibody at an EC80 concentration was incubated with cells for 30 min on ice protected from light. After incubation with anti-CD94 antibody, cells were washed and incubated with HLA-E tetramer PE (Creative Biolabs), anti-CD3 Pacific Blue labeled antibody, and anti-CD56 FITC labeled antibody (Biolegend) for 30 min on ice protected from light. Cells were then subjected to a final wash in FACS buffer before quantification on a Cytoflex (Beckman Coulter). All data acquisition and fluorescence compensation was performed using CytoFlex (Beckman Coulter, Atlanta, GA). Data analysis was performed using FlowJo software. NK cells were identified by gating lymphocytes based on forward and side scatter, followed by exclusion of doublets and dead cells, and gating on the CD3-CD56+ population. HLA-E expression was then quantified in the CD3-CD56+ NK cell population. Percent blocking was calculated as 100-((percent HLA-E positive for anti-CD94 antibody) / (percent HLA-E positive for isotype) x 100).

[0195] Antibody competition assay PBMCs from healthy donors were seeded in 96-well plates at a density of 100,000 cells per well and incubated with human Fc block (Biolegend) and cell viability dye (Thermo Fisher) for 30 min on ice protected from light. Cells were washed once with FACS buffer (PBS with 2% low IgG FBS). EC80 concentrations of unconjugated anti-CD94, CD3 FITC and CD56 PE antibodies, as well as APC-labeled anti-CD94 were incubated with cells for 30 min on ice protected from light. Cells were then subjected to a final wash in FACS buffer before quantification on a Cytoflex (Beckman Coulter). All data acquisition and fluorescence compensation were performed using CytoFlex (Beckman Coulter, Atlanta, GA). Data analysis was performed using FlowJo software. NK cells were identified by gating on lymphocytes based on forward and side scatter, followed by excluding doublets and dead cells, and gating on the CD3-CD56+ population. Anti-CD94 APCs were then quantified in the CD3-CD56+ NK cell population. All titration curves and EC50s were generated using Graphpad Prism.

[0196] Antibody internalization assay PBMCs from healthy donors were seeded in 96-well plates at a density of 100,000 cells per well in RPMI with 10% low IgG FBS and incubated with human Fc block (Biolegend) for 10 min at room temperature. Unconjugated anti-CD94 antibodies at EC80 concentrations were incubated with the cells for 30 min to 24 h at 4°C and 37°C. Cells were washed once with FACS buffer (PBS with 2% low IgG FBS) and kept on ice for the rest of the procedure. Goat anti-mouse Fcy-specific antibodies were incubated with the cells on ice for 30 min protected from light. Cells were then washed once and CD94 expression was quantified with a Cytoflex. All data acquisition and fluorescence compensation was performed using a CytoFlex (Beckman Coulter, Atlanta, GA). Data analysis was performed using FlowJo software. NK cells were identified by gating on lymphocytes based on forward and side scatter, followed by exclusion of doublets and dead cells, and gating on the CD3-CD56+ population. Anti-CD94 APCs were then quantified in the CD3-CD56+ NK cell population. The percent reduction in MFI was calculated by calculating the difference in MFI between 0.5 and 24 hours at 37°C and multiplying by 100.

[0197] Antibody-dependent cellular cytotoxicity assay Approximately 1×10 5 ~2×10 5 Fresh or frozen PBMCs were plated in tissue culture treated U-bottom 96-well plates in RPMI with 10% low IgG FBS. Cells were plated at 10:10 dilutions. 1 ~10 -6Cells were incubated overnight in ug / ml of human IgG1 isotype control antibody or 1E4 fucosylated and 18H3-KIF antibody. Cells were then stained with fluorescently labeled antibodies against CD3, CD56, and CD16 to identify remaining NK cells (see Flow Cytometry Analysis). A minimum of 10,000 events were collected on the flow cytometer in the lymphocyte population. The remaining NK / leukemia cell percentage was calculated by normalizing the absolute counts by the cell number under isotype treatment conditions. IC50 was determined using GraphPad Prism.

[0198] result Anti-CD94 antibodies were generated using standard hybridoma techniques as described above. Briefly, ATX-Gx Alloy transgenic mice were immunized with C-terminal His-tagged CD94 for 5 weeks. B cells from the mouse with the highest serum antibody titer after 5 weeks were used to generate hybridomas. Hybridomas were screened by ELISA to identify candidates specific for CD94.

[0199] Positive hits were verified by assessing binding to human NK cells by flow cytometry analysis. Binding of anti-CD94 antibody clones to primary human NK cells was assessed by flow cytometry analysis. Supernatants from anti-CD94 hybridomas were used to test binding of anti-CD94 antibody clones. Commercially available anti-CD94 antibody HP-3D9, and several IgG isotype controls were also tested for binding on NK cells.

[0200] The affinity of the antibody clones was also assessed by flow cytometry analysis. Figure 1 shows the titration curve generated for the anti-CD94 antibody clone 18H3. The anti-CD94 antibody 18H3 showed an affinity of 2.6 nM on human primary NK cells. The affinities determined for other anti-CD94 antibodies are listed in Figure 11. As shown in Figure 2, screening of hybridoma supernatants revealed that 18H3, 1M4, and 1E4 bind to human primary NK cells as measured by flow cytometry.

[0201] Anti-CD94 hybridoma supernatants were also tested for cross-reactivity to cynomolgus CD94. Hybridomas were screened for cross-reactivity to cynomolgus CD94 by flow cytometry using cynomolgus CD94-expressing HEK293 cells. As shown in Figure 3A, this analysis revealed that clones 18H3, 1M4, and 1E4 cross-reacted with cynomolgus CD94. Supernatant from clone 20F2 was reactive to human CD94 but did not cross-react with cynomolgus CD94. Cross-reactivity of commercially available anti-CD94 antibodies was also evaluated. As shown in Figure 3B, none of the commercially available anti-CD94 antibodies tested (HP-3D9, HP-3B1, 131412, 12K45, DX22) showed cross-reactivity to cynomolgus CD94.

[0202] These results indicate that the newly generated anti-CD94 antibodies bind to epitopes that are not shared with the HP-3D9, HP-3B1, 131412, 12K45, DX22, and 20F2 (non-commercial) antibodies. The cross-reactivity of the 18H3, 1M4, and 1E4 antibodies suggests that they may be useful in cynomolgus monkey toxicity studies prior to Phase I trials in human subjects.

[0203] Following validation, the VH and VL sequences were determined for the validated antibody clones. Table A summarizes the VH and VL sequences for the anti-CD94 clones used in Examples 1-3. Framework and CDR sequences (bold in Table A) were determined using the Kabat numbering scheme. [Table 3]

[0204] The validated anti-CD94 antibodies were further evaluated for their ability (or lack thereof) to block binding by HLA-E. HLA-E is the ligand for the CD94 / NKG2A heterodimer and plays an essential role in the inhibition of NK and CD8+ T cell activity when bound to CD94 / NKG2A. Without wishing to be bound by theory, it is believed that the interaction between HLA-E and CD94 / NKG2A may lead to the activation and proliferation of target cells. Therefore, in a diseased environment, it would be beneficial for a candidate anti-CD94 antibody not to block CD94:HLA-E interaction.

[0205] HLA-E blocking by anti-CD94 antibody clones and commercial anti-CD94 antibodies was evaluated by flow cytometry. As shown in Figure 4, 18H3 and 1E4 antibodies did not block HLA-E binding. More than 40% HLA-E blocking was observed for commercial anti-CD94 antibodies, whereas 0% blocking was observed for our anti-CD94 antibodies. Since CD94 expression levels vary from dim (approximately 10,000 receptors) to bright (>100,000 receptors), we propose that if an antibody blocks more than 20% of HLA-E binding, it can be considered a ligand blocking antibody. Overall, this HLA-E blocking assay demonstrates that 18H3 and 1E4 antibodies do not block the interaction of CD94 with HLA-E, whereas commercial CD94 antibodies do block this interaction.

[0206] To determine whether the newly identified antibodies bind to epitopes shared with themselves and existing antibodies, a competition assay was performed. Anti-CD94 antibodies 18H3, 1M4, and 1E4 were assayed for competition with each other and with commercially available antibodies. As shown in Figures 5A-5B, the 18H3 antibody binds to a unique epitope. 18H3 only partially competes with HP-3D9, and does not compete with DX22, HP-3B1, 131412, 12K45, 1E4, and 1M4 antibodies. Thus, the 18H3 antibody binds to an epitope that is not shared with commercially available antibodies.

[0207] Similarly, the 1M4 antibody did not compete with commercially available antibodies. As shown in Figures 6A-6B, 1M4 competed with the 1E4 antibody, but did not compete with DX22, 131412, HP-3D9, 12K45, and HP-3B1.

[0208] The 1E4 antibody did not compete with four of the five commercially available anti-CD94 antibodies tested. As shown in Figure 7, 1E4 partially competed with 12K45, but did not compete with HP-3D9, DX22, 131412, and HP-3B1.

[0209] An antibody internalization assay was performed to assess the internalization of the CD94 receptor when bound by an anti-CD94 antibody. In this assay, a cutoff of more than 50% is considered a high degree of internalization. Without wishing to be bound by theory, it is believed that a low level of internalization would be beneficial for antibody-dependent cellular cytotoxicity (ADCC) because the CD94 receptor would be retained on the cell surface and would maintain a high receptor density for effector cells to carry out ADCC. The results, illustrated in Figures 8A-8B, show that the CD94 receptor was internalized when bound to a commercial anti-CD94 antibody, but not when bound to the 18H3, 1M4, and 1E4 antibodies. The 18H3, 1M4, and 1E4 antibodies were not internalized more than 50% upon binding to CD94. In contrast, percentage reductions in MFI of 54%, 56%, 24%, 32%, and 18% were observed for HP-3D9, DX22, 18H3, 1M4, and 1E4, respectively, at 0.5 h compared to 24 h at 37°C.

[0210] To test the ability of anti-CD94 antibodies to induce ADCC of NK cells, an ADDC assay was performed using PBMCs from healthy donors. In this assay, the 18H3 antibody was generated in Expi-CHO cells cultured in the presence of kifunensine, a potent inhibitor of the mannosidase I enzyme, to generate 18H3-KIF, which mimics the non-fucosylated antibody. The fucosylated 1E4 antibody was also tested for ADCC. As shown in Figures 9A-9B, partially non-fucosylated human IgG1 18H3 and fucosylated 1E4 depleted human NK cells in a pool of PBMCs from a healthy donor in ex vivo culture after 24 hours of incubation. The depletion of NK cells by human IgG1 18H3 and 1E4 was concentration-dependent. The IC50 of 18H3-KIF was determined to be 0.02 μg / ml, while for the 1E4 antibody, the IC50 was undetermined.

[0211] Finally, the ability of the 18H3 antibody to induce ADCC of human leukemia cells was evaluated by ADCC assay using cells from patients with chronic NK cell lymphoproliferative disorder (CLPD-NK). In this assay, the 18H3 antibody was produced in Expi-CHO cells cultured in the presence of kifunensine to generate 18H3-KIF, which mimics non-fucosylated antibodies. As shown in Figure 10, the partially non-fucosylated human IgG1 18H3 depleted human CLPD-NK leukemia cells in a pool of PBMCs from CLPD-NK patients in ex vivo culture after 24 hours of incubation. The 18H3 antibody depleted human CLPD-NK leukemia cells in a concentration-dependent manner with an IC50 of 0.059 μg / ml. This depletion was selective, as other cell types were not affected.

[0212] FIG. 11 summarizes the properties and functional assessment of the anti-CD94 antibodies compared to commercial antibodies as described above.

[0213] Example 2: Characterization of anti-CD94 antibodies in in vivo mouse studies This example describes the characterization of antibodies specific for human CD94 using in vivo mouse studies.

[0214] Materials and Methods In vivo studies in humanized IL-15 transgenic mice Humanized NSG™-IL-15 transgenic mice were obtained from Jackson Lab. Mice were engrafted with PBMCs from healthy and LGLL donors for 3 and 28 days, respectively. Mice were injected with a single dose of human IgG1 isotype control or ATX-130 (5 mg / kg) and depletion of normal NK cells, normal CD8 T cells, and CD8 T leukemia cells in blood, spleen, bone marrow, and liver was assessed by flow cytometry 48 hours after dosing (Figure 12A).

[0215] result The ability (or lack thereof) of anti-CD94 antibodies to deplete human NK cells and CD8 T cells was assessed in IL-15 transgenic mice (NSG™-IL-15). Humanized NSG™-IL-15 transgenic mice are a mouse strain with a complete deletion of the mouse IL-2R gamma gene resulting in a defective adaptive and innate immune system. Expression of human IL-15 allows efficient engraftment of human immune cells, especially cytotoxic immune cells, from healthy donors and leukemia patients. As shown in FIG. 12B, a complete depletion of NK cells was observed with ATX-130 treatment (5 mg / kg) compared to isotype control. A partial depletion (approximately 50%) of CD8 T cells was observed. The remaining CD8 T cells were not depleted because they were CD94 negative. Overall, ATX-130 depletes CD94+ cells, including all human NK cells, and 50% of CD8 T cells.

[0216] The ability (or lack thereof) of ATX-130-KIF to deplete LGLL in blood, spleen, bone marrow, and liver was assessed in IL-15 transgenic mice (NSG™-IL-15). As shown in FIG. 13, more than 50% depletion of LGLL cells was observed with ATX-130 treatment (5 mg / kg) compared to isotype control. The remaining LGLL cells were CD94 negative and therefore not depleted. Overall, anti-CD94 antibodies could efficiently deplete CD94+ LGLL cells in mice in vivo. Example 3: Characterization of anti-CD94 antibodies in non-human primates

[0217] This example describes exploratory pharmacodynamics (PD) studies in cynomolgus monkeys to evaluate the efficacy of anti-CD94 antibody treatment in vivo.

[0218] Materials and Methods Exploratory studies in non-human primates in vivo Two healthy male cynomolgus monkeys, 6.5 and 9.2 years old and weighing 6.54 and 7.18 kg, respectively, were selected for the study, naive to all compounds and with no medical history prior to the start of the study. As shown in Figure 14A, two doses of ATX-130-KIF (2 mg / kg) were administered to cynomolgus monkey number 1 via intravenous (IV) infusion at a rate of approximately 0.25 mL / min over 60 minutes at weeks 0 and 8. Two doses of ATX-130-KIF (2 mg / kg) were administered to cynomolgus monkey number 2 via IV infusion at a rate of approximately 0.25 mL / min over 60 minutes at weeks 0 and 16. Blood, serum, and PBMC were collected at various time intervals before and after dosing, as shown in Figure 14A. PD was determined via depletion of target cells by flow cytometry analysis. The study endpoint was planned to be 22 weeks.

[0219] result To evaluate the efficacy of ATX-130-KIF in vivo, a PD study was completed in non-human primates (NHPs). Two healthy male cynomolgus monkeys (cynomolgus monkey #1 and cynomolgus monkey #2), aged 6.5 and 9.2 years and weighing 6.54 and 7.18 kg, respectively, were selected for the study, naive to all compounds and with no medical history prior to study initiation. Pharmacodynamic (PD) activity was determined via target cell depletion by flow cytometry analysis. All bioanalytical data are presented through 18 weeks, with a planned study endpoint of 22 weeks.

[0220] The ability (or lack thereof) of ATX-130-KIF to deplete cynomolgus NK cells in blood was assessed by flow cytometry analysis. As shown in FIG. 14B, the composition of NK cells in total PBMCs in cynomolgus monkey #1 (left panel) and cynomolgus monkey #2 (right panel) was 20% and 25%, respectively, at baseline. For each dose, depletion of target cells (NK cells) to <5% of PBMCs was detected in both animals 6 hours after administration of ATX-130-KIF. In cynomolgus monkey #1, a steady increase in NK cell percentage was observed from 6 hours to 336 hours, and then stabilized at 50% of baseline until the administration of the second dose at 1008 hours. Stabilization of NK cell numbers at 50% of baseline was observed 168 hours after the second dose. In cynomolgus monkey #2, NK cell recovery was slower than in cynomolgus monkey #1. NK cell numbers remained at low levels for 6 weeks following dose 1 and increased from weeks 8 to 14. Administration of ATX-130-KIF at week 16 reduced NK cell levels 6 hours after dosing and remained low until the final sample collection. Overall, the current results indicate that anti-CD94 antibodies can demonstrate efficacy in vivo in cynomolgus monkeys.

[0221] The ability (or lack thereof) of ATX-130-KIF to deplete blood cynomolgus CD4 T cells, CD8 T cells, and B cells was assessed by flow cytometry analysis. As shown in FIG. 15A, the composition of CD4+ T cells, CD8+ T cells, and B cells among total PBMCs in cynomolgus monkey #1 was 27%, 40%, and 4%, respectively, at baseline. As shown in FIG. 15B, the composition of CD4+ T cells, CD8+ T cells, and B cells among total PBMCs in cynomolgus monkey #2 was 26%, 40%, and 5%, respectively, at baseline. T cell and B cell depletion was detected in both animals 6 hours after administration of ATX-130-KIF. However, these immune cell populations returned to baseline levels 24 hours after dosing. The results from this study indicate that anti-CD94 antibodies only transiently deplete other immune cells without depleting non-CD94 expressing cells.

[0222] Cynomolgus monkey monocyte depletion was assessed by flow cytometry. As shown in Figure 16, an increase in monocytes was observed 6 hours after dosing, but returned to baseline levels 7 days later. The results from this study show that ATX-130-KIF transiently increases monocytes, suggesting that anti-CD94 antibody can bind to monocytes and perform ADCC, contributing to the targeted depletion of NK cells.

[0223] CD16 expression on cynomolgus monkey monocytes was assessed by flow cytometry. As shown in Figure 17, an increase in CD16 was observed 24 hours after dosing, but returned to baseline levels 7 days later. The results from this study showed that ATX-130-KIF transiently increased CD16 on monocytes, suggesting that anti-CD94 antibody binds to monocytes to perform ADCC and contribute to targeted depletion of NK cells.

[0224] Example 4: Characterization of anti-CD94 antibody variants Variants of the anti-CD94 antibody ATX-130 were generated and tested for binding to human CD94. The VH and VL domains of each variant antibody are shown in Table B. The sequences of these variable domains are provided in Table 2. [Table 4]

[0225] A Fab version of each antibody was generated and the binding of each Fab to human CD94 protein was assessed by the OCTET® Biolayer Interferometry (BLI) system. The results are shown in Table C. The variants showed similar binding to human CD94 compared to the original ATX-130 antibody, suggesting that the mutations introduced into the variant variable domains do not significantly affect binding (i.e., off-rates). [Table 5]

[0226] Binding of the variant antibodies to HEK cells expressing human CD94 was also tested. The variant and ATX-130 antibodies were produced in human IgG1 format and binding to HEK cells expressing human CD94 was assessed using flow cytometry. Experiments were performed in duplicate and the average EC50 of the two runs was calculated. Results are shown in Table D. Eleven of the thirteen variants showed similar binding (as measured by EC50) to cells expressing human CD94 compared to the original ATX-130 antibody, while two of the thirteen variants (Ab.23 and Ab.30) lost binding to cells expressing human CD94. [Table 6]

[0227] The present disclosure has been described in some detail by way of illustrative illustrations and examples for purposes of clarity of understanding, but these illustrations and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. An antibody that binds to human CD94, the antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; The VH domain has the amino acid sequence EVQLX １ ESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISX ２ SSX ３ X ４ IYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGRYYYYMDVWGKGTTVTVSS (in the sequence, X １ is L or V, and in the sequence ２ is T or S, and in the sequence, X ３ is N or S, and in the sequence, X ４ is F or Y) (SEQ ID NO:69), The VL domain has the amino acid sequence DIX １ MTQSPSSLSASVGDRVTITCRASQSISSWLAWX ２ QQKPX ３ K.X. ４ P.K.X. ５ LIYAASSLQSGVPSX ６ FSGSGSGTDFTLISSLQPEDX ７ ATYYCQX ８ YNSX ９ PFTFGPGTKVDIK (in the sequence, X １ is V or Q, and in the sequence ２ is Y or F, and in the sequence ３ is G or E, and in the sequence, X ４ is A or V, and in the sequence ５ is S or L, and in the sequence ６ is K or R, and in the sequence ７ is V or F, and in the sequence ８ is K or Q, and in the sequence ９ is A or Y) (SEQ ID NO:70), The antibody does not comprise a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 1), a CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO: 2), a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 3), a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6).

2. An antibody that binds to human CD94, the antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; the VH domain comprises a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:1); a CDR-H2 comprising an amino acid sequence selected from the group consisting of SISTSSNFIYYADSVKG (SEQ ID NO:2), SISSSSNFIYYADSVKG (SEQ ID NO:18), SISTSSSFIYYADSVKG (SEQ ID NO:21), and SISTSSNYIYYADSVKG (SEQ ID NO:24); and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:3); the VL domain comprises a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4); a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:5); and a CDR-L3 comprising an amino acid sequence selected from the group consisting of QKYNSAPFT (SEQ ID NO:6), QQYNSAPFT (SEQ ID NO:27), and QKYNSYPFT (SEQ ID NO:28); The antibody does not comprise a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 1), a CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO: 2), a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 3), a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6).

3. The VH domain is (a) a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 1), a CDR-H2 comprising the amino acid sequence SISTSSNFIYYADSVKG (SEQ ID NO: 2), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 3); (b) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 1), CDR-H2 comprising the amino acid sequence SISSSSNFIYYADSVKG (SEQ ID NO: 18), and CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 3); (c) a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 1), a CDR-H2 comprising the amino acid sequence SISTSSSFIYYADSVKG (SEQ ID NO: 21), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 3); or (d) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO: 1), CDR-H2 comprising the amino acid sequence SISTSSNYIYYADSVKG (SEQ ID NO: 24), and CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO: 3); The antibody of claim 1 or claim 2, comprising:

4. The VH domain (a) FR1 comprising an amino acid sequence selected from the group consisting of EVQLLESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 48) and EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 49); (b) FR2 comprising the amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO:50); (c) FR3 comprising the amino acid sequence of RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:51); and (d) FR4 comprising the amino acid sequence of WGKGTTVTVSS (SEQ ID NO:52); The antibody of any one of claims 1 to 3, further comprising:

5. The antibody of any one of claims 1 to 4, wherein the VH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 29 to 32.

6. The VL domain is (a) a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QKYNSAPFT (SEQ ID NO: 6); (b) a CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence QQYNSAPFT (SEQ ID NO: 27); or (c) CDR-L1 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO: 4), CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5), and CDR-L3 comprising the amino acid sequence QKYNSYPFT (SEQ ID NO: 28); The antibody according to any one of claims 1 to 5, comprising:

7. The VL domain is (a) FR1 comprising an amino acid sequence selected from the group consisting of DIVMTQSPSSLSASVGDRVTITC (SEQ ID NO: 53) and DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 54); (b) FR2 comprising an amino acid sequence selected from the group consisting of WYQQKPGKAPKSLIY (SEQ ID NO:55), WFQQKPGKAPKSLIY (SEQ ID NO:56), WYQQKPEKAPKSLIY (SEQ ID NO:57), WYQQKPGKVPKSLIY (SEQ ID NO:58), and WYQQKPGKAPKLLIY (SEQ ID NO:59); (c) a FR3 comprising an amino acid sequence selected from the group consisting of GVPSKFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 60), GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 61), and GVPSKFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 62); and (d) FR4 comprising the amino acid sequence of FGPGTKVDIK (SEQ ID NO:63); The antibody of any one of claims 1 to 6, further comprising:

8. The antibody of any one of claims 1 to 7, wherein the VL domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 33 to 41.

9. (a) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 33; (b) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 34; (c) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; (d) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; (e) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 37; (f) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 38; (g) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 39; (h) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 40; (i) the VH domain comprises the amino acid sequence of SEQ ID NO: 16 and the VL domain comprises the amino acid sequence of SEQ ID NO: 41; (j) the VH domain comprises the amino acid sequence of SEQ ID NO: 29 and the VL domain comprises the amino acid sequence of SEQ ID NO: 17; (k) the VH domain comprises the amino acid sequence of SEQ ID NO: 29 and the VL domain comprises the amino acid sequence of SEQ ID NO: 33; (l) the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:34; (m) the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:35; (n) the VH domain comprises the amino acid sequence of SEQ ID NO: 29 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; (o) the VH domain comprises the amino acid sequence of SEQ ID NO: 29 and the VL domain comprises the amino acid sequence of SEQ ID NO: 37; (p) the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:38; (q) the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:39; (r) the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:40; (s) the VH domain comprises the amino acid sequence of SEQ ID NO:29 and the VL domain comprises the amino acid sequence of SEQ ID NO:41; (t) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 17; (u) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 33; (v) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 34; (w) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; (x) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; (y) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 37; (z) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 38; (aa) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 39; (bb) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 40; (cc) the VH domain comprises the amino acid sequence of SEQ ID NO: 30 and the VL domain comprises the amino acid sequence of SEQ ID NO: 41; (dd) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 17; (ee) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 33; (ff) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 34; (gg) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; (hh) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; (ii) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 37; (jj) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 38; (kk) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 39; (ll) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 40; (mm) the VH domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL domain comprises the amino acid sequence of SEQ ID NO: 41; (nn) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 17; (oo) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 33; (pp) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 34; (qq) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; (rr) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; (ss) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 37; (tt) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 38; (uu) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 39; (vv) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 40; or (ww) the VH domain comprises the amino acid sequence of SEQ ID NO: 32 and the VL domain comprises the amino acid sequence of SEQ ID NO: 41; The antibody according to claim 1 or claim 2.

10. The antibody of any one of claims 1 to 9, wherein the antibody is an antigen-binding antibody fragment or a single chain antibody.

11. The antibody of any one of claims 1 to 9, wherein the antibody further comprises an Fc region.

12. The antibody of claim 11 , wherein the Fc region is a human IgG1 Fc region.

13. The antibody of claim 11 or claim 12, wherein the antibody comprises a non-fucosylated human Fc region.

14. The antibody of any one of claims 11 to 13, wherein the antibody binds to human cellular Fc gamma receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region.

15. The antibody according to any one of claims 11 to 14, which is capable of inducing antibody-dependent cellular cytotoxicity (ADCC) against cells expressing human CD94 on their surface.

16. A polynucleotide encoding the antibody according to any one of claims 1 to 15.

17. A vector comprising the polynucleotide of claim 16.

18. The vector of claim 17 , wherein the vector is an expression vector.

19. 19. An isolated host cell comprising a polynucleotide according to claim 16 or a vector according to claim 17 or claim 18.

20. 20. A method for producing an antibody, comprising culturing a host cell according to claim 19 under conditions suitable for the production of said antibody.

21. 21. The method of claim 20, further comprising recovering the antibody from the host cell.

22. A pharmaceutical composition comprising the antibody of any one of claims 1 to 15 and a pharma- ceutically acceptable carrier.

23. A method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an antibody of any one of claims 1 to 15 or a composition of claim 22.

24. 24. The method of claim 23, wherein administration of the antibody results in a reduction in the number of peripheral blood LGL cells or NK cells in the subject.

25. 24. The method of claim 23, wherein the disease or disorder is Felty Syndrome and administration of the antibody to the subject results in a reduction of one or more symptoms of Felty Syndrome in the subject.

26. 24. The method of claim 23, wherein the disease or disorder is inclusion body myositis and administration of the antibody to the subject results in a reduction of one or more symptoms of inclusion body myositis in the subject.

27. 24. The method of claim 23, wherein the disease or disorder is aggressive NK leukemia and administration of the antibody to the subject results in a reduction of one or more symptoms of aggressive NK leukemia in the subject.

28. 24. The method of claim 23, wherein the disease or disorder is rheumatoid arthritis and administration of the antibody to the subject results in a reduction of one or more symptoms of rheumatoid arthritis in the subject.

29. 24. The method of claim 23, wherein the disease or disorder is LGL leukemia and administration of the antibody to the subject results in a reduction of one or more symptoms of LGL leukemia in the subject.

30. 24. The method of claim 23, wherein the disease or disorder is CLPD-NK and administration of the antibody to the subject results in a reduction of one or more symptoms of CLPD-NK in the subject.

31. 24. The method of claim 23, wherein the disease or disorder is natural killer (NK) cell lymphoma or T cell lymphoma, and administration of the antibody to the subject results in a reduction of one or more symptoms of lymphoma in the subject.

32. 32. The method of claim 31 , wherein the NK cell lymphoma or the T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma / delta TCL, or subcutaneous panniculitis TCL.

33. 33. The method of claim 32, wherein the NK cell lymphoma or the T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

34. 24. The method of claim 23, wherein the disease or disorder is microscopic colitis, and administration of the antibody to the subject results in a reduction of one or more symptoms of microscopic colitis in the subject.

35. A method for reducing the number of peripheral blood LGL cells and / or NK cells in a subject, comprising administering to the subject an effective amount of an antibody described in any one of claims 1 to 15 or a composition described in claim 22.

36. A method for inducing ADCC activity in a subject, the method comprising administering to the subject an effective amount of an antibody described in any one of claims 1 to 15 or a composition described in claim 22.

37. A method for treating CLPD-NK in a human subject in need of such treatment, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 15 or a composition according to claim 22.

38. The method of claim 37, wherein administration of the antibody results in amelioration of one or more symptoms of CLPD-NK in the subject.

39. 23. A method for enhancing chimeric antigen receptor T cell (CAR-T) therapy in a human subject in need thereof, comprising administering to the subject an effective amount of the antibody of any one of claims 1 to 15 or the composition of claim 22.

40. The method of claim 39, wherein said administration of said antibody or said composition results in depletion of NK cells in said subject prior to administration of said CAR-T treatment.

41. 23. A method for depleting CD8+ CD94+ T cells in a human subject in need of depletion of said CD8+ CD94+ T cells, comprising administering to said subject an effective amount of an antibody of any one of claims 1 to 15 or a composition of claim 22.

42. The method of any one of claims 23 to 41, further comprising administering to the subject an IL-2 polypeptide.

43. The method of any one of claims 23 to 42, wherein the subject is a human.

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