Anti-CD94 antibodies and methods of use thereof

Abstract

The present disclosure relates to antibodies that bind to human CD94, as well as methods, uses, polynucleotides, vectors, host cells, and pharmaceutical compositions related to the antibodies. In some embodiments, the antibodies are human antibodies or humanized antibodies that bind to human CD94 and do not block the binding between CD94 and HLA-E. In some embodiments, the antibodies cross-react with cynomolgus monkey CD94. In some embodiments, the antibodies do not promote internalization of surface expressed CD94 to the extent of existing antibodies. In some embodiments, the antibodies facilitate ADCC targeting, for example, cells expressing human CD94 on the cell surface.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 323,959, filed on March 25, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (186542000740SEQLIST.xml; size: 69,347 bytes; and creation date: March 20, 2023) are incorporated herein by reference in their entirety. Field of the Invention

[0005] The present disclosure relates to antibodies that bind to CD94 and the use of said antibodies for treating diseases and disorders associated with NK cells and / or T cells. Background of the Invention

[0007] Natural killer (NK) / T cell lymphoma and leukemia are characterized by clonal expansion of NK and / or CD8 + and CD4 + T cells. NK / T cell lymphoma accounts for a small part of non-Hodgkin's lymphoma (NHL) and has a 5-year survival rate of less than 50% (Kwong, 2012). There are about 4,000-7,000 new patients each 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 lymphoma can occur at any age, and more than half of NHL patients are 65 years old or older (Bajaj, 2019). There are also NK and T cell-based leukemias, such as LGL and aggressive NK leukemia. Other examples of diseases and disorders in which NK cells play a role include LGL leukemias (e.g., T-cell LGL leukemia), chronic lymphoproliferative disorder of NK cells (CLPD-NK, formerly known as NK-LGL), rheumatoid arthritis, Felty's syndrome, aggressive NK leukemias (e.g., aggressive natural killer leukemia (ANKL) and extranodal NKL nasal type (ENKL)), IBM, and IBD.

[0008] There are 13 different 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 γ / δ TCL, and subcutaneous panniculitis TCL (Bajaj, 2019). The main subtypes of NK / T cell lymphoma are mainly cytotoxic cell driven (NK / CD8+ T cells), including extranodal NK / T cell lymphoma, hepatosplenic TCL, enteropathy-associated TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous γ / δ TCL, and subcutaneous panniculitis TCL. NK / T cell lymphoma affects multiple organs, such as the skin, gastrointestinal (GI) tract, liver, spleen, and bone marrow. Symptoms include swollen lymph nodes in the neck. Most NK / T cell lymphoma subtypes are driven by Epstein bar virus infection.

[0009] Current treatment includes chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone) followed by stem cell transplantation; however, progression-free survival remains at 40-50%. Treatments currently under investigation include alemtuzumab, carfilzomib (proteasome inhibitor), romidepsin (HDAC inhibitor), bevacizumab, belentuzumab vedotin (antibody-drug conjugate), bortezomib (proteasome inhibitor), belinostat (HDAC inhibitor), pralatrexate, vorinostat (HDAC inhibitor), and avelumab. Despite the wide variety of drugs in development, many are often used off-label with mixed results.

[0010] There are currently no effective therapies for the treatment of NK / T-cell lymphoma, and no therapies that selectively target NK / T-cell lymphoma have been developed. Current therapies for NK / T-cell lymphoma can produce off-target effects and are not completely effective. The high disease mortality rate coupled with the lack of effective treatments underscores the need for treatment advances for NK / T-cell lymphoma.

[0011] Therefore, there is a need in the art to develop safe and effective therapies to treat diseases mediated by NK cells and / or T cells expressing CD94, such as NK / T cell lymphoma.

[0012] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the invention

[0013] To meet these and other needs, the present disclosure provides, among other things, antibodies that specifically bind to CD94 (e.g., human CD94), methods of treating diseases or injuries associated with NK cells and / or T cells that express CD94 (e.g., NK / T cell lymphomas and leukemias (e.g., LGL leukemias)), and methods of depleting or reducing the number of NK cells and / or T cells that express CD94 in a subject following administration of an antibody that specifically binds to CD94. These antibodies of the present disclosure may have one or more of the following characteristics: high affinity binding to human CD94 (e.g., cells that express human CD94 on the surface), cross-reactivity with cynomolgus CD94 (useful in preclinical studies), the ability to bind to human CD94 without blocking its interaction with HLA-E, minimal internalization after binding to CD94, and / or induction of ADCC against cells that express CD94 (such as leukemia cells). These characteristics are believed to be advantageous, for example, to test, develop, and use in the treatment of NK / T cell-based diseases such as lymphomas and leukemias. For example, without wishing to be bound by theory, it is believed that targeting CD94 on NK cells for ADCC can induce cancer cells (eg, LGL leukemia or lymphoma cells) to kill other cancer cells.

[0014] In some aspects, provided herein are antibodies that bind to (e.g., specifically bind to) human CD94, wherein the antibody comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises the amino acid sequence EVQLX1ESGGGLVKPGGS LRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISX2SSX3X4IYY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGRYY YYMDVWGKGTTVTVSS, wherein X1 is L or V; wherein X2 is T or S; wherein X3 is N or S; and wherein X4 is F or Y (SEQ ID NO: 69); wherein the VL domain comprises the amino acid sequence DIX1MTQSPSSLSASVGDRVTITCRASQS ISSWLAWX2QQKPX3KX4PKX5LIYAASSLQSGVPSX6FSGSGSGTDFTLTISSLQPEDX7ATYYCQX8YNSX9PFTFGPGTKVDIK, wherein X1 is V or Q; wherein X2 is Y or F; wherein X3 is G or E; wherein X4 is A or V; wherein X5 is S or L; wherein X6 is K or R; wherein X7 is V or F; wherein X8 is K or Q; and wherein X9 is A or Y (SEQ ID NO:70); and 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-H4 comprising the amino acid sequence RASQSISSWLA (SEQ ID NO:4). In some embodiments, the antibody comprises a CDR-L1 comprising the amino acid sequence AASSLQS (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.

[0015] In some aspects, provided herein are antibodies that bind to (e.g., specifically bind to) human CD94, wherein the antibody comprises 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), SISTSSSFIYY ADSVKG (SEQ ID NO: 21), and SISTSSNYIYYADSVKG (SEQ ID NO: 24); and a CDR-H3 comprising the amino acid sequence DLGR YYYYMDV (SEQ ID NO: 3); wherein 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 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 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 SISSSSNFIY YADSVKG (SEQ ID NO: 18), and a CDR-H3 comprising the amino acid sequence DLGR YYYYMDV (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 SISTSSSFIYYADSVK G (SEQ ID NO: 21), and a CDR-H3 comprising the amino acid sequence DLGRYYYYMD V (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) 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 an amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO:50); (c) FR3 comprising an amino acid sequence of RFTISRDNAKNSL YLQMNSLRAEDTAVYYCAR (SEQ ID NO:51); and (d) FR4 comprising an amino acid sequence of WGKGTTVTVSS (SEQ ID NO:52). In some embodiments, the VH domain further comprises: (a) FR1 comprising the amino acid sequence of EVQLLES GGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO:48); (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).In some embodiments, the VH domain further comprises: (a) a FR1 comprising the amino acid sequence of EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 49); (b) a FR2 comprising the amino acid sequence of WVRQAPGKG LEWVS (SEQ ID NO: 50); (c) a FR3 comprising the 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 antibody comprises a VL domain comprising a CDR-L1 comprising the amino acid sequence of RASQSISSWLA (SEQ ID NO: 4), a CDR-L2 comprising the amino acid sequence of AASSLQS (SEQ ID NO: 5), and a CDR-L3 comprising the amino acid sequence of 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 QQY NSAPFT (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 further comprises: (a) an FR1 comprising an amino acid sequence selected from the group consisting of DIVMTQSPSSLSASVGDRVTITC (SEQ ID NO: 53) and DIVMTQSPSSLSASVGDRVTITC (SEQ ID NO: 54); (b) an 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) an FR3 comprising an amino acid sequence selected from the group consisting of GVPSKFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 60), GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 61). and (d) an FR4 comprising an amino acid sequence of FGPGTKVDIK (SEQ ID NO: 63). In some embodiments, the antibody is a human antibody.

[0016] In some aspects, provided herein are antibodies that bind to (e.g., specifically bind to) human CD94, wherein the antibody comprises 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 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); wherein 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-H4 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). ID NO:28); and 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.

[0017] In some aspects, provided herein are antibodies that bind to (e.g., specifically bind to) human CD94, wherein the antibody comprises 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), SSSS NF (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); a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 5); and a CDR-H4 comprising an amino acid sequence selected from the group consisting of QKYNSAPFT (SEQ ID NO: 6), QQYNSAPFT (SEQ ID NO: 7), and QQYNSAPFT (SEQ ID NO: 8). QKYNSYPFT (SEQ ID NO:27) and QKYNSYPFT (SEQ ID NO:28); and 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 DLG RYYYYMDV (SEQ ID NO: 15). In some embodiments, the antibody comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence GFTF SSY (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 QKYNSAP FT (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.

[0018] 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 NOs: 16 and 29-32 and / or the VL domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.

[0019] 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, such as 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) lacking an α-1,6-fucosyltransferase gene, such as FUT8. In some embodiments, the antibody binds to human cell Fcγ receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. In some embodiments, the antibody is capable of inducing antibody-dependent cellular toxicity (ADCC) against cells expressing human CD94 on the surface.

[0020] In other aspects, polynucleotides encoding antibodies according to any one of the above embodiments are provided herein. A vector (e.g., an expression vector) comprising a polynucleotide according to any one of the above embodiments is further provided herein. A host cell (e.g., a separated host cell or cell line) comprising a polynucleotide or a vector according to any one of the above embodiments is further provided herein. A method for producing an antibody is further provided herein, the method comprising cultivating 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 a host cell.

[0021] Further provided herein are compositions (eg, pharmaceutical compositions) comprising an antibody according to any of the above embodiments and a pharmaceutically acceptable carrier.

[0022] In other aspects, provided herein is a method for treating a disease or condition in a subject, 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, the administration of the antibody results in a decrease in the number of peripheral blood LGL or NK cells in the subject. In some embodiments, the disease or condition is Felty's syndrome, and wherein administering the antibody to the subject results in alleviation of one or more symptoms of Felty's syndrome in the subject. In some embodiments, the disease or condition is inclusion body myositis, and wherein administering the antibody to the subject results in alleviation of one or more symptoms of inclusion body myositis in the subject. In some embodiments, the disease or condition is aggressive NK leukemia, and wherein administering the antibody to the subject results in alleviation of one or more symptoms of aggressive NK leukemia in the subject. In some embodiments, the disease or condition is rheumatoid arthritis, and wherein administering the antibody to the subject results in alleviation of one or more symptoms of rheumatoid arthritis in the subject. In some embodiments, the disease or condition is LGL leukemia, and wherein administering the antibody to the subject results in alleviation of one or more symptoms of LGL leukemia in the subject. In some embodiments, the disease or condition is CLPD-NK, and wherein the antibody is administered to the subject resulting in one or more CLPD-NK symptoms of the subject being alleviated. In some embodiments, the disease or condition is a natural killer (NK) cell or T-cell lymphoma, and wherein the antibody is administered to the subject resulting in one or more lymphoma symptoms of the subject being alleviated. In some embodiments, the NK cell or T-cell lymphoma is an extranodal NK / T-cell lymphoma, hepatosplenic T-cell lymphoma (TCL), enteropathy-associated TCL, skin TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, adult TCL, monomorphic epithelial intestinal TCL, epidermotropic CD8+ skin TCL, primary skin γ / δTCL or subcutaneous membrane inflammation TCL. In some embodiments, the NK cell or T-cell lymphoma is an extranodal NK / T-cell lymphoma, hepatosplenic TCL or enteropathy-associated TCL. In some embodiments, the disease or disorder is microscopic colitis, and wherein administering the antibody to the subject results in a reduction in one or more symptoms of microscopic colitis in the subject.

[0023] In other aspects, provided herein is a method for reducing the number of peripheral blood LGL and / or NK cells of a subject, the method comprising administering to the subject an effective amount of an antibody or composition according to any one of the above embodiments. In other aspects, provided herein is a method for inducing ADCC activity of a subject, the method comprising administering to the subject an effective amount of an antibody or composition according to any one of the above embodiments. In other aspects, provided herein is a method for treating CLPD-NK of a human subject in need, 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, the administration of the antibody results in an improvement in one or more CLPD-NK symptoms of the subject.

[0024] In other aspects, provided herein is a method for treating natural killer (NK) cells or T cell lymphomas, the method comprising administering an effective amount of an antibody or composition according to any one of the above embodiments to a subject in need thereof. 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 an affinity greater than that of anti-CD94 antibody clones HP-3D9, HP-3B1, DX22, 131412 and 12K45. In some embodiments, the NK cell or T cell lymphoma is an extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-related TCL, skin TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL or adult TCL. In some embodiments, the NK cell or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

[0025] In other aspects, the present invention provides a method for enhancing chimeric antigen receptor T cell (CAR-T) therapy of a human subject in need, the method comprising administering an effective amount of an antibody or composition according to any one of the above embodiments to the subject, followed by administering CAR-T therapy to the subject. In some embodiments, the administration of the antibody or composition results in NK cell depletion of the subject before administering CAR-T therapy.

[0026] In other aspects, provided herein is a method for 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.

[0027] In some embodiments according to any of the embodiments described herein, the method further comprises administering an IL-2 polypeptide to the subject.

[0028] In some embodiments according to any of the embodiments described herein, the subject is a human.

[0029] In other aspects, antibodies or compositions according to any one of the above embodiments are provided herein for: treating a disease or disorder of a subject, reducing the number of peripheral blood LGL and / or NK cells of a subject, treating CLPD-NK of a human subject in need, treating natural killer (NK) cells or T-cell lymphomas, treating microscopic colitis in a subject, or enhancing CAR-T therapy in a subject in need. In some embodiments, the NK cell or T-cell lymphoma is an extranodal NK / T cell lymphoma, hepatosplenic T-cell lymphoma (TCL), enteropathy-associated TCL, skin TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, adult TCL, monomorphic epithelial intestinal TCL, epidermotropic CD8+ skin TCL, primary skin γ / δ TCL, or subcutaneous panniculitis TCL. In some embodiments, the NK cell or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

[0030] In other aspects, the present invention provides an antibody or composition according to any one of the above embodiments for the manufacture of a medicament, for example, for treating a disease or disorder of a subject, reducing the number of peripheral blood LGL and / or NK cells of a subject, treating CLPD-NK of a human subject in need, treating natural killer (NK) cells or T cell lymphomas, treating microscopic colitis in a subject, or enhancing CAR-T therapy for a subject in need. In some embodiments, the NK cell or T cell lymphoma is an extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, skin TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, or adult TCL. In some embodiments, the NK cell or T cell lymphoma is an extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

[0031] In other aspects, provided herein is a kit or article comprising an antibody or composition according to any one of the above embodiments. In some embodiments, the kit also includes instructions for using the kit, such as for treating a disease or disorder of a subject, reducing the number of peripheral blood LGL and / or NK cells of a subject, treating CLPD-NK of a human subject in need, treating natural killer (NK) cells or T-cell lymphomas, treating microscopic colitis in a subject, or enhancing CAR-T therapy for a subject in need. In some embodiments, the NK cell or T-cell lymphoma is an extranodal NK / T cell lymphoma, hepatosplenic T-cell lymphoma (TCL), enteropathy-associated TCL, skin TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL, angioimmunoblastic TCL, adult TCL, monomorphic epithelial intestinal TCL, epidermal CD8+ skin TCL, primary skin γ / δTCL or subcutaneous panniculitis TCL. In some embodiments, the NK cell or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL, or enteropathy-associated TCL.

[0032] It should be understood that one, some or all of the properties 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 become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The novel features of the present invention are particularly set forth 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 of illustrative embodiments, in which the principles of the invention are used, and in which the accompanying drawings are:

[0034] Figure 1 The affinity of anti-CD94 antibody 18H3 to human primary natural killer (NK) cells as measured by flow cytometry is shown. Peripheral blood mononuclear cells (PBMC) of healthy donor HD-40 were used for antibody staining. The 18H3 antibody was titrated from 100nM to 0.046nM at a dilution of 1:3 and incubated with PBMC. CD3 and CD56 antibodies were used to identify NK cells on flow scatter. The affinity of 18H3 was assessed using the 18H3 antibody binding on CD3+ and CD56 bright NK cells. Titration curves and EC50 were generated using Graphpad Prism. 18H3 binds to CD3+CD56 bright NK cells with an affinity of 2.6nM. Human IgG1 isotype control (hIgG1) with secondary antibody and only goat (Fab) 2 fragment anti-human Fcy specific secondary antibody AF647 (secondary Ab) were used as controls.

[0035] Figure 2 The binding of anti-CD94 antibodies to human primary NK cells as measured by flow cytometry is shown. Anti-CD94 hybridoma supernatants were screened on human primary NK cells by flow cytometry. HP-3D9 is a commercial anti-CD94 antibody, which was used as a positive control. Mouse IgG1, IgG2a, IgG2b, and IgG3 were used as negative controls. Anti-CD94 antibodies 18H3, 1M4, and 1E4 bind to CD94 expressed on human primary NK cells.

[0036] Figure 3A-3B Cross-reactivity of anti-CD94 antibodies with cynomolgus monkey CD94 is shown. Figure 3A Cross-reactivity of hybridoma antibodies to cynomolgus CD94 is shown. Anti-CD94 hybridoma supernatants were screened by flow cytometry on HEK293 cells expressing cynomolgus CD94 to determine cross-reactivity with cynomolgus CD94. HP-3D9 is a commercial anti-CD94 antibody that was used as a negative control. Mouse IgG1, IgG2a, IgG2b, and IgG3 were used as negative controls. 18H3, 1M4, and 1E4 cross-react with cynomolgus CD94, while 20F2 is an example of another CD94 antibody clone that reacts with human CD94 but not with cynomolgus CD94. Figure 3B The cross-reactivity of commercial antibodies to cynomolgus CD94 is shown. Cross-reactivity in cynomolgus monkeys was tested using commercially available anti-CD94 antibodies. 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.

[0037] Figure 4 The results of HLA-E tetramer blocking analysis performed using flow cytometry with anti-CD94 and commercially available anti-CD94 antibodies are shown. Healthy donor PBMCs were incubated with anti-CD94 antibodies. Then PE-labeled HLA-E tetramers were incubated with the cells and antibody mixture and detected using flow cytometry. For HP-3B1, 131412, 12K45, DX22, 1M4 and 1E4, 2.5 μl HLA-E tetramer reagents were used, while 5 μl was used for 18H3 and HP-3D9. The saturation concentration of each antibody was used in this analysis. The blocking percentage was calculated as 100–((HLA-E positive percentage of anti-CD94 antibodies) / (HLA-E positive percentage of isotypes)*100). The 18H3 and 1E4 antibodies do not block the binding of HLA-E to CD94.

[0038] Figure 5A-5BShown are the results of competition assays 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 5A Competition analysis between 18H3 and a commercial anti-CD94 antibody is shown. A commercial anti-CD94 antibody was titrated and incubated with PBMCs simultaneously with 18H3. Figure 5B Competition analysis of 18H3 and other hybridoma anti-CD94 antibodies disclosed herein (1M4 and 1E4) is 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 labeled with AF647 and incubated with cells simultaneously with 18H3-AF647. 18H3 only partially competes with HP-3D9 and does not compete with DX22, HP-3B1, 131412, 12K45, 1E4 and 1M4. These results indicate that the epitope bound by the 18H3 antibody is not shared with commercially available antibodies.

[0039] Figure 6A-6B Shown are the results of competition assays 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. Fig. 6A Competition analysis between 1M4 and a commercial anti-CD94 antibody is shown. A commercial anti-CD94 antibody was titrated and incubated with PBMCs simultaneously with 1M4. Figure 6B Competition analysis of 1M4 and 1E4 antibodies is shown. To test the competition between 1M4 and 1E4, 1E4 was incubated with cells at a concentration of 11 μg / ml. Simultaneously with 1E4, 1M4 with an anti-mouse secondary antibody was incubated with cells. The 1M4 antibody did not compete with the commercially available antibody, but competed with 1E4.

[0040] Figure 7 The results of competition assays performed to evaluate the 1E4 anti-CD94 antibody are shown. For all competition assays, 1E4 was incubated with PBMCs at a concentration of 11 μg / ml. Commercial anti-CD94 antibodies were titrated and incubated with PBMCs simultaneously with 1E4. The 1E4 antibody did not compete with four of the five commercial anti-CD94 antibodies tested, but partially competed with 12K45.

[0041] Figures 8A-8B Results of anti-CD94 antibody internalization analysis are shown. Healthy donor peripheral blood mononuclear cells (PBMCs) were incubated with unconjugated anti-CD94 antibodies at various time points ranging from 30 minutes to 24 hours. Cells were maintained at 4°C to prevent internalization or at 37°C to induce internalization. Fig. 8AResults for commercial antibodies HP-3D9 and DX22 are shown. Commercial anti-CD94 antibodies were internalized in a time-dependent manner. Figure 8B Results for 18H3, 1M4, and 1E4 antibodies are shown. 18H3, 1M4, and 1E4 antibodies were not significantly internalized after binding to CD94.

[0042] Figures 9A-9B The results of antibody-dependent cellular cytotoxicity (ADCC) analysis using healthy donor PBMCs against anti-CD94 antibodies are shown. The 18H3 antibody was produced in Expi-CHO cells cultured in the presence of kifunine, a potent inhibitor of the mannosidase I enzyme, to produce 18H3-KIF, which mimics a non-fucosylated antibody. ADCC analysis was performed using the fucosylated 1E4 antibody. PBMCs were seeded in 96-well plates and plated between 1×10 -6 The cells were incubated overnight in the presence of anti-CD94 antibody in the range of 10 to 10 μg / ml. The number of NK cells was quantified by flow cytometry. The number of NK cells in human IgG1 and anti-CD94 antibody treatment conditions was normalized to the number of NK cells in human IgG1 treated wells. Fig.9A The results of ADCC analysis using human IgG1 18H3 antibody are shown. Fig. 9B The results of ADCC analysis using fucosylated 1E4 antibody are shown. Both human IgG1 18H3 and fucosylated 1E4 depleted human primary NK cells in a concentration-dependent manner.

[0043] Fig.10 The results of anti-CD94 antibody ADCC analysis using PBMCs from CLPD-NK patients are shown. 18H3 was produced in Expi-CHO cells cultured in the presence of kifunine, a potent inhibitor of the mannosidase I enzyme, to produce 18H3-KIF, which mimics a non-fucosylated antibody. PBMCs were seeded in 96-well plates and incubated overnight in the presence of anti-CD94 antibodies ranging from 1×10-6 to 10ug / ml in 10-fold dilutions. The number of CD3-CD16+ leukemic cells was quantified by flow cytometry. The number of leukemic cells under human IgG1 and anti-CD94 antibody treatment conditions was normalized to the number of leukemic cells in human IgG1-treated wells. Partially non-fucosylated human IgG1 18H3 depleted human CLPD-NK leukemic cells in a concentration-dependent manner.

[0044] Fig.11 Depicting an overview of anti-CD94 antibody characteristics and functional evaluation relative to commercially available anti-CD94 antibodies.

[0045] Fig. 12AShown is the study design examining the effects of ATK-130 or isotype control on humanized IL-15 transgenic mice engrafted with healthy human donor PBMCs.

[0046] Fig. 12B Show the depletion of normal human NK and CD8 T cells in IL-15 transgenic mice. Mice were implanted with healthy donor PBMC for three days. A dose of human IgG1 isotype control or ATX-130 (5 mg / kg) was injected into mice (5 mice per group), and 48 hours after administration, the depletion of NK cells (upper figure) and CD8 T cells (lower figure) in blood, spleen and bone marrow were assessed by flow cytometry. The number of NK and CD8 T cells remaining in each sample was quantified for depletion.

[0047] Fig.13 Depletion of LGLL cells in the blood, spleen, bone marrow and liver of IL-15 transgenic mice is shown. LGLL PBMCs were implanted into mice for 28 days. A dose of human IgG1 isotype control or ATX-130 (5 mg / kg) was injected into mice (5 mice per group), and 48 hours after administration, depletion of LGLL cells in blood, spleen, bone marrow and liver was assessed by flow cytometry. Depletion was quantified by the number of CD94+LGLL cells remaining in each sample.

[0048] Fig.14A Shown is the design of an exploratory pharmacodynamics (PD) study conducted in non-human primates to evaluate the in vivo efficacy of ATK-130.

[0049] Fig. 14B Depletion of NK cells in cynomolgus monkeys in vivo when ATX-130-KIF was administered at 2 mg / kg as assessed by flow cytometry. ATX-130-KIF (2 mg / kg) was administered to two naive cynomolgus monkeys (Cyno#1 and Cyno#2) via intravenous (IV) infusion for 60 minutes. Additional 2 mg / kg ATX-130-KIF was administered to Cyno#1 at 8 weeks and to Cyno#2 at 16 weeks. Body weights and clinical observations were recorded regularly. PBMCs were isolated from 5 mL of blood drawn from each animal at each time point. 0.3×10 6 Cells were stained with antibody panels. The CD3-CD8+ gating strategy was used to identify cynomolgus monkey NK cells. NK cells were quantified by calculating the percentage of cell colonies to total PBMCs. Arrows indicate the time point of administration of the dose.

[0050] Figures 15A-15BEvaluation of CD4 T, CD8 T, and B cell depletion in cynomolgus monkeys is shown. PBMCs were isolated from 5 mL of blood drawn at each time point for Cyno#1 and Cyno#2. 0.3×10 6 Cells were stained with a panel of antibodies. CD3+CD4+, CD3+CD8+ and CD3-CD20+ gating strategies were used to identify Cyno#1 ( Fig.15A ) and Cyno#2( Fig. 15B )CD4+ T cells, CD8+ T cells and B cells. Cells were quantified by calculating the percentage of cell populations to total PBMCs.

[0051] Fig.16 Results of monocyte depletion assessed by flow cytometry are shown. PBMCs were isolated from 5 mL of blood drawn at each time point for Cyno#1 (left panel) and Cyno#2 (right panel). 0.3×10 6 Cells were counted and stained with a panel of antibodies. A CD3-CD14+ gating strategy was used to identify monocytes. Cells were quantified by calculating the percentage of the cell population to total PBMCs.

[0052] Fig.17 Results of evaluating CD16 expression on monocytes by flow cytometry are shown. For Cyno#1 (left) and Cyno#2 (right), PBMCs were isolated from 5 mL of blood drawn at each time point. 0.3×10 6 cells and stained with a panel of antibodies. A CD3-CD14+ gating strategy was used to identify monocytes. CD16 MFI was assessed on monocytes. DETAILED DESCRIPTION

[0053] Several aspects are described below with reference to exemplary applications for illustration. It should be understood that numerous specific details, relationships and methods have been stated to provide a comprehensive understanding of the features described herein. However, those of ordinary skill in the relevant art will readily recognize that the features described herein can be practiced without one or more specific details or using other methods. The features described herein are not limited by the described actions or event sequences, because some actions can occur in different orders and / or occur simultaneously with other actions or events. In addition, not all described actions or events are necessary for implementing the methods according to the features described herein.

[0054] Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well. In addition, where the terms "including / includes", "having / has / "with" or variations thereof are used in 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.

[0055] Unless otherwise indicated, any reference to "or" herein is intended to include "and / or". As used herein, the term "about" with respect to a number means the number plus or minus 10% of the number. The term "about" with respect to a range means the range minus 10% of its lower value and plus 10% of its upper value.

[0056] I. Antibodies

[0057] 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 a modified immunoglobulin Fc portion, the modifications comprising reduced fucosylation, non-fucosylation, or mutations that enhance ADCC activity and / or improve the 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 characteristics: binding to human CD94 and cynomolgus CD94, not blocking the binding of HLA-E to CD94 / NKG2A heterodimers, having a low degree of target (e.g., CD94) internalization, non-fucosylation or having reduced fucosylation, and / or inducing or promoting ADCC activity.

[0058] A. Antibody Targets and Affinity

[0059] 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 to cynomolgus CD94. In some embodiments, the antibodies of the present disclosure bind to CD94 on the surface of NK cells and / or T cells.

[0060] In some embodiments, the antibodies of the present disclosure bind to a human CD94 protein or a portion thereof, or a protein that is 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%) homologous to a human CD94 protein or a portion thereof. The amino acid sequences of exemplary human CD94 proteins are provided in the sequences of SEQ ID NOs: 64-66:

[0061] MAVFKTTLWRLISGTLGIICLSLMSTLGILLKNSFTKLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESRHLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI(SEQ ID NO:64)

[0062] MAVFKTTLWRLISGTLGIICLSLMSTLGILLKNSFTKLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESRHLCASQKSSLLQLQNTDELQDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI(SEQ ID NO:65)

[0063] MAAFTKLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESRHLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALS QYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLISYSEEHTAWLWENGSALSQYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI(SEQ ID NO:66)

[0064] In some embodiments, the terms binding, specifically binding to, or specific for 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 greater affinity, avidity, more readily, and / or for a 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) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. D ). In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In another embodiment, specific binding may include but does not require exclusive binding.

[0065] 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).

[0066] In some embodiments, the antibodies of the present disclosure bind to a cynomolgus CD94 protein or a portion thereof, or a protein that is at least 80% (e.g., any of 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%) homologous to a cynomolgus CD94 protein or a portion thereof. The amino acid sequence of the cynomolgus CD94 protein is known in the art, e.g., UniProtKB identifier: Q68VD4.

[0067] In certain embodiments, the affinity of an antibody for its target (e.g., CD94) can be measured by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, such as flow cytometry or Western blotting, and using the assays described herein (e.g., in the Examples). In some embodiments, K is measured using a radiolabeled antigen binding assay (RIA). D , the analysis is performed with the Fab form of the antibodies of the present disclosure and their targets (e.g., CD94). In some embodiments, surface plasmon resonance analysis is used to measure K DExemplary assays are described, for example, in Drake, AW and Klakamp, ​​SL (2007) J. Immunol. Methods 318: 147-152.

[0068] In some embodiments, any method known in the art can be used to assess the binding of the disclosed antibodies to CD94 (e.g., human CD94 and / or cynomolgus CD94). For example, peripheral blood mononuclear cells (PMBCs) and / or NK cells can be used in an ex vivo flow cytometry-based analysis to assess the binding of the disclosed antibodies to human CD94, for example, as described in the Examples. Titration curves and EC50s can be generated and assessed using methods known in the art, such as using Graphpad prism. In another embodiment, cells expressing cynomolgus CD94 (such as HEK293 cells expressing cynomolgus CD94) can be used in an ex vivo or in vitro flow cytometry-based analysis to assess the binding of the disclosed antibodies to cynomolgus CD94, for example, as described in the Examples.

[0069] In certain embodiments, the antibodies of the present disclosure have a K for binding to their targets (e.g., human and / or cynomolgus monkey CD94) of less than about 10 μM. D In certain embodiments, the antibodies of the present disclosure have a K binding to their targets (e.g., human and / or cynomolgus monkey CD94) of less than about 1 μM. D . In certain embodiments, the antibodies of the present disclosure have a K for binding to their 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, 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. D In some embodiments, the antibodies of the present disclosure have a K for binding to their target (e.g., human and / or cynomolgus monkey CD94) 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. D .

[0070] In certain embodiments, the antibodies of the present disclosure have a K for binding to human CD94 on human NK cells or T cells 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. D In certain embodiments, the antibodies of the present disclosure have a K for binding to human CD94 on human NK cells or T cells 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. D In certain embodiments, the antibodies of the present disclosure have a K for binding to human CD94 on human NK cells or T cells between about 2 nM and about 80 nM. D .

[0071] In certain embodiments, the antibodies of the present disclosure have a Kp for binding to cynomolgus monkey CD94 on cells expressing cynomolgus monkey CD94 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. D In certain embodiments, the antibodies of the present disclosure have a K for binding to cynomolgus monkey CD94 on cells expressing cynomolgus monkey CD94 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. D In certain embodiments, the antibodies of the present disclosure have a K for binding to cynomolgus monkey CD94 on cells expressing cynomolgus monkey CD94 between about 2 nM and about 80 nM.D .

[0072] In some embodiments, the antibodies of the present disclosure bind to the same or different epitopes on the target as antibodies known in the art for their targets (e.g., CD94). In some embodiments, the antibodies of the present disclosure bind to different epitopes than antibodies known in the art. In some embodiments, the antibodies of the present disclosure specifically bind to human CD94, wherein the antibodies do 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 antibodies of the present disclosure specifically bind to human CD94, wherein the antibodies do not bind to the same epitope on human CD94 as anti-CD94 antibody clones DX22, HP-3B1 or 131412. In some embodiments, the antibodies of the present disclosure specifically bind to human CD94, wherein the antibodies 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 antibodies of the present disclosure specifically bind to human CD94, wherein the antibodies bind to the same epitope on human CD94 as the anti-CD94 antibody clone HP-3D9. In some embodiments, the antibodies of the present disclosure specifically bind to human CD94, wherein the antibodies bind to the same epitope on human CD94 as the anti-CD94 antibody clone 12K45.

[0073] In some embodiments, if an antibody of the present disclosure does not bind to the same epitope on its target (e.g., CD94) as another antibody against the target (e.g., a commercially available antibody against the target or an antibody known in the art), then the antibody of the present disclosure does not block the 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.

[0074] In some embodiments, the antibodies of the present disclosure bind to their targets (e.g., CD94) with a higher affinity than antibodies known in the art for the target. In some embodiments, the antibodies of the present disclosure bind to their targets (e.g., CD94) with a higher affinity than anti-CD94 antibody clones HP-3D9, DX22, HP-3B1, 131412, or 12K45. In some embodiments, the antibodies of the present disclosure bind to their targets (e.g., CD94) with a higher affinity than anti-CD94 antibody clones HP-3B1, 131412, or 12K45.

[0075] In some embodiments, the antibodies of the present disclosure specifically bind to human CD94, wherein the antibodies bind to human CD94 with a higher affinity than anti-CD94 antibody clones HP-3D9, DX22, HP-3B1, 131412, or 12K45. In some embodiments, the antibodies of the present disclosure specifically bind to human CD94, wherein the antibodies bind to human CD94 with a higher affinity than anti-CD94 antibody clones HP-3B1, 131412, or 12K45.

[0076] In some embodiments, an antibody of the present disclosure binds to its target (e.g., CD94) with any of at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, or more greater affinity than another antibody known in the art to the target.

[0077] B. Exemplary Anti-CD94 Antibodies

[0078] Certain aspects of the present disclosure relate to antibodies that bind to (e.g., specifically bind to) human CD94, wherein the antibody comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the antibody is a human antibody. In some embodiments, variants of the anti-CD94 antibody ATX-130 are provided herein, which are described in International Application No. PCT / US2021 / 052668, filed September 29, 2021.

[0079] In some embodiments, the antibody comprises a VH domain comprising the amino acid sequence EVQLX1ESGGGLVKPGGSLRLSCAASGFTFSSYSM NWVRQAPGKGLEWVSSISX2SSX3X4IYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGRYYYYMDVWGKGTTVTVSS, wherein X1 is L or V; wherein X2 is T or S; wherein X3 is N or S; and wherein X4 is F or Y (SEQ ID NO:69); and / or a VL domain comprising the amino acid sequence DIX1MTQSPSSLSASVGDRVTITCRASQSISSWL AWX2QQKPX3KX4PKX5LIYAASSLQSGVPSX6FSGSGSGTDFTLTIS SLQPEDX7ATYYCQX8YNSX9PFTFGPGTKVDIK, wherein X1 is V or Q; wherein X2 is Y or F; wherein X3 is G or E; wherein X4 is A or V; wherein X5 is S or L; wherein X6 is K or R; wherein X7 is V or F; wherein X8 is K or Q; and wherein 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 AAS SLQS (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 1, 2, 3 or 4 amino acid substitutions present 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 comprising 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions present in SEQ ID Nos: 33-41 relative to the amino acid sequence of SEQ ID NO: 17.

[0080] 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 an amino acid sequence selected from the group consisting of SISTSSNFIYYADSVKG (SEQ ID NO: 2), SISSSSNFIYYA DSVKG (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 / or 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-H4 comprising an amino acid sequence selected from the group consisting of QKYNSA PFT (SEQ ID NO: 6), QQYNSAPFT (SEQ ID NO: 27), and QKYNS YPFT (SEQ ID NO:28); wherein the antibody does not include 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).

[0081] 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 VH domain or a set of three CDRs in a VH domain of the present disclosure can be combined with any VL domain or a set of three CDRs in a VL domain of the present disclosure.

[0082] As known in the art, the variable domains of the heavy and light chains of antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 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, CDR and FR sequences generally appear in 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., in VH or VL domains).

[0083] In some embodiments, the VH domain further comprises: (a) FR1 comprising an amino acid sequence selected from the group consisting of EVQLLESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO:48) and EVQL VESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO:49); (b) FR2 comprising an amino acid sequence of WVRQAPGKGLEWVS (SEQ ID NO:50); (c) FR3 comprising an amino acid sequence of RFTISRDNAKNSLYLQMNSLRAEDT AVYYCAR (SEQ ID NO:51); and (d) FR4 comprising an amino acid sequence of WG KGTTVTVSS (SEQ ID NO:52). In some embodiments, the VH domain further comprises: (a) FR1 comprising the amino acid sequence of EVQLLESGGGLVKPGGSLR LSCAASGFTFS (SEQ ID NO:48); (b) FR2 comprising the amino acid sequence of WV RQAPGKGLEWVS (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). In some embodiments, the VH domain further comprises: (a) FR1 comprising the amino acid sequence of 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 RFTISRDNAKNSLYLQMNSLR AEDTAVYYCAR (SEQ ID NO:51); and (d) FR4 comprising the amino acid sequence of WGKGTTVTVSS (SEQ ID NO:52).

[0084] 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 VL domain or a set of three CDRs in a VL domain disclosed herein can be combined with any VH domain or a set of three CDRs in a VH domain disclosed herein.

[0085] In some embodiments, the VL domain further comprises: (a) an FR1 comprising an amino acid sequence selected from the group consisting of DIVMTQSPSSLSASVGDRVTITC (SEQ ID NO:53) and DIQMTQSPSSL SASVGDRVTITC (SEQ ID NO:54); (b) an 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 WYQQKPGKAPKL LIY (SEQ ID NO:59); (c) an FR2 comprising an amino acid sequence selected from the group consisting of GVPSKFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO:60), GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO:61). (d) an FR4 comprising an amino acid sequence of FGPGTKVDIK (SEQ ID NO: 63).

[0086] In some embodiments, the VH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 29-32 and / or the VL domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 33-41. 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.

[0087] 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.

[0088] In some embodiments, the antibodies of the present disclosure comprise a VH domain comprising 1, 2 or 3 CDRs from a single VH domain listed in Table 1; and / or a VL domain comprising 1, 2 or 3 CDRs from a single VL domain listed in Table 1. In some embodiments, the antibodies of the present disclosure comprise a VH domain comprising 1, 2 or 3 CDRs from a single VH domain listed in Table 2; and / or a VL domain comprising 1, 2 or 3 CDRs from a single VL domain listed in Table 2. In some embodiments, the antibodies of the present disclosure comprise the VH and / or VL domains listed in Table 2. As shown below and described herein, in some embodiments, in the antibodies of the present disclosure, any set of 3 VH CDRs shown in Table 1 can be combined with any set of 3 VL CDRs shown in Table 1. In some embodiments, in the antibodies of the present disclosure, any set of 3 CDRs in the VH domain shown in Table 2 can be combined with any set of 3 CDRs in the VL domain shown in Table 2. In some embodiments, in the antibodies of the present disclosure, any VH domain shown in Table 2 can be combined with any VL domain shown in Table 2.

[0089] Table 1. Anti-CD94 antibody CDR sequences

[0090]

[0091]

[0092]

[0093]

[0094] Table 2. Anti-CD94 antibody variable domain sequences

[0095]

[0096]

[0097]

[0098] Many definitions of the CDR sequences of antibody variable domains are known in the art and can be used to describe antibodies of the present disclosure, for example, by CDR sequences. In some embodiments, antibody CDR sequences are as defined in Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991). In some embodiments, antibody CDR sequences are as defined in Chothia (see, for example, Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). In some embodiments, antibody CDR sequences are as defined 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 mixing two or more definitions (e.g., Kabat, Chothia and / or IMGT).

[0099] 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-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 an amino acid sequence GFTFSSYS (SEQ ID NO:13); 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), 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).

[0100] 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-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 an amino acid sequence GFTFSSY (SEQ ID NO: 14); NO:13), CDR-H1 comprising the amino acid sequence STSSNF (SEQ ID NO:14), CDR-H3 comprising the amino acid sequence DLGRYYYYMDV (SEQ ID NO:15), 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 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).

[0101] In some embodiments, the antibodies of the present disclosure comprise an Fc region. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the antibodies comprise a non-fucosylated human Fc region. In some embodiments, the antibody binds to human cell Fcγ receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. In some embodiments, the antibodies are capable of inducing antibody-dependent cellular toxicity (ADCC) against cells expressing human CD94 on the surface.

[0102] In some embodiments, the term antibody is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen binding activity. In some embodiments, the antibodies of the present disclosure are separated antibodies. An "isolated" antibody is an antibody that has been identified and separated and / or recovered from a component of its natural environment. The contaminant components of its natural environment are materials that will interfere with the research, diagnosis, and / or therapeutic use of the antibody, and may include enzymes, hormones, and other proteins or non-protein solutes. In some embodiments, the antibody is purified (1) to more than 95% by weight of the antibody, as determined by, for example, the Lowry method, and in some embodiments, to more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using, for example, a spinning cup sequencer, or (3) to homogeneity found by SDS-PAGE under reducing or non-reducing conditions using, for example, Coomassie blue or silver staining. Separated antibodies include antibodies in situ within recombinant cells, because at least one component of the natural environment of the antibody will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0103] In some embodiments, monoclonal antibody is an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or appearing during the production of monoclonal antibody preparations, and such variants are generally present in trace amounts. In contrast to polyclonal antibody preparations typically including different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is for a single determinant on an antigen. Therefore, in some embodiments, monoclonal antibodies are obtained from a substantially homogeneous antibody population. Monoclonal antibodies can be produced using any method known in the art. For example, monoclonal antibodies to be used according to the present disclosure can be prepared 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 human immunoglobulin loci.

[0104] C. Blockade of HLA-E Binding

[0105] Major histocompatibility complex class I E (HLA-E) is a ligand of CD94 / NKG2A heterodimers and plays a vital role in inhibiting NK cell and CD8+T cell activity when bound to CD94 / NKG2A heterodimers. Therefore, without wishing to be bound by theory, blocking the interaction between HLA-E and CD94 / NKG2A can lead to activation and proliferation of target cells (e.g., cells expressing CD94). Therefore, it may be beneficial for anti-CD94 antibodies not to block the interaction between HLA-E and CD94 / NKG2A heterodimers, for example, in subjects with NK / T cell lymphomas to whom anti-CD94 antibodies are administered. In some embodiments, HLA-E is human HLA-E, also known as QA1 and HLA-6.2. For exemplary HLA-E genes, see, e.g., NCBI Gene ID No. 3133; for exemplary HLA-E polypeptides, see, e.g., NP_005507.3.

[0106] In some embodiments, blocking the binding of HLA-E to the CD94 / NKG2A heterodimer refers to blocking the binding of HLA-E to the CD94 / NKG2A heterodimer, blocking the binding of HLA-E to CD94, and / or blocking the binding of HLA-E to NKG2A.

[0107] Any method known in the art can be used to assess the blocking of the binding of the antibodies of the present disclosure to HLA-E and CD94 / NKG2A heterodimers. For example, the blocking of the binding of the antibodies of the present disclosure to HLA-E and CD94 / NKG2A heterodimers can be assessed using an in vitro analysis using PBMC and / or NK cells, for example, as described in the Examples. In an exemplary analysis, PBMCs, for example, obtained from healthy donors, are incubated on ice for 30 minutes with human Fc blockers (Biolegend, San Diego, CA) and cell viability dyes (Thermo Fisher, Carlsbad, CA) and protected from light. The cells are then washed once with FACS buffer (PBS containing 2% IgG low FBS). Saturated concentrations of anti-CD94 antibodies or isotype control antibodies are incubated on ice for 30 minutes with cells and protected from light. The cells are then washed and incubated on ice for 30 minutes and protected from light with HLA-E tetramer PE (Creative Biolabs, Shirley, NY), CD3 Pacific Blue and CD56 FITC antibodies (Biolegend, San Diego, CA). Then, before quantification on a flow cytometer, cells are washed for the last time in FACS buffer. Methods known in the art can be used, such as using a CytoFlex flow cytometer (Beckman Coulter, Chaska, MN) to perform data acquisition and fluorescence compensation. Any method known in the art can be used, such as using FlowJo software to perform data analysis. By gating lymphocytes on forward and side scatter, then double and dead cell exclusion, and gating on CD3-CD56+ colonies to identify NK cells. Then HLA-E is quantified on CD3-CD56+NK cell colonies. The percentage of blocking of HLA-E binding to CD94 / NKG2A heterodimers by anti-CD94 antibodies compared to a control antibody (e.g., an isotype control antibody) is calculated as: 100–((percentage of HLA-E positive for anti-CD94 antibodies) / (percentage of HLA-E positive for isotype)*100).

[0108] In some embodiments, the antibody blocks binding of HLA-E to the CD94 / NKG2A heterodimer if, for example, the antibody blocks more than about 20% of the binding of HLA-E to the CD94 / NKG2A heterodimer compared to an isotype control antibody. In some embodiments, the antibody blocks binding of HLA-E to the CD94 / NKG2A heterodimer if, for example, the antibody 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 the binding of HLA-E to the CD94 / NKG2A heterodimer compared to a control antibody (e.g., an isotype control antibody).

[0109] In some embodiments, an antibody does not block HLA-E binding to CD94 / NKG2A heterodimers if, for example, the antibody blocks about 20% or less of HLA-E binding to CD94 / NKG2A heterodimers compared to an isotype control antibody. In some embodiments, an antibody does not block HLA-E binding to CD94 / NKG2A heterodimers if, for example, the antibody 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 CD94 / NKG2A heterodimers compared to a control antibody (e.g., an isotype control antibody).

[0110] In some embodiments, the antibodies provided herein do not block the binding of HLA-E 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 the binding of HLA-E to CD94 / NKG2A heterodimers compared to a control antibody (e.g., an isotype control antibody). In some embodiments, an antibody provided herein blocks 0% of HLA-E binding to the CD94 / NKG2A heterodimer compared to a control antibody (eg, an isotype control antibody).

[0111] D. Enhanced ADCC activity

[0112] In some embodiments, antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular toxicity, antibody-directed cytotoxicity or ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer cells (NK cells), neutrophils and macrophages) that produce Fc receptors recognize antibodies bound to target cells and then cause the lysis of 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 trigger an ADCC reaction.

[0113] In some embodiments, the antibodies provided herein have enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. In some embodiments, enhanced ADCC activity refers to that the Fc region of an antibody or an antibody in the presence of effector cells in vitro or in vivo is more efficiently and / or more effectively mediates or induces ADCC than the native or wild-type antibody and / or the native or wild-type Fc region of an antibody, which can be determined using ADCC analysis, for example, as described herein or as generally known in the art. In some embodiments, effector cells are leukocytes that produce one or more Fc receptors and perform effector functions. In some embodiments, such cells produce at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes mediated by ADCC include peripheral blood mononuclear cells (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells, and neutrophils.

[0114] In some embodiments, ADCC activity can be assessed directly using in vitro assays using 51 Cr release analysis uses peripheral blood mononuclear cells (PBMC) and / or NK effector cells, 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 after ADCC analysis, or the concentration of the antibody or Fc fusion protein when the lysis of the target cell reaches half maximum (e.g., EC50 or IC50). In some embodiments, ADCC activity is determined using PBMC, LGL cells and / or NK cells using an in vitro analysis, e.g., as described in the Examples, and the ADCC activity of the antibodies of the present disclosure is described as the percentage of target cells remaining after ADCC analysis and / or the IC50 or EC50 of the antibody (i.e., the concentration of the antibodies of the present disclosure when achieving half maximum target cell depletion or cell lysis). Any method known in the art can be used, such as using a dose response curve and GraphPad Prism to determine the IC50 or EC50 of the antibody.

[0115] In some embodiments, the antibodies provided herein induce ADCC activity with an IC50 or EC50 measured using an ex vivo assay between 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). In some embodiments, the antibodies provided herein induce ADCC activity, wherein the IC50 or EC50 measured using an in vitro assay is about 20 ng / ml or less. In some embodiments, the antibodies provided herein induce ADCC activity, wherein the IC50 or EC50 measured using an in vitro assay is about 60 ng / ml or less. In some embodiments, the antibodies of the present disclosure exhibit an IC50 or EC50 lower than that of a control antibody (e.g., a wild-type control antibody, or an antibody known or commercially available in the art for the same target) by 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% IC50 or EC50.

[0116] In some embodiments, IC50 or EC50 refers to the concentration of a compound (e.g., antibody) that induces an intermediate response between baseline and maximum after a specified exposure time. For example, IC50 or EC50 can be used to measure the efficacy of antibody-mediated and / or induced effector functions (e.g., ADCC activity). In some embodiments, the IC50 or EC50 of a dose-response curve represents the concentration of a compound (e.g., antibody) at which 50% of its maximum effect is observed.

[0117] In some embodiments, the antibodies of the present disclosure have a higher maximum target cell lysis than a control antibody (e.g., a wild-type control antibody, or an antibody against the same target known or commercially available in the art). For example, an antibody of the present disclosure may exhibit a maximum target cell lysis 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 maximum target cell lysis of a control antibody (e.g., a wild-type control antibody, or an antibody against the same target known or commercially available in the art).

[0118] (i) Enhanced binding to Fc receptors

[0119] In some embodiments, the antibodies provided herein include a human immunoglobulin Fc region with 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., γ receptors); such receptors include subclasses of FcγRI, FcγRII, and FcγRIII, as well as their allelic variants and alternative splicing events. For an overview of Fc receptors, see Ravetch and China: Annu. Port. Immunol. 9, 457 (1991); Capel et al. Immunomethods, 4, 25 (1994); and deHaas et al., J. Leg. Clin. Med. 126, 330 (1995).

[0120] In some embodiments, the antibodies provided herein bind to human cell Fcγ receptor IIIA to a greater extent than antibodies comprising a wild-type Fc region. In some embodiments, the human cell Fcγ receptor IIIA comprises a valine residue or a phenylalanine residue at amino acid residue position 158. See, e.g., UniProt accession P08637 or VAR_003960. In some embodiments, the human cell Fcγ receptor IIIA comprises a sequence of SEQ ID NO: 67 or 68.

[0121] Human Fcγ receptor IIIA 158F

[0122] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK(SEQ ID NO:67)

[0123] Human cell Fcγ receptor IIIA 158V

[0124] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK(SEQ ID NO:68)

[0125] 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 isotypes. 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 Fcγ receptor IIIA (FcγRIIIA) to a greater extent than antibodies comprising a wild-type human IgG1 Fc region. In some embodiments, the human cell Fcγ receptor IIIA comprises a valine residue or a phenylalanine residue at amino acid residue position 158. Exemplary assays for determining binding to human cellular Fcγ 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.

[0126] In some embodiments, the Fc region is the C-terminal region of at least a portion of the constant region in the immunoglobulin heavy chain. 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) in the Fc region may or may not be present. In some embodiments, the numbering of the amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991. In some embodiments, the wild-type Fc region or native Fc region is an Fc region comprising an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. In some embodiments, the variant Fc region is an Fc region comprising an amino acid sequence different from the native or wild-type sequence of the Fc region in at least one amino acid. In some embodiments, the variant Fc region has at least one amino acid substitution, such as 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 the effector function of the variant Fc region compared to the native or wild-type Fc region. In some embodiments, the effector function is a biological activity attributable to the Fc region of the antibody, which varies with 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); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0127] The binding affinity of the antibody to the Fc receptor can be assessed using any method known in the art, such as using surface plasmon resonance and / or ELISA, for example, as described in Shields et al. (2001) J. Biol. Chem., 276: 6591-6604. In some embodiments, the affinity of the antibodies of the present disclosure for FcγRIIIA can be at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or more than the affinity of the wild-type control.

[0128] In some embodiments, affinity refers to the combined strength 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 can generally be expressed by the dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein.

[0129] In some embodiments, a statement that a molecule (e.g., an antibody and / or Fc region) binds to a target (e.g., an Fc receptor, a cell surface protein) more tightly (e.g., with a lower dissociation constant) than another molecule (e.g., an antibody and / or Fc region) in a binding assay (e.g., as described herein and / or as generally known in the art), under substantially the same conditions, a molecule (e.g., an antibody and / or Fc region) binds to a target (e.g., an Fc receptor, a cell surface protein) more tightly (e.g., with a lower dissociation constant) than another molecule (e.g., an antibody and / or Fc region). For example, a statement that antibody "X" binds to an Fc receptor to a greater extent than antibody "Y" indicates that in a binding assay (e.g., as described herein and / or as generally known in the art), under substantially the same conditions, antibody "X" binds to an Fc receptor more tightly (e.g., with a lower dissociation constant) than antibody "Y". In another example, a statement that antibody "X" has a greater binding affinity to a target (e.g., a cell surface protein, such as CD94) than antibody "Y" indicates that antibody "X" binds to the target (e.g., a cell surface protein, such as CD94) more tightly (e.g., with a lower dissociation constant) than antibody "Y" under substantially identical conditions in a binding assay (e.g., as described herein and / or as generally known in the art).

[0130] (ii) Reduction of fucosylation

[0131] In some embodiments, the antibodies of the present disclosure are non-fucosylated or fucose-deficient, such as glycosylated antibody variants comprising an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, antibodies with reduced fucose or lack of fucose have improved ADCC function. Relative to the amount of fucose on the same antibody produced in a cell line, the non-fucosylated or fucose-deficient antibody has reduced fucose. In some embodiments, the 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.

[0132] In some embodiments, fucosylation (fucosylation / fucosylated) refers to a fucose residue within an oligosaccharide attached to the peptide backbone of an antibody of the present disclosure. Specifically, a fucosylated antibody comprises α(l,6) linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or two N-linked oligosaccharides in the Fc region of the antibody, such as position Asn 297 (EU numbering of Fc region residues) of the human IgG1 Fc domain. Due to minor sequence variations in immunoglobulins, Asn297 may also be located at approximately +3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300.

[0133] In some embodiments, the degree of fucosylation is the percentage of fucosylated oligosaccharides relative to all oligosaccharides, for example, as identified by methods known in the art, such as in an N-glycosidase F-treated antibody composition 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 substantially all oligosaccharides contain fucose residues, i.e., fucosylated. Therefore, individual antibodies in such compositions typically contain fucose residues 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% or substantially no oligosaccharides are fucosylated, and individual antibodies in such compositions do not contain fucose residues 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. Individual antibodies in such compositions may comprise fucose residues in zero, one, or two N-linked oligosaccharides in the Fc region, provided that the composition does not comprise substantially all individual antibodies lacking fucose residues in the N-linked oligosaccharides in the Fc region, or substantially all individual antibodies containing fucose residues in both N-linked oligosaccharides in the Fc region. In one embodiment, the 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%).

[0134] In some embodiments, the glycosylated antibody variant comprises an Fc region, wherein 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; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

[0135] Antibodies with reduced fucosylation or non-fucosylated antibodies can be produced using any method known in the art. In some embodiments of the antibodies of the present disclosure, at least one or two heavy chains of the antibody may be non-fucosylated. For example, antibodies of the present disclosure with reduced fucosylation or non-fucosylated antibodies of the present disclosure can be produced in cell lines with α1,6-fucosyltransferase (Fut8) knockout and / or overexpression of β1,4-N-acetylglucosaminyltransferase III (GnT-III) and / or overexpression of Golgi μ-mannosidase II (ManII). Antibodies with reduced fucosylation or non-fucosylated antibodies can also be produced using cell lines lacking 'FUT8' (α-1,6 fucosyltransferase), the enzyme that catalyzes the transfer of fucose; using Chinese hamster ovary (CHO) cells, such as cells lacking FUT8 (Yamane-Ohnuki et al., 2004); or using small interfering RNA (siRNA) to block the expression of the FUT8 gene (Mori et al., 2004). Other cell lines that can be used to produce non-fucosylated or defucosylated antibodies or antibodies with reduced fucosylation are known in the art, including, for example, Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)), and cells overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi μ-mannosidase II (ManII).

[0136] In some embodiments, the antibodies of the present disclosure have reduced fucose relative to the amount of fucose on the same antibody produced in wild-type CHO cells. For example, the antibody may have a lesser amount of fucose relative to the fucose that the antibody would have if produced by a native CHO cell (e.g., a CHO cell that produces a native glycosylation pattern, such as a CHO cell containing a native FUT8 gene). In some embodiments, the antibodies provided herein are antibodies wherein less than about 50%, 40%, 30%, 20%, 10%, 5% or 1% of the N-linked glycans thereon contain fucose. In certain embodiments, the antibodies provided herein are antibodies wherein none of the N-linked glycans thereon contain fucose, i.e., wherein the antibody is completely free of fucose, or does not have fucose, or is non-fucosylated, or non-fucosylated. The amount of fucose can be determined by one skilled in the art, for example, by calculating the average amount of fucose at Asn297 within a sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. In some embodiments, at least one or both heavy chains of the antibody are non-fucosylated.

[0137] Antibodies lacking 1,6-fucose on their heavy chain glycosylation can have enhanced binding affinity for 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 include modified Fc regions, the modifications including reduced fucosylation, non-fucosylation, and / or mutations that enhance ADCC activity and / or improve the 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 NK cells and / or T cells expressing CD94 to a greater extent than fucosylated or wild-type antibodies.

[0138] 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., antibodies provided herein) can induce antibody-directed cytotoxicity (ADCC) and deplete or reduce the number of NK cells and / or T cells expressing CD94 to a greater extent than fucosylated or wild-type antibodies. In some embodiments, at least one or two heavy chains of the antibodies of the present disclosure are non-fucosylated. In some embodiments, the antibodies of the present disclosure are modified so that the carbohydrates of the antibodies are non-fucosylated. In some embodiments, the antibodies of the present disclosure are modified so that less than about 90% (e.g., less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5% or about 1%) of the carbohydrates of the antibodies contain fucose. In some embodiments, the antibodies of the present disclosure are modified so that less than about 40% of the carbohydrates of the antibodies contain fucose. In some embodiments, the antibodies provided herein are afucosylated.

[0139] In some embodiments, the molecules (e.g., antibodies) provided herein induce antibody-directed cytotoxicity (ADCC) and deplete or reduce the number of NK cells and / or T cells expressing CD94 to a greater extent than fucosylated or wild-type antibodies.

[0140] (iii) Mutations that enhance ADCC activity

[0141] The antibodies of the present disclosure may include variant Fc regions. In some embodiments, the variant Fc regions include at least one amino acid substitution in the Fc region that improves ADCC activity. For example, the antibodies of the present disclosure may have variant IgG1 Fc regions that include one or more Fc mutations selected from S239D, A330L, I332E, F243L, and G236A. In another embodiment, the antibodies of the present disclosure may have human IgG1 Fc variant regions that include one or more Fc mutations selected from S239D, A330L, I332E, F243L, and G236A. Other amino acid substitutions known to enhance ADCC activity may 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).

[0142] (iv) Reduced internalization

[0143] In some embodiments, the antibodies of the present disclosure have a low degree of internalization, e.g., receptor-induced internalization or target internalization (i.e., internalization of surface-expressed CD94), e.g., as compared to a wild-type control antibody or an antibody against the same target known or commercially available in the art. Antibodies with lower internalization have a higher occupancy of receptors (e.g., CD94) on the cell surface and a higher level of receptor-antibody complexes on the cell surface, which may enhance ADCC activity. The internalization ability of the antibodies of the present disclosure can be tested in vitro. The internalization ability of the antibodies of the present disclosure can be tested in an ex vivo assay, e.g., using PBMCs and / or NK cells, e.g., as described in the Examples. The internalization of a target (e.g., CD94) can be expressed as a percentage decrease in mean fluorescence intensity (MFI) over a period of time using a flow cytometry-based analysis, e.g., as described in the Examples. For example, internalization of a target (i.e., the internalization ability of an antibody of the present disclosure) can be expressed as a percent reduction in MFI by calculating the difference in MFI over a 24 hour period (e.g., between 0.5 and 24 hours) in cells incubated with the antibody at 37 degrees Celsius and multiplying by 100, e.g., as described in the Examples.

[0144] In some embodiments, an antibody has a high degree of internalization if it causes a greater than 50% reduction in MFI, calculated by calculating the difference in MFI over a 24 hour period (e.g., between 0.5 and 24 hours) in cells incubated with the antibody at 37°C and multiplying by 100, as measured by an ex vivo assay, e.g., using PBMCs and / or NK cells, as described in the Examples. In some embodiments, an antibody is incubated with cells expressing human CD94 on the surface at 37°C for 24 hours, assessed using methods known in the art and / or as described above, and if surface antibody staining due to internalization is reduced by more than 50%, then the antibody has a high degree of internalization.

[0145] In some embodiments, an antibody has a low degree of internalization if it causes a decrease in MFI of 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%), as measured by an ex vivo assay, e.g., using PBMCs and / or NK cells, as described in the Examples. In some embodiments, the antibodies of the present disclosure have a low degree of internalization and result in a reduction in MFI of 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% calculated as described above. In some embodiments, the antibody has a low degree of internalization if there is less than a 50% reduction in surface antibody staining due to internalization when the antibody is incubated with cells expressing human CD94 on the surface for 24 hours at 37°C and assessed using methods known in the art and / or as described above.

[0146] In some embodiments, an antibody of the present disclosure has or causes 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 internalization of a control antibody (e.g., a commercial or wild-type control antibody, or an isotype control antibody), wherein internalization is assessed as described above and / or using any suitable method known in the art.

[0147] Antibody candidates with no or low internalization activity can be further tested for binding to targets from cynomolgus monkeys and / or from humans (e.g., cynomolgus monkeys and / or human CD94). Antibodies that bind to cynomolgus monkey and / or human targets can be used for in vitro and in vivo cell killing assays (e.g., ADCC assays). The cell killing activity (e.g., ADCC activity) of the selected antibodies can be compared with commercially available antibodies or antibodies known in the art.

[0148] E. Antibody Production

[0149] The antibodies disclosed herein can be produced using any technique and / or method known in the art. Techniques for preparing antibodies (e.g., monoclonal antibodies (mAbs)) against almost any target antigen are well known in the art. See, e.g., and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, Vol. 1, pp. 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen (e.g., CD94 or a portion thereof), removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, culturing clones that produce antibodies to the antigen, and isolating antibodies from the hybridoma cultures. One of ordinary skill will recognize that where the antibody is administered to a human subject, the antibody will bind to a human antigen (e.g., human CD94 or a portion thereof).

[0150] MAbs can be isolated and purified from hybridoma cultures by various established techniques. Such separation techniques include affinity chromatography, size exclusion chromatography, and ion exchange chromatography using protein-A or protein-G agarose. See, for example, pages 2.7.1-2.7.12 and 2.9.1-2.9.3 of Coligan. See also Baines et al., "Purification of Immunoglobulin G (IgG)," METHODS IN MOLECULAR BIOLOGY, Vol. 10, pp. 79-104 (The Humana Press, Inc. 1992).

[0151] After initial production of antibodies to an immunogen (eg, CD94 or a portion thereof), the antibodies can be sequenced and subsequently produced by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below.

[0152] In an exemplary method of producing the antibodies disclosed herein, a recombinant target (e.g., CD94) can be used for immunization of mice. Antibodies generated after mouse immunization (e.g., as described above) can be analyzed by ELISA and flow cytometry for specific or selective binding to their target (e.g., CD94). Antibodies can be selected based on their ability to bind to a target (e.g., CD94).

[0153] In some embodiments, non-human primate antibodies can be generated.General techniques for generating therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., WO 91 / 11465 (1991) and Losman et al., Int. J. Cancer 46:310 (1990).

[0154] 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 corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. Such antibodies can be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenges (e.g., CD94 or a portion thereof). Methods for producing fully human antibodies using a combination of methods 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. Phamacol. 3: 544-50). In certain embodiments, the claimed methods and procedures may utilize human antibodies produced by such techniques. Other methods for producing fully human antibodies include phage display, e.g., as described in Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4: 126-40; generating antibodies in normal humans or humans exhibiting a particular disease state, e.g., as described in Dantas-Barbosa et al., 2005; or using transgenic animals (e.g., mice) that have been genetically engineered to produce human antibodies using standard immunization protocols as discussed above, e.g., as 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).

[0155] (i) In vitro cell killing assay

[0156] The generation of 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 as described above, as described in the examples and / or using methods known in the art. The improved cell killing or ADCC activity of the antibodies of the present disclosure may be tested for the depletion of NK cells and / or T cells expressing CD94. Exemplary in vitro models that reproduce human activity may be used to test the depletion of NK cells and / or T cells expressing CD94 (Tomasevic et al., Growth Factors, 2014; 32(6): 223–235; Huang et al., JCI insights, 2016; 1(7): e86689). Peripheral blood lymphocytes (PBLs) isolated from the blood of normal (ie, healthy) donors are incubated with antibodies having human Fc regions (with and without fucose and / or with and without Fc region mutations). The level of killing of NK cells and / or T cells expressing CD94 in PBL (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 antibody can be tested as described above, for example, using the above-mentioned analysis, using a variety of biological samples, such as blood, synovial fluid, bone marrow and spleen whole cell homogenates from patients, the patients suffering from diseases such as 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, NK cell chronic lymphoproliferative disorder (CLPD-NK), LGL leukemia, Felty's syndrome, CLPD-NK, IBM, and RA with LGL and / or aggressive NK leukemia.

[0157] In addition to the cell killing assays described above, in vitro ADCC and antibody-dependent cellular phagocytosis (ADCP) assays can also be performed to analyze the cell killing, ADCC and / or ADCP activity of the antibodies of the present disclosure, using the antibodies of the present 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). Cell killing, ADCC and / or ADCP assays and other analytical methods known in the art can be used, for example, 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. The in vitro activities of the antibodies of the present disclosure can be compared with commercially available antibodies or antibodies known in the art directed to the same target.

[0158] (ii) In vivo cell killing assay

[0159] The generation of antibodies of the present disclosure may involve testing the in vivo ADCC activity of the antibodies, for example, to show the activity of the selected antibody candidate in depleting or reducing the level of NK cells and / or T cells expressing CD94 in vivo. Any method known in the art can be used to determine the in vivo cell killing activity (e.g., ADCC and / or ADCP activity) of the antibodies of the present disclosure. For example, the ability of the antibodies of the present disclosure to deplete or reduce NK cells and / or T cells expressing CD94 in vivo can be tested in cynomolgus monkeys using methods known in the art. For example, in an exemplary method of testing the in vivo cell killing activity (e.g., ADCC and / or ADCP activity) of the antibodies of the present disclosure, a group of cynomolgus monkeys is bled one day before administering a single dose of the antibodies of the present disclosure (e.g., antibody treatment) to identify the pre-dose level of NK cells and / or T cells expressing CD94 by flow cytometry. After administration of the antibodies of the present disclosure, for example, after treatment with the antibodies of the present disclosure, the monkeys are bled at the following time points: 1 hour, 1 day, 7 days, 14 days, and 30 days. At each time point, the level of NK cells and / or T cells expressing CD94 in blood and other biological samples (such as synovial fluid, bone marrow, and spleen) is determined by flow cytometry. The in vivo activity of the antibodies of the present disclosure can be compared with commercially available antibodies or antibodies against the same target known in the art. The anti-CD94 antibodies of the present disclosure can be compared with anti-CD94 antibody clones DX22, HP-3D9, HP-3B1, 131412, or 12K45.

[0160] The skilled artisan will readily appreciate that other methods known in the art for testing in vivo ADCC activity can be used to analyze the in vivo ADCC activity of the antibodies of the present disclosure (eg, transgenic animals, such as transgenic mice).

[0161] Other known antibodies against targets (e.g., CD94) can also be used in the methods provided herein. For example, the anti-CD94 antibodies of the present disclosure can be tested together with the following anti-CD94 antibodies (e.g., in vitro or in vivo ADCC activity, or any other characteristics described herein): HP-3D9 (LSBio catalog # LS-C134679-100; Abnova catalog #: MAB6947); 2I2; 131412 (R&D Systems catalog #: MAB1058); 13B146 (US Biological catalog #: 030068); 13B147 (USBiological catalog #: 030069); 1H1 (Abnova catalog #: MAB10543); 3G2 (Biorbyt catalog #: orb69389); DX22 (Biolegend catalog # 305502); REA113 (Miltenyi Biotec catalog #: 130-098-967); KP43; EPR21003; AT13E3 (ATGen catalog: ATGA0487); HP-3B1; 12K45; and B-D49.

[0162] (iii) Humanization

[0163] The antibody of the present disclosure can 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 and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those HVRs of non-human antibodies, and all or substantially all of the FRs correspond to those FRs of human antibodies. A humanized antibody optionally may 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 been humanized.

[0164] For example, monoclonal antibodies can be humanized by transferring the mouse CDRs of the variable heavy and light chains from mouse immunoglobulins to the corresponding variable domains of human antibodies. The mouse framework regions (FRs) in chimeric monoclonal antibodies can also be replaced by human FR sequences. In order to maintain the stability and antigenic specificity of the humanized monoclonal antibodies, one or more human FR residues can be replaced by mouse counterpart residues. Humanized monoclonal antibodies can be used for therapeutic treatment of subjects. Techniques for producing humanized monoclonal antibodies are well known in the art, for example, as described 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.

[0165] The antibodies of the present disclosure may have one or more of the characteristics described above, 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 afucosylation, cross-reactivity (e.g., binding to human CD94 and cynomolgus CD94), no blocking of HLA-E binding to CD94 / NKG2A heterodimers, competition with commercially available antibodies (e.g., commercially available anti-CD94 antibodies), low internalization, and / or a desired affinity for its target protein (e.g., CD94, human CD94, and / or cynomolgus CD94).

[0166] In some embodiments, the antibodies of the present disclosure are soluble at concentrations above about 10 mg / mL. In some embodiments, the antibodies of the present disclosure form 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 antibodies of the present disclosure have the ability to maintain binding to their targets (e.g., CD94) during storage, for example, 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 at any one of about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C. In some embodiments, the antibodies of the present disclosure are stable (e.g., lack of degradation products, e.g., as measured by SDS-PAGE) during storage, e.g., 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 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.

[0167] In some embodiments, the toxicology of the antibody of the present disclosure is tested. Any method known in the art can be used to perform toxicological analysis of the antibody of the present disclosure. In an exemplary toxicological analysis, the toxicity of the antibody of the present disclosure is tested in cynomolgus monkeys at a dosage higher than 5 times (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 any one of higher) than the dosage expected for human subjects.

[0168] In some embodiments, the antibodies of the present disclosure are capable of depleting or reducing the levels of NK cells and / or T cells expressing CD94 in vitro and / or in vivo. The depletion or reduction of the levels of NK cells and / or T cells expressing CD94 can be measured using any method known in the art. For example, the depletion of NK cells and / or T cells expressing CD94 can be measured using cell killing, ADCC and / or ADCP assays, e.g., as described above and / or as described in the Examples.

[0169] In some embodiments, the antibodies of the present disclosure are soluble at concentrations above 10 mg / mL, have low levels of soluble aggregates (<5%), maintain their binding to the target as measured by ELISA (>90% potency), and have no degradation products as measured by SDS PAGE when incubated for 3 months at 2-8° C. In some embodiments, toxicology analysis of the antibodies of the present disclosure can be performed in cynomolgus monkeys at doses more than 5-fold higher than the dose expected for use in human subjects.

[0170] In some embodiments, antibodies that bind to CD94 can deplete or reduce the levels of NK cells and / or T cells expressing CD94 and may have significant benefits to patients (e.g., human patients) suffering from diseases or conditions such as LGL leukemia, rheumatoid arthritis, Felty's syndrome, CLPD-NK, aggressive NK leukemia, IBM, IBD, or NK / T cell lymphomas, 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. In addition, the antibody treatment may have better tolerability and fewer side effects than first-line and second-line therapies, including chemotherapy such as 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 berentuzumab vedotin. The antibody treatment may prove to be more selective in depleting disease-inducing cells than current non-selective therapies. Non-limiting examples of diseases and disorders in which NK cells and / or T cells expressing CD94 can 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 present invention provides a method of reducing the number of IELs expressing CD94 or depleting IELs in a human subject by administering 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.

[0171] II. Uses and Methods of Treatment

[0172] 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 variety of diseases and disorders such as NK / T cell lymphomas. Many of these disorders or diseases are characterized by clonal expansion of NK cells and / or CD8+ and CD4+T cells.

[0173] In some embodiments, provided herein are molecules (e.g., antibodies) that bind to CD94 (e.g., expressed on the surface of NK cells or T cells). Also provided herein are molecules (e.g., antibodies) that bind to CD94 and have reduced fucosylation, non-fucosylation (e.g., having a modified or mutated immunoglobulin Fc portion, the modification or mutation reduces or eliminates fucosylation). Also provided herein are molecules (e.g., antibodies) that bind to CD94 and have modifications or mutations that enhance ADCC activity and / or improve the affinity of the Fc region for Fc receptors 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 features: binding to human CD94 and cynomolgus CD94, not blocking the binding of HLA-E to CD94 / NKG2A heterodimers, having a low degree of internalization, non-fucosylation or having reduced fucosylation, and / or inducing, promoting or enhancing ADCC activity.

[0174] In some embodiments, provided herein is a method for treating a disease or condition in a subject, the method comprising administering to the subject an effective amount of an antibody described herein, the antibody specifically binding to human CD94. In some embodiments, provided herein is a method for treating a disease or condition in a subject, the method comprising administering to the subject an effective amount of an antibody specifically binding to human CD94, wherein the antibody has one or more of the following characteristics: does not block the binding of HLA-E to CD94 / NKG2A heterodimers, 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 NK cell chronic 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, 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 γ / δ TCL, subcutaneous panniculitis TCL or microscopic colitis. In some embodiments, administration of the antibody results in a decrease in the number of NK cells and / or T cells expressing CD94. In some embodiments, the administration of the antibody results in a decrease in the number of peripheral blood NK cells, CD8+, CD4+ or CD8+ / CD4+T cells and / or LGL cells (e.g., expressing CD94) of the subject. In some embodiments, the administration of the antibody results in a decrease in the number of peripheral blood LGL cells (e.g., expressing CD94) of the subject. In some embodiments, the administration of the antibody results in a decrease in the number of peripheral blood NK cells (e.g., expressing CD94) of the subject. In some embodiments, the administration of the antibody results in a decrease in the number of peripheral blood CD8+ / CD4+T cells (e.g., expressing CD94) of the subject. In some embodiments, the administration of the antibody results in a decrease in the number of peripheral blood CD8+T cells (e.g., expressing CD94) of the subject. In some embodiments, the administration of the antibody results in a decrease in the number of peripheral blood CD4+T cells (e.g., expressing CD94) of the subject.

[0175] Also provided herein is a method for reducing the number of peripheral blood NK cells and / or T cells expressing CD94, the method comprising administering to a subject an effective amount of an antibody of the present disclosure, the antibody specifically binding to human CD94. In some embodiments, the antibody has one or more of the following features: does not block the binding of HLA-E to CD94 / NKG2A heterodimers, 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 the group consisting of chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, inflammatory bowel disease, 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+ skin TCL, primary cutaneous γ / δ TCL, subcutaneous panniculitis TCL, or microscopic colitis. In some embodiments, administration of the antibody to the subject results in a decrease in the number of peripheral blood NK cells and / or T cells expressing CD94 compared to before administration of the antibody.

[0176] Also provided herein is a method for inducing ADCC activity in a subject, the method comprising administering to the subject an effective amount of an antibody of the present disclosure, the antibody specifically binding to human CD94. In some embodiments, the antibody has one or more of the following features: does not block the binding of HLA-E to CD94 / NKG2A heterodimers, 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 the group consisting of chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, inflammatory bowel disease, 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+ skin TCL, primary cutaneous γ / δ TCL, subcutaneous panniculitis TCL, or microscopic colitis. In some embodiments, administration of the antibody to the subject results in a decrease in the number of peripheral blood NK cells and / or T cells expressing CD94 compared to before administration of the antibody.

[0177] In some embodiments, the disease or condition is CLPD-NK. In some embodiments, the disease or condition is LGL leukemia. In some embodiments, the disease or condition is Felty's syndrome. In some embodiments, the disease or condition is rheumatoid arthritis. In some embodiments, the disease or condition is aggressive NK leukemia. In some embodiments, the disease or condition is inclusion body myositis. In some embodiments, the disease or condition is inflammatory bowel disease. In some embodiments, the disease or condition is T-large granular lymphocytic leukemia (T-LGLL). In some embodiments, the disease or condition is natural killer-large granular lymphocytic leukemia (NK-LGLL). In some embodiments, the disease or condition is NK / T cell lymphoma. In some embodiments, the disease or condition is extranodal NK / T cell lymphoma. In some embodiments, the disease or condition is hepatosplenic T cell lymphoma (TCL). In some embodiments, the disease or condition is enteropathy-associated TCL. In some embodiments, the disease or condition is skin TCL. In some embodiments, the disease or condition is anaplastic large cell lymphoma (ALK+). In some embodiments, the disease or condition is anaplastic large cell lymphoma (ALK-). In some embodiments, the disease or condition is peripheral TCL (not otherwise specified). In some embodiments, the disease or condition is angioimmunoblastic TCL. In some embodiments, the disease or condition is adult TCL. In some embodiments, the disease or condition is monomorphic epithelial-tropic intestinal TCL. In some embodiments, the disease or condition is epidermotropic CD8+ skin TCL. In some embodiments, the disease or condition is primary cutaneous gamma / delta TCL. In some embodiments, the disease or condition is subcutaneous panniculitis TCL. In some embodiments, the disease or condition is microscopic colitis.

[0178] 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, administering the antibody to a human subject results in an improvement in CLPD-NK symptoms in the person.

[0179] Also provided herein is a method for treating NK / T cell lymphoma in a human subject in need thereof, the method comprising administering an effective amount of an antibody of the present disclosure to the human subject, wherein the antibody specifically binds to human CD94. In some embodiments, administering the antibody to a human subject results in an improvement in the NK / T cell lymphoma symptoms of this person. In some embodiments, the NK / T cell lymphoma is selected from extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, skin TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, adult TCL, monomorphic epithelial intestinal TCL, epidermotropic CD8+ skin TCL, primary skin γ / δ TCL or subcutaneous panniculitis TCL.

[0180] 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, wherein the antibody has one or more of the following characteristics: does not block the binding of HLA-E to CD94 / NKG2A heterodimers, 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 an improvement in the CLPD-NK symptoms of the person.

[0181] 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, wherein the antibody specifically binds to human CD94, wherein the antibody has one or more of the following characteristics: does not block the binding of HLA-E to CD94 / NKG2A heterodimers, 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 an improvement in the 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 epithelial-tropic intestinal TCL, epidermotropic CD8+ skin TCL, primary cutaneous γ / δ TCL or subcutaneous panniculitis TCL. In some embodiments, the NK cell or T cell lymphoma is extranodal NK / T cell lymphoma, hepatosplenic TCL or enteropathy-associated TCL. In some embodiments, the NK cell or T cell lymphoma is characterized by NK cells and / or T cells expressing CD94 (e.g., human CD94).

[0182] Also provided herein is a method for treating microscopic colitis 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, wherein the antibody has one or more of the following characteristics: does not block the 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 an improvement in the symptoms of microscopic colitis in the person. Microscopic colitis is a gastrointestinal disease characterized by inflammation of the colon leading to persistent, non-bloody, watery diarrhea. It is called microscopic because tissue destruction can only be seen under a microscope, not by naked eye examination. The colon of these patients appears normal or has near-normal colonic mucosa macroscopically. 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 the colon tissue. Currently, it affects 100 / 100,000 people worldwide, with 39.3% of cases belonging to the lymphocytic subtype (Hisamatsu et al. (2016) Inflamm. Intest. Dis. 2: 52-62). The cause of microscopic colitis is currently unknown, but common factors include medications, bacterial and viral infections, autoimmune diseases (such as rheumatoid arthritis, celiac disease or psoriasis), and bile acid accumulation. The current diagnostic method is histological examination of intestinal tissue, because the disease cannot be diagnosed without histopathological examination of biopsy material. Common symptoms of microscopic colitis include persistent watery diarrhea, leading to weight loss, swelling, anemia, malnutrition, etc. There is no cure or adequate treatment for microscopic colitis, except anti-diarrhea medications, a low-fat, low-fiber and low-dairy diet, steroids, bile acid blockers, anti-inflammatory drugs, TNF inhibitors, or in rare cases surgery to remove part or all of the colon. Although it is not clear what the main pathogenic cells of microscopic colitis are, studies have shown that CD8+ intraepithelial lymphocytes (IELs) are elevated in the colon of patients with microscopic colitis (Goranzon et al. (2013) J. Crohns Colitis 7: e434-442). There is evidence in the literature 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) may perform ADCC on IEL cells via cannibalism.The literature also shows that CD16 is highly elevated in microscopic colitis biopsies compared to normal controls, suggesting that IELs may engage anti-CD94 to induce ADCC on 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 may act as effector cells as well as target cells.

[0183] Also provided herein is a method for enhancing chimeric antigen receptor T cell (CAR-T) therapy for a human subject in need, the method comprising administering an effective amount of an antibody of the present disclosure to the human subject, followed by administering CAR-T therapy to the subject, wherein the antibody specifically binds to human CD94, wherein the antibody has one or more of the following characteristics: does not block the binding of HLA-E to CD94 / NKG2A heterodimers, 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 before administration of CAR-T therapy. In some embodiments, the CAR-T therapy is used to treat cancer, that is, the subject has been / is undergoing cancer treatment. The short half-life of CAR-T cells used to treat various blood cancers and solid cancers significantly hinders the development of cell therapy. Lymphocyte depletion chemotherapy is typically used before CAR-T therapy, such as cyclophosphamide combined with or without fludarabine (Flu). However, these standard chemotherapies do not fully deplete NK cells, resulting in the elimination of readily available CAR-T cells shortly after infusion. Without wishing to be bound by theory, it is believed that anti-CD94 antibody therapy may temporarily deplete NK cells, thereby enabling the implantation of HLA class I-deficient cells ("universal CAR T cells") or organs to prevent NK cell-mediated rejection. The temporary depletion of NK cells should provide a window for the implantation of HLA class I-negative cells without posing a safety risk. After the CD94 depletion antibody disappears, the number of NK cells may return to normal. The newly emerged NK cells should be tolerant to the transferred HLA class I-negative cells (based on mixed bone marrow chimera experiments). Therefore, anti-CD94 antibody therapy may provide an opportunity to enhance CAR-T therapy, for example in cancer patients.

[0184] In some embodiments, the terms treat / treating / treatment, ameliorate / ameliorating, reducing one or more symptoms, reducing symptoms, reducing one or more symptoms, reducing symptoms, and other grammatical equivalents refer to relieving, eliminating, or ameliorating one or more symptoms of a disease or condition, preventing additional symptoms, ameliorating or preventing the underlying cause of a symptom, inhibiting a disease or condition, for example, preventing the development of a disease or condition, alleviating a disease or condition, causing regression of a disease or condition, alleviating an illness caused by a disease or condition, or stopping symptoms of a disease or condition, and are intended to include prevention. In some embodiments, the terms also include achieving a therapeutic benefit and / or a preventive benefit. In some embodiments, a therapeutic benefit refers to eradicating or ameliorating the underlying disease or condition being treated. In addition, a therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disease or condition, such that an improvement is observed in the patient, although in some embodiments, the patient still suffers from the underlying disease or condition. For prophylactic benefit, the pharmaceutical compositions are administered to a patient at risk of developing a particular disease or condition, or to a patient reporting one or more of the physiological symptoms of a disease or condition, even if a diagnosis of said disease or condition has not yet been made.

[0185] In some embodiments, an effective amount, a therapeutically effective amount, or a pharmaceutically effective amount can be a sufficient amount of at least one pharmaceutical composition or compound (e.g., an antibody of the disclosure) administered that will relieve to some extent one or more of the symptoms of the disease or condition being treated.

[0186] In some embodiments, a decrease 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 22 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, following administration of the antibody to the subject.

[0187] 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 limit of clinical diagnosis of the disease or disorder. In some embodiments, the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject is reduced to less than or equal to 2×10 9 Cells / L (e.g., in a peripheral blood sample obtained from a subject). See, e.g., Lamy, T. et al. (2017) Blood 129:1082-1094. In some embodiments, after administration of the antibody to the subject, the number of peripheral blood NK cells and / or T cells expressing CD94 in the subject is reduced to below the limit of clinical diagnosis of the disease or disorder 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 any of longer. In some embodiments, after administration of the antibody to the subject, the number of peripheral blood NK cells and / or T cells expressing CD94 in the subject is reduced to less than or equal to 2×10 9 cells / L (e.g., in a peripheral blood sample obtained from the subject) is present in the subject for at least about 1 week, such as 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.

[0188] In some embodiments, the number of peripheral blood NK cells and / or T cells expressing CD94 in the subject is reduced to below the detection limit of peripheral blood NK cells and / or T cells expressing CD94 in the subject. In some embodiments, after administering the antibody to the subject, the number of peripheral blood NK cells and / or T cells expressing CD94 in the subject is reduced to below the detection limit of peripheral blood NK cells and / or T cells expressing CD94 in the subject for at least about 1 week, for example, 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 more. In some embodiments, peripheral blood NK cells and / or T cells expressing CD94 are detected by flow cytometry (e.g., as performed on a peripheral blood sample from a subject).

[0189] In some embodiments, the peripheral blood NK cells expressing CD94 are CD3+CD56bright. In some embodiments, the peripheral blood T cells expressing CD94 are CD4+, CD8+, or CD8+ and CD4+.

[0190] In some embodiments, the reduction in the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject is reversible. In 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 after administration of the antibody to the subject.

[0191] In some embodiments, a statement that a cell or cell population is positive (+) 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 in the cell. In some embodiments, a statement that a cell or cell population is positive (+) or expresses a surface marker (e.g., a cell surface protein) refers to the presence of cell surface expression of the particular marker, e.g., as detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is substantially higher than the staining detected by the same procedure under otherwise identical conditions using isotype matched controls and / or fluorescence minus one (FMO) gated controls, and / or at a level that is substantially similar to that of cells known to be positive for the marker, and / or at a level that is substantially higher than that of cells known to be negative for the marker.

[0192] In some embodiments, a statement that a cell or cell population is negative (-) 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 in the cell. In some embodiments, a statement that a cell or cell population is negative (-) 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, e.g., as detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is substantially similar to or lower than the staining detected by the same procedure with isotype-matched controls and / or fluorescence minus one (FMO) gated controls under otherwise identical conditions, and / or at a level that is lower than the level of cells known to be positive for the marker, and / or at a level that is substantially similar to or lower than the level of cells known to be negative for the marker.

[0193] In some embodiments, the EC50 or IC50 of the antibody for reducing peripheral blood NK cells and / or T cells expressing CD94 in a subject is between about 1 ng / ml and about 100 ng / ml, such as 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. In some embodiments, the IC50 or EC50 of the antibody for reducing peripheral blood NK cells and / or T cells expressing CD94 in a subject is between about 10 ng / ml and about 80 ng / ml. In some embodiments, the IC50 or EC50 of the antibody for reducing peripheral blood NK cells and / or T cells expressing CD94 in a subject is about 20 ng / ml. In some embodiments, the IC50 or EC50 of the antibody for reducing peripheral blood NK cells and / or T cells expressing CD94 in a subject is about 60 ng / ml. EC50 or IC50 can be measured using any method known in the art, for example, as described in the Examples.

[0194] In some embodiments, the subject is a human, a primate, a non-human primate (e.g., an African green monkey, a cynomolgus monkey, a rhesus monkey, etc.), a farm mammal, a hunting mammal, or a domesticated 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 an elderly person. 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, 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 γ / δ TCL, or subcutaneous panniculitis TCL.

[0195] In some embodiments, administration of the antibody to a subject does not result in tumor lysis syndrome in the subject. Tumor lysis syndrome can 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.)

[0196] In some embodiments, the antibodies of the present disclosure bind to CD94. In some embodiments, the antibodies of the present disclosure deplete and / or reduce the level of NK cells and / or T cells expressing CD94. In some embodiments, the antibodies of the present disclosure have a demonstrated benefit to a patient (e.g., a human patient) having a disease or condition such as CLPD-NK, LGL leukemia, rheumatoid arthritis, Felty's syndrome, 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, subcutaneous panniculitis TCL, or other diseases associated with LGL, T cells, and / or NK cells. In some embodiments, the antibodies of the present disclosure have a higher affinity than those for the disease or disorder (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, 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). The invention provides better tolerability and fewer side effects for first-line and second-line therapies for advanced NSCLC (eg, TCL, angioimmunoblastic TCL, or adult TCL), such as chemotherapy, such as 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 berentuzumab vedotin. In some embodiments, the antibodies of the present disclosure demonstrate more selective depletion of disease-inducing cells, e.g., NK cells and / or T cells expressing CD94, compared to current non-selective therapies (such as chemotherapy, such as 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 berentuzumab vedotin).Thus, in some embodiments, the present disclosure provides a method of reducing the number of NK cells and / or T cells expressing CD94 in a human subject or depleting such cells after administration of a molecule (e.g., an antibody of the present disclosure) that binds to a cell surface protein (such as CD94) on NK cells and / or T cells and comprises (a) a region that specifically binds to a target and (b) an immunoglobulin Fc region.

[0197] In some embodiments, the methods provided herein further comprise administering to the subject an IL-2 polypeptide, e.g., a therapeutic IL-2 polypeptide. IL-2 polypeptides (e.g., therapeutic IL-2 polypeptides) suitable for administration to a subject (e.g., a subject suffering from a disease or condition 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.

[0198] A. Administration and Dosing Regimen

[0199] (i) Route of administration

[0200] In some embodiments, administration (administer / administering / administration) etc. refers to a method for enabling treatment or pharmaceutical composition to be delivered to a desired biological site of action. In some embodiments, the antibodies of the present disclosure (and any additional therapeutic agent) used in any method provided herein can be administered to a subject (e.g., a person) by any suitable means including parenteral, intrapulmonary, intranasal, and intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, antibodies of the present disclosure are administered by intravenous infusion. Administration of antibodies of the present disclosure can be by any suitable route, for example, by injection (such as intravenous or subcutaneous injection), which depends in part on whether administration is short-term or long-term.

[0201] (ii) Dosage regimen

[0202] Various dosing schedules or regimens can be used, including but not limited to single or multiple administrations, bolus administrations, and pulse infusions through multiple time points, to administer the antibodies of the present disclosure used in any method provided herein to the subject. The specific dosage of the antibody of the present disclosure to be administered will vary according to the specific target specificity, the type of disease or condition, the nature and severity of the subject and the disease, the physical condition of the subject, the treatment regimen (e.g., whether a combination therapeutic agent is used), and the selected route of administration. In some embodiments, the dosage of the antibody of the present disclosure can be within the range of about 0.0001 mg / kg to 100 mg / kg of the subject's body weight. The exemplary dosage regimen of the antibody of the present disclosure requires the administration of the antibody with multiple doses over an extended period of time (e.g., at least six months).

[0203] Other known antibodies against CD94 can also be used in the methods provided herein. For example, the following anti-CD94 antibodies can be used: HP-3D9 (LSBio catalog # LS-C134679-100; Abnova catalog #: MAB6947); 2I2; 131412 (R&D Systems catalog #: MAB1058); 13B146 (USBiological catalog #: 030068); 13B147 (USBiological catalog #: 030069); 1H1 (Abnova catalog #: MAB10543); 3G2 (Biorbyt catalog #: orb69389); DX22 (Biolegend catalog # 305502); REA113 (Miltenyi Biotec catalog #: 130-098-967); KP43; EPR21003; AT13E3 (ATGen catalog: ATGA0487); HP-3B1; 12K45; and B-D49.

[0204] B. Disease

[0205] There are 9 different diseases involving NK / T cell lymphoma: extranodal NK / T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, skin TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL and adult TCL. See, for example, Bajaj, 2019. NK / T cell lymphoma affects multiple organs, such as skin, GI, liver, spleen, bone marrow. Symptoms of NK / T cell lymphoma include cervical lymphadenopathy. Advantageously, the methods described herein can be used, for example, 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 via the mechanism of using NK cells (such as ADCC), essentially using pathological cells to eliminate each other.

[0206] The methods described herein can also be used to reduce the number of abnormal or pathological NK and / or T cells that express CD94 and are associated with NK / T cell lymphomas (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), for example, via mechanisms using NK cells (such as ADCC), essentially eliminating each other using the pathological cells.

[0207] There are three different diseases involving LGL: T-cell LGL (T-LGL) leukemia; chronic lymphoproliferative disorder of NK cells (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).

[0208] In addition to NK or T LGL leukemia, NK or LGL cells also play a key 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 conditions in which LGL 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 can be used to reduce the number of abnormal or pathological NK cells (e.g., CLPD-NK, ANKL, or ENKL cells), for example, via mechanisms using NK cells (such as ADCC), essentially eliminating each other using the pathological cells. For exemplary descriptions of the symptoms of these diseases, see, e.g., Lamy et al., Blood, 2017x Vol. 129, No. 9; Loughran Blood, Vol. 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.

[0209] (i) CLPD-NK

[0210] Chronic lymphoproliferative disorder of NK cells (CLPD-NK) is 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 and is generally characterized by a persistent (e.g., 6 months or longer) increase in peripheral blood NK cells (e.g., ≥2×10 9 / L).

[0211] Symptoms of CLPD-NK include various cytopenias, such as neutropenia and anemia, fatigue, fever, night sweats, recurrent infections, rheumatoid arthritis, lymphadenopathy, hepatosplenomegaly, skin lesions, hematological tumors, vasculitis, neuropathy, and autoimmune disorders.

[0212] In some embodiments of the methods provided herein, the disease or disorder is CLPD-NK, and the administration of the antibody results in alleviation of one or more CLPD-NK symptoms in the subject. In some embodiments, a decrease in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in alleviation of one or more CLPD-NK symptoms in the subject.

[0213] Symptoms of CLPD-NK can 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 (e.g., to determine hepatosplenomegaly), bone marrow examination, and flow cytometry. Methods for measuring symptoms of CLPD-NK are described in, for example, Swerdlow, SH et al. (2016) Blood 127: 2375-2390.

[0214] (ii) LGL leukemia

[0215] Large granular lymphocytic (LGL) leukemia is a chronic lymphoproliferative disorder that presents with a chronic elevation of large granular lymphocytes (LGL) in the peripheral blood and is known as T-cell LGL leukemia.

[0216] Symptoms of LGL leukemia include an enlarged spleen, B symptoms (eg, systemic symptoms such as fever, night sweats, and weight loss), anemia, neutropenia, and recurrent infections. Rheumatoid arthritis is also common in people with T-cell LGL leukemia.

[0217] In some embodiments of the methods provided herein, the disease or disorder is LGL leukemia, and administration of the antibody results in alleviation of one or more LGL leukemia symptoms in the subject. In some embodiments, a decrease in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in alleviation of one or more LGL leukemia symptoms in the subject.

[0218] Symptoms of LGL leukemia can be measured by any method known in the art, such as using laboratory tests to measure anemia, neutropenia and other cytopenias, complete blood cell counts, magnetic resonance imaging (MRI), CT scans, palpation or ultrasound (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.

[0219] (iii) Felty's syndrome

[0220] Felty's syndrome is an autoimmune disease characterized by rheumatoid arthritis, an enlarged spleen (e.g., inflammatory splenomegaly), and a decreased number of neutrophils in the blood. Symptoms of Felty's syndrome include joint pain, stiffness, and / or swelling, physical manifestations associated with rheumatoid arthritis, an enlarged spleen, neutropenia, infections, keratoconjunctivitis sicca, fever, weight loss, fatigue, skin discoloration, sores (e.g., ulcers), hepatomegaly, anemia, thrombocytopenia, abnormal liver function, swollen lymph nodes, and vasculitis.

[0221] In some embodiments of the methods provided herein, the disease or condition is Felty's syndrome, and the administration of the antibody results in a reduction in one or more symptoms of Felty's syndrome in the subject. In some embodiments, a reduction in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in a reduction in one or more symptoms of Felty's syndrome in the subject. Symptoms of Felty's syndrome include, but are not limited to, joint inflammation, joint pain, and splenomegaly.

[0222] Symptoms of Felty's syndrome can be measured by any method known in the art, such as using laboratory tests to measure anemia, neutropenia, thrombocytopenia, and other cytopenias, complete blood counts, magnetic resonance imaging (MRI), CT scans, or ultrasound (e.g., to determine splenomegaly and / or hepatomegaly), laboratory tests for abnormal liver function, palpation to determine splenomegaly and / or hepatomegaly, flow cytometry, Disease Activity Score-28 (DAS-28, e.g., as used to monitor rheumatoid arthritis symptoms), and DAS-28 combined with erythrocyte sedimentation rate (ESR).

[0223] (iv) Rheumatoid arthritis

[0224] Rheumatoid arthritis is an autoimmune disorder that primarily affects the joints but can also affect other organs and can be associated with cardiovascular disease, osteoporosis, interstitial lung disease, infection, cancer, fatigue, and depression. Symptoms of rheumatoid arthritis include joint swelling, tenderness, and warmth, 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, pulmonary lobar panniculitis, atrophy of the finger skin, palmar erythema, skin fragility, diffuse alopecia areata, pulmonary fibrosis, Kaplan's syndrome, exudative pleural effusion, atherosclerosis, myocardial infarction, stroke, pericarditis, heart Endocarditis, left heart failure, valvulitis, heart and / or blood vessel fibrosis, anemia, increased platelet count, low white blood cell count, renal amyloidosis, episcleritis, scleritis, keratoconjunctivitis sicca, keratitis, vision loss, liver problems, 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.

[0225] In some embodiments of the methods provided herein, the disease or condition is rheumatoid arthritis, and the administration of the antibody results in a reduction in one or more rheumatoid arthritis symptoms in the subject. In some embodiments, a reduction in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in a reduction in one or more rheumatoid arthritis symptoms in the subject.

[0226] In some embodiments, the symptoms and disease state / progression of rheumatoid arthritis are measured according to the 2010 ACR / EULAR classification criteria for rheumatoid arthritis (see, e.g., Aletaha et al., (2010) Annals of Rheumatic Diseases, 69(9): 1580-8). Symptoms of rheumatoid arthritis can also be measured by any method known in the art, such as using laboratory tests for measuring erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, anti-citrullinated protein antibodies, anemia and other cytopenias, increased platelet counts, low white blood cell counts, complete blood cell counts, renal amyloidosis, medical imaging (such as X-rays, MRI), CT scans, ultrasound (e.g., ultrasound examination using a high-frequency transducer; Doppler ultrasound), flow cytometry, disease activity score-28 (DAS-28), and DAS-28 combined with erythrocyte sedimentation rate (ESR).

[0227] (v) Aggressive NK leukemia

[0228] Aggressive NK cell leukemia is an aggressive disease with systemic hyperplasia of NK cells and a rapidly declining clinical course. Aggressive NK leukemia may also be referred to as aggressive NK cell lymphoma. The symptoms of aggressive NK cell leukemia include systemic symptoms (e.g., discomfort, weight loss, fatigue), hepatosplenomegaly, lymphadenopathy, coagulopathy, hemophagocytic syndrome, multiple organ failure, infections such as Epstein-Barr virus, allergic reactions (e.g., allergic reactions to insect bites, such as mosquito bites), which may cause necrosis and systemic symptoms, such as fever, lymphadenopathy, abdominal pain, diarrhea, and allergies.

[0229] In some embodiments of the methods provided herein, the disease or disorder is an aggressive NK cell leukemia, and the administration of the antibody results in alleviation of one or more symptoms of an aggressive NK cell leukemia in the subject. In some embodiments, a reduction in the number of peripheral blood LGLs and / or NK cells in the subject after administration of the antibody results in alleviation of one or more symptoms of an aggressive NK cell leukemia in the subject.

[0230] The symptoms of aggressive NK leukemia can be measured by any method known in the art, such as using laboratory tests and / or magnetic resonance imaging (MRI), CT scans, palpation or ultrasound (e.g., to determine splenomegaly), bone marrow examination, and flow cytometry to measure anemia, neutropenia and other cytopenias, complete blood cell counts. Methods for measuring the symptoms of aggressive NK leukemia are described in, for example, Swerdlow, SH et al. (2016) Blood 127: 2375-2390.

[0231] (vi) Inclusion body myositis

[0232] Inclusion body myositis (IBM), also known as sporadic inclusion body myositis, is an inflammatory muscle disease characterized by an autoimmune and degenerative process that results in progressive weakness and emaciation of distal and / or proximal muscles. In general, IBM is characterized by the invasion of immune cells into muscle tissue. In some cases, the level of creatine kinase in the blood of IBM patients is elevated. Symptoms of IBM include progressive muscle weakness, muscle wasting / atrophy, frequent stumbling and falling, difficulty with finger manipulation, foot drop, limited activity, impaired balance, muscle pain, difficulty swallowing, and fatigue.

[0233] In some embodiments of the methods provided herein, the disease or disorder is IBM, and the 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 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.

[0234] Symptoms of IBM can be measured by any method known in the art, such as muscle biopsy, blood tests (e.g., measuring creatine kinase), electromyography (EMG) studies, blood tests to measure NT5C1A antibodies, flow cytometry, and myositis disease activity assessment tools, including but not limited to the Myositis Intent to Treat Activity Index (MITAX) and the Myositis Disease Activity Assessment Visual Analog Scale (MYOACT).

[0235] (vii) Inflammatory bowel disease

[0236] Inflammatory bowel disease (IBD) refers to a group 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, blood in the stool, loss of appetite, and weight loss.

[0237] In some embodiments of the methods provided herein, the disease or disorder is IBD, and the administration of the antibody results in alleviation of one or more IBD symptoms in the subject. In some embodiments, a reduction in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in alleviation of one or more IBD symptoms in the subject.

[0238] Symptoms of IBD can be measured by any method known in the art, such as laboratory blood tests for anemia, other cytopenias, or infection, fecal occult blood tests, colonoscopy, flexible sigmoidoscopy, upper gastrointestinal endoscopy, capsule endoscopy, balloon-assisted enteroscopy, X-rays, CT scans, MRI scans, ultrasound, and flow cytometry.

[0239] III. Pharmaceutical Preparations

[0240] In some embodiments, a pharmaceutical composition, composition or pharmaceutical preparation refers to a biologically active compound (e.g., an antibody of the present disclosure), optionally mixed with at least one pharmaceutically acceptable chemical component, such as but not limited to a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, etc.

[0241] Pharmaceutical compositions, pharmaceutical preparations and / or compositions of any of the antibodies of the present disclosure for use in any of the methods as described herein can be prepared by mixing such antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of a lyophilized formulation or an aqueous solution.

[0242] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: 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 methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residual yl) polypeptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinyl pyrrolidone; amino acid, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharide, disaccharide and other carbohydrate, including glucose, mannose or dextrin; chelating agent, such as EDTA; sugar, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterion, such as sodium; metal complex (e.g., Zn-protein complex); and / or nonionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more other glycosaminoglycanases, such as chondroitinases.

[0243] The formulations herein may also contain more than one active ingredient as necessary for the particular indication (eg, disease or condition) being treated, preferably those with complementary activities that do not adversely affect each other.

[0244] The active ingredient can be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) 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).

[0245] Sustained release preparations can 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 (eg, films, or microcapsules).

[0246] Preparations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0247] IV. Kits and Articles

[0248] In another aspect of the present disclosure, a kit or article containing materials useful for the methods provided herein (e.g., treating the above-mentioned diseases or conditions, reducing the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject, or inducing ADCC activity in a subject) is provided. The kit or article may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed of a variety of materials (such as glass or plastic). The container holds a composition by itself or in combination with another composition that can be effectively used in the methods provided herein (e.g., treating the above-mentioned diseases or conditions, 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 may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the present disclosure. The label or package insert indicates that the composition is used for the methods provided herein (e.g., treating the above-mentioned diseases or conditions, reducing the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject, or inducing ADCC activity in a subject). In addition, the kit or article of manufacture may include (a) a first container containing a composition, wherein the composition comprises an antibody of the present disclosure; and (b) a second container containing a composition, wherein the composition comprises another therapeutic agent. The kit or article of manufacture in this embodiment of the invention may also include a package insert indicating that the composition can be used to treat a specific disease or condition (e.g., as described herein), reduce the number of peripheral blood NK cells and / or T cells expressing CD94 in a subject, or induce ADCC activity in a subject. Alternatively or in addition, the kit or article of manufacture may also include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. It may also include other materials that may be desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0249] The following description is provided to enable one of ordinary skill in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications of the embodiments described herein will be apparent to one of ordinary skill in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the embodiments described and shown herein, but to conform to the scope consistent with the claims.

[0250] Example

[0251] Example 1: Anti-CD94 Antibody Generation and Evaluation

[0252] This example describes the generation and characterization of antibodies specific for human CD94.

[0253] Materials and methods

[0254] Anti-CD94 antibody generation and screening

[0255] The ATX-Gx Alloy transgenic mice (mice producing human antibodies) of 4 weeks old were subcutaneously immunized with CD94 labeled with C-terminal His for five weeks, with antigen boosting once a week. Before and after boosting, the antibody titer in the mouse serum was assessed via ELISA and flow cytometry. The mice with the highest serum antibody titer were selected to provide the B cells for producing hybridomas.

[0256] Before cell fusion, mice were administered an additional boost of CD94-His antigen. Mice were then sacrificed and their spleens were collected. Splenocytes and SP2 / 0-Ag14 myeloma cells were mixed and then fused by incubation or electroporation at 37°C in the presence of polyethylene glycol (PEG). Cells were then collected and inoculated into 96-well plates in the case of limiting dilution to achieve one cell per well. Cells were subsequently treated with hypoxanthine, aminopterin and thymidine (HAT) culture medium and cells were selected to culture for more than 2 weeks.

[0257] Hybridoma supernatants were screened using ELISA and flow cytometry to identify candidates with specificity for CD94. For the ELISA, CD94-His antigen was immobilized on a plate and 100 μl of each supernatant was incubated with the antigen. Antibodies captured on the ELISA plate were detected using a fluorescently labeled secondary antibody, and positive hits were validated by analyzing antibody binding on human primary NK cells using flow cytometry. Cynomolgus CD94 cross-reactivity was assessed by antibody binding to BaF3 cells expressing cyno-CD94 using flow cytometry.

[0258] Verification of the VH and VL sequences was performed using standard RNA extraction of hybridomas, followed by reverse transcription of the RNA into cDNA and PCR using Alloy ATC-Gx specific primers.

[0259] Healthy donor and patient samples

[0260] Fresh healthy donor buffy coats were obtained from Stanford Blood Center. Peripheral blood mononuclear cells (PBMCs) were separated via ficoll-paque (GE Healthcare, Chicago, IL) and cryopreserved in Bambanker cell freezing medium (Bulldog-Bio, Portsmouth, NH). In brief, buffy coats were diluted in phosphate buffered saline (PBS) at a ratio of 1:1, followed by layering of the diluted buffy coats and centrifugation at 760 g in ficoll. Before downstream analysis, the PBMC layer was separated and washed in PBS. Peripheral blood leukocytes (PBLs) were separated by erythrocyte lysis.

[0261] Antibody affinity analysis

[0262] Healthy donor PBMCs were seeded in 96-well plates at a density of 100,000 cells per well and incubated on ice for 30 minutes with human Fc blockers (Biolegend) and cell viability dyes (Thermo Fisher) and protected from light. Wash the cells once with FACS buffer (PBS containing 2% IgG low FBS). Anti-CD94 antibodies with a concentration of 100nM to 0.046nM and 1:3 times dilution were incubated on ice with cells for 30 minutes and protected from light. The cells were then washed and incubated on ice for 30 minutes with goat IgG anti-mouse Fcy specific AF647 secondary antibodies or goat (Fab) 2 fragment anti-human Fcy specific AF647 secondary antibodies (Jackson immunoresearch), anti-CD3 Pacific Blue markers and anti-CD56 FITC-labeled antibodies (Biolegend) and protected from light. After incubation, the cells were washed for the last time in FACS buffer and then quantified on 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 lymphocytes on forward and side scatter, followed by double and dead cell exclusion, and gating on CD3-CD56+ populations. CD94 expression was then quantified on the CD3-CD56+ NK cell population. Antibody titration curves and EC50 were generated using Graphpad Prism.

[0263] Antibody cross-reactivity analysis of cynomolgus monkey CD94

[0264] HEK293 cells expressing cynomolgus CD94 were seeded in 96-well plates at a density of 100,000 cells per well and incubated on ice for 30 minutes with cell viability dye (Thermo Fisher) and protected from light. Wash the cells once with FACS buffer (PBS containing 2% IgG low FBS). Hybridoma supernatants, anti-CD94 antibodies, and isotype controls were incubated with cells on ice for 30 minutes and protected from light. The cells were then washed and incubated on ice for 30 minutes with goat IgG anti-mouse Fcy-specific AF647 secondary antibodies or goat (Fab) 2 fragment anti-human Fcy-specific AF647 secondary antibodies (Jackson ImmunoResearch) . After incubation, the cells received a final wash in FACS buffer and were then quantified on Cytoflex (Beckman Coulter). All data acquisition and fluorescence compensation were performed using CytoFlex (Beckman Coulter, Atlanta, GA). Data analysis was performed using FlowJo software. HEK293 cells were identified by gating the main cell population using forward and side scatter, followed by doublet and dead cell exclusion. CD94 expression was then quantified on this population. MFI bar graphs were generated using Graphpad Prism.

[0265] HLA-E blocking assay

[0266] Healthy donor PBMCs were seeded in 96-well plates at a density of 100,000 cells per well and incubated on ice for 30 minutes with human Fc blockers (Biolegend) and cell viability dyes (Thermo Fisher) and protected from light. Wash the cells once with FACS buffer (PBS containing 2% IgG low FBS). Anti-CD94 antibodies at EC80 concentrations were incubated with cells on ice for 30 minutes and protected from light. After incubation with anti-CD94 antibodies, the cells were washed and incubated on ice for 30 minutes with HLA-E tetramer PE (CreativeBiolabs), anti-CD3 Pacific Blue markers and anti-CD56 FITC-labeled antibodies (Biolegend) and protected from light. The cells were then washed for the last time in FACS buffer and subsequently quantified on Cytoflex (BeckmanCoulter). 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 on forward and side scatter, followed by doublet and dead cell exclusion, and gating on the CD3-CD56+ population. HLA-E expression was then quantified on 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)*100).

[0267] Antibody competition assay

[0268] Healthy donor PBMCs were seeded in 96-well plates at a density of 100,000 cells per well, incubated on ice for 30 minutes with human Fc blockers (Biolegend) and cell viability dyes (Thermo Fisher) and protected from light. Wash the cells once with FACS buffer (PBS containing 2% IgG low FBS). Unconjugated anti-CD94 antibodies, CD3 FITC and CD56 PE antibodies at EC80 concentrations, and APC-labeled anti-CD94 antibodies were incubated with cells on ice for 30 minutes and protected from light. The cells were then washed for the last time in FACS buffer and subsequently quantified on 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 lymphocytes on forward and side scatter, followed by double and dead cell exclusion, and gating on CD3-CD56+ populations. Anti-CD94 APCs were then quantified on the CD3-CD56+ NK cell population. All titration curves and EC50s were generated using Graphpad Prism.

[0269] Antibody internalization assay

[0270] Healthy donor PBMCs were seeded in 96-well plates at a density of 100,000 cells per well in RPMI containing 10% IgG low FBS and incubated with human Fc blocker (Biolegend) for 10 minutes at room temperature. Unconjugated anti-CD94 antibodies at EC80 concentrations were incubated with cells for 30 minutes to 24 hours at 4°C and 37°C. The cells were washed once with FACS buffer (PBS containing 2% IgG low FBS) and kept on ice for the remaining procedures. Goat anti-mouse Fcy specific antibodies were incubated with cells on ice for 30 minutes and protected from light. The cells were then washed once and CD94 expression was quantified on Cytoflex. 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 lymphocytes on forward and side scatter, followed by doublet and dead cell exclusion, and gating on the CD3-CD56+ population. Anti-CD94 APCs were then quantified on 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.

[0271] Antibody-dependent cellular cytotoxicity assay

[0272] Approximately 1×10 5 – 2×10 5 Fresh or frozen PBMCs were plated in RPMI containing 10% low IgG FBS in a tissue culture treated 96-well U-bottom plate. 1 –10 -6 ug / ml of human IgG1 isotype control antibody or 1E4 fucosylation and 18H3-KIF antibody 10 times dilutions were incubated overnight. The cells were then stained with fluorescently labeled antibodies for CD3, CD56 and CD16 to identify the remaining NK cells (see flow cytometry analysis). In the lymphocyte population, the flow cytometer collected a minimum of 10,000 events. The remaining NK / leukemia cell percentage was calculated by standardizing the absolute counts with the number of cells under isotype treatment conditions. IC50 was determined using GraphPad Prism.

[0273] result

[0274] As described above, anti-CD94 antibodies were generated using standard hybridoma technology. Briefly, ATX-Gx Alloy transgenic mice were immunized for five weeks with CD94 labeled with C-terminal His. Mouse B cells with the highest serum antibody titers after five weeks were used to generate hybridomas. Hybridomas were screened by ELISA to identify candidates specific for CD94.

[0275] Positive hits were verified by evaluating binding to human NK cells using flow cytometry analysis. Anti-CD94 antibody clones were evaluated for binding to primary human NK cells by flow cytometry analysis. Supernatants from anti-CD94 hybridomas were used to test the 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.

[0276] The affinity of the antibody clones was also assessed by flow cytometric analysis. Figure 1 The titration curve generated by anti-CD94 antibody clone 18H3 is shown. Anti-CD94 antibody 18H3 showed an affinity of 2.6 nM for human primary NK cells. The affinities determined for other anti-CD94 antibodies were Fig.11 Listed in Figure 2 As shown, screening of hybridoma supernatants revealed binding of 18H3, 1M4, and 1E4 to human primary NK cells as measured by flow cytometry.

[0277] The anti-CD94 hybridoma supernatants were also tested for cross-reactivity with cynomolgus CD94. The hybridomas were screened for cross-reactivity with cynomolgus CD94 by flow cytometry using HEK293 cells expressing cynomolgus CD94. Figure 3A As shown, this analysis revealed that clones 18H3, 1M4, and 1E4 cross-reacted with cynomolgus monkey CD94. Supernatant from clone 20F2 did not cross-react with cynomolgus monkey CD94, but was reactive with human CD94. The cross-reactivity of commercially available anti-CD94 antibodies was also assessed. Figure 3B As shown, none of the commercially available anti-CD94 antibodies tested (HP-3D9, HP-3B1, 131412, 12K45, DX22) exhibited cross-reactivity with cynomolgus monkey CD94.

[0278] These results indicate that the newly generated anti-CD94 antibodies bind to epitopes that are not shared with 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 used in cynomolgus monkey toxicity studies prior to Phase I trials in human subjects.

[0279] After validation, the VH and VL sequences of the validated antibody clones were determined. Table A summarizes the VH and VL sequences of the anti-CD94 clones used in Examples 1-3. Framework and CDR sequences (in bold in Table A) were determined using the Kabat numbering scheme.

[0280] Table A. Sequences of anti-CD94 antibody clones

[0281]

[0282] The ability (or lack thereof) of the validated anti-CD94 antibodies to block HLA-E binding is further evaluated. HLA-E is a ligand of the CD94 / NKG2A heterodimer and, when bound to CD94 / NKG2A, plays a vital role in inhibiting NK cell and CD8+T cell activity. Without wishing to be bound by theory, it is believed that the interaction between HLA-E and CD94 / NKG2A can lead to activation and proliferation of target cells. Therefore, it is beneficial for anti-CD94 antibody candidates not to block CD94:HLA-E interactions in a disease setting.

[0283] The blocking of HLA-E by anti-CD94 antibody clones and commercial anti-CD94 antibodies was evaluated by flow cytometry. Figure 4As shown, 18H3 and 1E4 antibodies do not block HLA-E binding. Over 40% HLA-E blocking was observed for commercially available anti-CD94 antibodies, while 0% blocking was observed for the anti-CD94 antibodies. Because the expression level of CD94 varies from dim (approximately 10,000 receptors) to bright (>100,000 receptors), we suggest that if an antibody blocks more than 20% of HLA-E binding, it can be considered a ligand blocking antibody. In summary, this HLA-E blocking analysis shows that 18H3 and 1E4 antibodies do not block the interaction of CD94 with HLA-E, while commercially available CD94 antibodies block this interaction.

[0284] Competition analysis was performed to determine whether the newly identified antibodies bind to epitopes shared with self and existing antibodies. The anti-CD94 antibodies 18H3, 1M4, and 1E4 were analyzed for competition with each other and with commercially available antibodies. Figure 5A-5B As shown, 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. Therefore, the epitope bound by the 18H3 antibody is not shared with commercially available antibodies.

[0285] Likewise, the 1M4 antibody did not compete with commercially available antibodies. Figure 6A-6B As shown, 1M4 competed with the 1E4 antibody, but not with DX22, 131412, HP-3D9, 12K45, and HP-3B1.

[0286] The 1E4 antibody did not compete with four of the five commercially available anti-CD94 antibodies tested. Figure 7 As shown, 1E4 partially competed with 12K45, but not with HP-3D9, DX22, 131412, and HP-3B1.

[0287] 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 value of more than 50% would be considered high 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 remain on the cell surface and maintain a high receptor density on the effector cells to perform ADCC. Figures 8A-8BThe results in show that the CD94 receptor becomes internalized when bound to the commercial anti-CD94 antibody, but not when bound to the 18H3, 1M4, and 1E4 antibodies. The 18H3, 1M4, and 1E4 antibodies did not internalize more than 50% after binding to CD94. In contrast, a 54%, 56%, 24%, 32%, and 18% reduction in MFI was observed for HP-3D9, DX22, 18H3, 1M4, and 1E4, respectively, compared to 0.5 hours to 24 hours at 37°C.

[0288] To test the ability of anti-CD94 antibodies to induce ADCC of NK cells, an ADDC assay was performed using healthy donor PBMCs. For this assay, the 18H3 antibody was produced in Expi-CHO cells cultured in the presence of kifunidine, a potent inhibitor of the mannosidase I enzyme, to produce 18H3-KIF, which mimics the non-fucosylated antibody. The fucosylated 1E4 antibody was also tested for ADCC. Figures 9A-9B As shown, partially non-fucosylated human IgG1 18H3 and fucosylated 1E4 depleted human NK cells in ex vivo cultured PBMC pools from healthy donors after 24 hours of incubation. Human IgG1 18H3 and 1E4 depleted NK cells in a concentration-dependent manner. The IC50 of 18H3-KIF was determined to be 0.02 μg / ml, while the IC50 of the 1E4 antibody was not determined.

[0289] Finally, the ability of 18H3 antibody to induce ADCC of human leukemia cells was evaluated by ADCC assay using cells from patients with chronic lymphoproliferative disorder of NK cells (CLPD-NK). For this assay, 18H3 antibody was produced in Expi-CHO cells cultured in the presence of kifunine to produce 18H3-KIF, which mimics the non-fucosylated antibody. Fig.10 As shown, partially non-fucosylated human IgG1 18H3 depleted human CLPD-NK leukemia cells in ex vivo cultured PBMC pools of CLPD-NK patients 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 no other cell types were affected.

[0290] Fig.11 The anti-CD94 antibodies were summarized for characterization and functional evaluation relative to commercially available antibodies as described above.

[0291] Example 2: In vivo mouse studies to characterize anti-CD94 antibodies

[0292] This example describes the use of in vivo mouse studies to characterize antibodies specific for human CD94.

[0293] Materials and methods

[0294] In vivo study of humanized IL-15 transgenic mice

[0295] Obtain humanized NSG from Jackson Lab TM -IL-15 transgenic mice. Mice were engrafted with PBMCs from healthy and LGLL donors for 3 and 28 days, respectively. A dose of human IgG1 isotype control or ATX-130 (5 mg / kg) was injected into mice, and 48 hours after administration, the depletion of normal NK cells, normal CD8 T cells, and CD8 T leukemic cells in the blood, spleen, bone marrow, and liver was evaluated by flow cytometry ( Fig. 12A ).

[0296] result

[0297] In IL-15 transgenic mice (NSG TM -IL-15) to evaluate the ability (or lack thereof) of anti-CD94 antibodies to deplete human NK and CD8 T cells. TM -IL-15 transgenic mice are a mouse strain that completely lacks the mouse IL-2Rγ gene, resulting in defects in both adaptive and innate immune systems. The expression of human IL-15 enables efficient engraftment of human immune cells, particularly cytotoxic immune cells, from healthy donors and leukemia patients. Fig. 12B As shown, complete NK cell depletion was observed with ATX-130 treatment (5 mg / kg) relative to isotype control. Partial depletion of CD8 T cells (approximately 50%) was observed. The remaining CD8 T cells were CD94 negative and therefore not depleted. In summary, ATX-130 depletes CD94+ cells, including all human NK cells and 50% of CD8 T cells.

[0298] In IL-15 transgenic mice (NSG TM -IL-15) were used to evaluate the ability (or lack thereof) of ATX-130-KIF to deplete LGLL in blood, spleen, bone marrow, and liver. Fig.13 As shown, more than 50% depletion of LGLL cells was observed with ATX-130 treatment (5 mg / kg) relative to isotype control. The remaining LGLL cells were CD94 negative and therefore not depleted. In conclusion, anti-CD94 antibodies were able to effectively deplete CD94+ LGLL cells in mice.

[0299] Example 3: Characterization of anti-CD94 antibodies in non-human primates

[0300] This example describes exploratory pharmacodynamics (PD) in cynomolgus monkeys to evaluate the efficacy of anti-CD94 antibody treatment in vivo.

[0301] Materials and methods

[0302] In vivo non-human primate exploratory studies

[0303] Two healthy male cynomolgus monkeys, aged 6.5 and 9.2 years and weighing 6.54 and 7.18 kg, respectively, were selected for the study. They were naive to all compounds and had no past medical history before the start of the study. Fig.14A As shown, at Weeks 0 and 8, two doses of ATX-130-KIF (2 mg / kg) were administered to Cyno#1 via intravenous (IV) infusion at a rate of approximately 0.25 mL / min over 60 minutes. At Weeks 0 and 16, two doses of ATX-130-KIF (2 mg / kg) were administered to Cyno#2 via IV infusion at a rate of approximately 0.25 mL / min over 60 minutes. Fig.14A Blood, serum and PBMC were collected before and after dosing at different time intervals as shown. PD was determined by flow cytometry analysis via target cell depletion. The study endpoint is expected to be 22 weeks.

[0304] result

[0305] In order to evaluate the efficacy of ATX-130-KIF in vivo, PD studies were completed in non-human primates (NHPs). Two healthy male cynomolgus monkeys were selected for the study, and their ages were 6.5 years old and 9.2 years old and weighed 6.54 and 7.18 kg (Cyno#1 and Cyno#2), and they had not been exposed to all compounds before the start of the study, and there was no previous medical history. Pharmacodynamic (PD) activity was determined by flow cytometry analysis via target cell depletion. All bioanalysis data were presented to 18 weeks, and the study endpoint was expected to be 22 weeks.

[0306] The ability (or lack thereof) of ATX-130-KIF to deplete cynomolgus monkey NK cells in blood was assessed by flow cytometry analysis. Fig. 14BAs shown, the NK cell composition in the total PBMC in Cyno#1 (left figure) and Cyno#2 (right figure) was 20% and 25% at baseline, respectively. For each dose, 6 hours after ATX-130-KIF was applied, target cells (NK cells) were detected in both animals to be depleted to <5% of PBMC. In Cyno#1, a steady increase in the percentage of NK cells was observed from 6 hours to 336 hours, followed by stabilization at 50% of baseline until the second dose was applied at 1008 hours. After 168 hours after the second dose, NK cell counts were observed to stabilize at 50% of baseline. In Cyno#2, NK cell recovery was slower than in Cyno#1. NK cell counts remained at a low level for 6 weeks after the first dose, and increased in the 8th to 14th week. The application of ATX-130-KIF in the 16th week reduced the NK cell levels 6 hours after administration, and remained low until the latest sample collection. In short, the current results show that anti-CD94 antibodies can show efficacy in the body of cynomolgus monkeys.

[0307] The ability (or lack thereof) of ATX-130-KIF to deplete cynomolgus monkey CD4 T, CD8 T, and B cells in blood was assessed by flow cytometry analysis. Fig.15A As shown in Figure 2, the composition of CD4+T cells, CD8+T cells, and B cells in total PBMCs in Cyno#1 were 27%, 40%, and 4%, respectively, at baseline. Fig. 15B As shown, the composition of CD4+T cells, CD8+T cells, and B cells of total PBMCs in Cyno#2 was 26%, 40%, and 5% at baseline, respectively. Six hours after ATX-130-KIF administration, depletion of T and B cells was detected in both animals. However, these immune cell populations returned to baseline levels 24 hours after administration. The results of this study show that anti-CD94 antibodies only transiently deplete other immune cells without depleting non-CD94 expressing cells.

[0308] Depletion in cynomolgus monkeys was assessed by flow cytometry. Fig.16 As shown, an increase in monocytes was observed 6 hours after administration, but returned to baseline levels after 7 days. The results of the study showed that ATX-130-KIF transiently increased monocytes, suggesting that anti-CD94 antibodies can engage monocytes in performing ADCC and contribute to targeted depletion of NK cells.

[0309] Cynomolgus CD16 expression on monocytes was assessed by flow cytometry. Fig.17As shown, an increase in CD16 on monocytes was observed 24 hours after administration, but returned to baseline levels after 7 days. The results of the study showed that ATX-130-KIF transiently increased CD16 on monocytes, indicating that anti-CD94 antibodies engage monocytes to perform ADCC and contribute to targeted depletion of NK cells.

[0310] Example 4: Characterization of anti-CD94 antibody variants

[0311] 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.

[0312] Table B. Anti-CD94 antibody variants tested.

[0313] Antibody VH domain VL domain Ab.22 SEQ ID NO:29 SEQ ID NO:17 Ab.23 SEQ ID NO:30 SEQ ID NO:17 Ab.24 SEQ ID NO:31 SEQ ID NO:17 Ab.25 SEQ ID NO:32 SEQ ID NO:17 Ab.26 SEQ ID NO:16 SEQ ID NO:33 Ab.27 SEQ ID NO:16 SEQ ID NO:34 Ab.28 SEQ ID NO:16 SEQ ID NO:35 Ab.29 SEQ ID NO:16 SEQ ID NO:36 Ab.30 SEQ ID NO:16 SEQ ID NO:37 Ab.31 SEQ ID NO:16 SEQ ID NO:38 Ab.32 SEQ ID NO:16 SEQ ID NO:39 Ab.33 SEQ ID NO:16 SEQ ID NO:40 Ab.34 SEQ ID NO:16 SEQ ID NO:41

[0314] The Fab form of each antibody was generated and The biolayer interferometry (BLI) system was used to evaluate the binding of each Fab to human CD94 protein. The results are shown in Table C. As compared with the original ATX-130 antibody, the variants showed similar binding to human CD94, indicating that the mutations introduced in the variant variable domains did not significantly affect the binding (i.e., the dissociation rate).

[0315] Table C. Binding of anti-CD94 Fab variants to human CD94 protein.

[0316] Sample ID k dissociation (1 / s) ATX-130 2.90E-03 Ab.22 2.38E-03 Ab.23 3.37E-03 Ab.24 6.95E-03 Ab.25 3.87E-03 Ab.26 4.46E-03 Ab.27 4.21E-03 Ab.28 4.25E-03 Ab.29 3.38E-03 Ab.30 2.82E-03 Ab.31 3.52E-03 Ab.32 3.10E-03 Ab.33 4.81E-03 Ab.34 5.76E-03

[0317] The variant antibodies were also tested for binding to HEK cells expressing human CD94. Variants and ATX-130 antibodies in the form of human IgG1 were generated, and flow cytometry was used to evaluate binding to HEK cells expressing human CD94. The experiment was performed in duplicate, and the average EC50 of the 2 experiments was calculated. The results are shown in Table D. As compared to the original ATX-130 antibody, 11 of the 13 variants showed similar binding to cells expressing human CD94 (as measured by EC50), but 2 of the 13 variants (Ab.23 and Ab.30) lost binding to cells expressing human CD94.

[0318] Table D. Binding of anti-CD94 human IgG1 variant antibodies to cells expressing human CD94.

[0319]

[0320] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the present 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, wherein the antibody comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises the amino acid sequence EVQLX1ESGGGLVKPGGS LRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISX2SSX3X4IYY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGRYYYYMDVWGKGTTVTVSS, wherein X1 is L or V; wherein X2 is T or S; wherein X3 is N or S; and wherein X4 is F or Y (SEQ ID NO: 69); wherein the VL domain comprises the amino acid sequence DIX1MTQSPSSLSASVGDR VTITCRASQSISSWLAWX2QQKPX3KX4PKX5LIYAASSLQSGVPSX6FSGSGSGTDFTLTISSLQPEDX7ATYYCQX8YNSX9PFTFGPGTKVDI K, wherein X1 is V or Q; wherein X2 is Y or F; wherein X3 is G or E; wherein X4 is A or V; wherein X5 is S or L; wherein X6 is K or R; wherein X7 is V or F; wherein X8 is K or Q; and wherein X9 is A or Y (SEQ ID NO:70); and 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).

2. An antibody that binds to human CD94, wherein the antibody comprises 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); wherein 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); and 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).

3. The antibody of claim 1 or claim 2, wherein the VH domain comprises: (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) 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); (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).

4. The antibody of any one of claims 1-3, wherein the VH domain further comprises: (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).

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

6. The antibody of any one of claims 1-5, wherein the VL domain comprises: (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) 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).

7. The antibody of any one of claims 1-6, wherein the VL domain further comprises: (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), WF QQKPGKAPKSLIY (SEQ ID NO:56), WYQQKPEKAPKSLIY (SEQ ID NO:57), WYQQKPGKVPKSLIY (SEQ ID NO:58), and WYQQKPGKAPKLLIY (SEQ ID NO: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) FR4 comprising the amino acid sequence of FGPGTKVDIK (SEQ ID NO: 63).

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

9. The antibody of claim 1 or claim 2, wherein: (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; (1) 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; (11) 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.

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

11. The antibody of any one of claims 1-9, 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 11-13, wherein the antibody binds to human cellular Fcγ receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region.

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

16. A polynucleotide encoding the antibody of any one of claims 1-15. A vector comprising the polynucleotide according to claim 16 .

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

19. An isolated host cell comprising the polynucleotide of claim 16 or the vector of claim 17 or claim 18.

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

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 pharmaceutically 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 the antibody of any one of claims 1-15 or the composition of claim 22.

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

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

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

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

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

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

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

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

32. The method of claim 31, wherein the NK cell 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 intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous γ / δ TCL, or subcutaneous panniculitis TCL.

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

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

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

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

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

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

39. 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 subject an effective amount of an antibody as described in any one of claims 1-15 or a composition as described in claim 22.

40. The method of claim 39, wherein administration of the antibody or composition results in NK cell depletion in the subject prior to administration of the CAR-T therapy.

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

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

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

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