Anti-CD161 antibodies and uses thereof

Abstract

The present invention relates generally to anti-CD161 antibodies, pharmaceutical compositions comprising such antibodies, and methods of using such antibodies to treat disorders associated with or mediated by CD161, such as certain cancers. Additionally, the present invention also relates to expression vectors and host cells for producing these antibodies.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 236,122, filed August 23, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference in its entirety. The XML file, created on August 11, 2022, is named IMT-001WO_SL.xml and is 206,992 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates generally to anti-CD161 antibodies, pharmaceutical compositions comprising these antibodies, and methods of using these antibodies to treat disorders associated with or mediated by CD161, such as certain cancers. Additionally, the present invention also relates to expression vectors and host cells for producing these antibodies. [Background technology]

[0004] Modulation of immune cell checkpoint receptors using antibody-directed therapeutic approaches has attracted interest over the past decade. Many of these receptors are involved in T cell checkpoint modulation. However, checkpoint modulation of B cells, natural killer (NK) cells, and myeloid cells in particular is highly desirable and has attracted considerable attention. NK cells are part of the innate immune system, recognizing and inducing cytotoxicity against a wide range of target cells, such as tumor cells or virus-infected cells. Activated NK cells typically kill target cells by means similar to cytotoxic T cells, namely, via cytolytic granules containing perforin and granzymes, as well as via death receptor pathways. Activated NK cells also secrete proinflammatory cytokines, such as IFN-γ, and chemokines, which promote the recruitment of other leukocytes to target tissues.

[0005] NK cell receptors (NKRs) are divided into two major structural classes: the immunoglobulin superfamily and the C-type lectin-like (CLR) superfamily. NKR-protein 1 (NKR-P1) (e.g., CD161) is a key immunoregulatory gene and a family of CLR transmembrane molecules expressed on various cell types, including splenic dendritic cells, subsets of T cells, and granulocytes. Lectin-like transcript 1 (LLT1), C-type lectin domain family 2 member D (CLEC2D), or osteoclast inhibitory lectin (OCIL) molecules, are cognate ligands for the CD161 receptor, and this interaction inhibits NK cell and T cell function. There are six splice variants of CLEC2D, with isoform 1 being the canonical sequence expressed on NK cells, T cells, monocytes / macrophages, activated B cells, and dendritic cells and functioning as a human NK cell-activating receptor. The polypeptide chain of CLEC2D contains multiple domains, including an N-terminal cytoplasmic domain, a transmembrane domain, a stalk region, and a C-terminal CLR extracellular domain, and has two predicted N-glycosylation sites.

[0006] The interaction between CLEC2D and CD161 can lead to certain disorders, such as certain cancers, that evade the subject's immune system. Such immune evasion or escape has been reported in human glioblastoma and other diseases. Furthermore, CLEC2D expression on germinal center B cells is thought to regulate crosstalk between NK cells and antigen-presenting cells (APCs). Therefore, blocking the CLEC2D-CD161 interaction provides a new therapeutic option for treating various cancers. Summary of the Invention [Problem to be solved by the invention]

[0007] Although the intracellular signaling downstream of CLEC2D-CD161 interaction is not well defined, the interaction between CLEC2D and CD161 has been shown to inhibit the function of both NK cells and T cells. Despite the progress that has been made in the treatment of certain cancers, there remains a need for new and innovative therapies to treat certain cancers, particularly those that evade the immune system. [Means for solving the problem]

[0008] Summary of the Invention The present invention is based in part on the discovery of high affinity anti-CD161 antibodies that disrupt the CLEC2D-CD161 interaction, which can be used, inter alia, to prevent certain cancers from evading a subject's immune system.

[0009] Accordingly, in one aspect, the disclosure provides an isolated anti-CD161 antibody comprising a light chain variable region; and a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences, wherein (a) the CDR-H1 sequence is FX1FX2X3X4AMS (SEQ ID NO: 1); (b) the CDR-H2 sequence is AISX5X6GGX7TX8YADSVKG (SEQ ID NO: 2); and (c) the CDR-H3 sequence is AKPLDSSX9WADFX 10 X 11(SEQ ID NO: 3); X1 is T or A; X2 is G, S, or E; X3 is Q, T, P, or R; X4 is Y or F; X5 is A or G; X6 is A, V, or S; X7 is T or S; X8 is K, A, or Y; X9 is Q, F, or L; 10 is D or Q; and X 11 is L or A. In certain embodiments, the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 sequences, and (d) the CDR-L1 sequence is RASQX 12 IX 13 SWLA (SEQ ID NO: 4); (e) the CDR-L2 sequence is X 14 ASX 15 LQX 16 (SEQ ID NO: 5); and (f) the CDR-L3 sequence is QQX 17 X 18 X 19 LPIT (SEQ ID NO: 6); X 12 is G, D, or T; X 13 is D, S, or Y; X 14 is A, Y, or F; X 15 is S, A, G, or F; X 16 is D or S; X 17 is A, H, or Q; X 18 is S, D, W, or L; and X 19 is V, D, Y, or K.

[0010] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 8; (b) the CDR-H2 sequence is SEQ ID NO: 9; and (c) the CDR-H3 sequence is SEQ ID NO: 10. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 12; (e) the CDR-L2 sequence is SEQ ID NO: 13; and (f) the CDR-L3 sequence is SEQ ID NO: 14.

[0011] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 16; (b) the CDR-H2 sequence is SEQ ID NO: 17; and (c) the CDR-H3 sequence is SEQ ID NO: 18. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 20; (e) the CDR-L2 sequence is SEQ ID NO: 21; and (f) the CDR-L3 sequence is SEQ ID NO: 22.

[0012] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 24; (b) the CDR-H2 sequence is SEQ ID NO: 25; and (c) the CDR-H3 sequence is SEQ ID NO: 26. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 28; (e) the CDR-L2 sequence is SEQ ID NO: 29; and (f) the CDR-L3 sequence is SEQ ID NO: 30.

[0013] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 32; (b) the CDR-H2 sequence is SEQ ID NO: 33; and (c) the CDR-H3 sequence is SEQ ID NO: 34. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 36; (e) the CDR-L2 sequence is SEQ ID NO: 37; and (f) the CDR-L3 sequence is SEQ ID NO: 38.

[0014] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 40; (b) the CDR-H2 sequence is SEQ ID NO: 41; and (c) the CDR-H3 sequence is SEQ ID NO: 42. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 44; (e) the CDR-L2 sequence is SEQ ID NO: 45; and (f) the CDR-L3 sequence is SEQ ID NO: 46.

[0015] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 48; (b) the CDR-H2 sequence is SEQ ID NO: 49; and (c) the CDR-H3 sequence is SEQ ID NO: 50. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 52; (e) the CDR-L2 sequence is SEQ ID NO: 53; and (f) the CDR-L3 sequence is SEQ ID NO: 54.

[0016] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 56; (b) the CDR-H2 sequence is SEQ ID NO: 57; and (c) the CDR-H3 sequence is SEQ ID NO: 58. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 60; (e) the CDR-L2 sequence is SEQ ID NO: 61; and (f) the CDR-L3 sequence is SEQ ID NO: 62.

[0017] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 64; (b) the CDR-H2 sequence is SEQ ID NO: 65; and (c) the CDR-H3 sequence is SEQ ID NO: 66. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 68; (e) the CDR-L2 sequence is SEQ ID NO: 69; and (f) the CDR-L3 sequence is SEQ ID NO: 70.

[0018] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 72; (b) the CDR-H2 sequence is SEQ ID NO: 73; and (c) the CDR-H3 sequence is SEQ ID NO: 74. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 76; (e) the CDR-L2 sequence is SEQ ID NO: 77; and (f) the CDR-L3 sequence is SEQ ID NO: 78.

[0019] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 80; (b) the CDR-H2 sequence is SEQ ID NO: 81; and (c) the CDR-H3 sequence is SEQ ID NO: 82. Alternatively or additionally, in some embodiments, (d) the CDR-L1 sequence is SEQ ID NO: 84; (e) the CDR-L2 sequence is SEQ ID NO: 85; and (f) the CDR-L3 sequence is SEQ ID NO: 86.

[0020] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 88; (b) the CDR-H2 sequence is SEQ ID NO: 89; and (c) the CDR-H3 sequence is SEQ ID NO: 90. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 92; (e) the CDR-L2 sequence is SEQ ID NO: 93; and (f) the CDR-L3 sequence is SEQ ID NO: 94.

[0021] In some embodiments, the disclosure provides an isolated anti-CD161 antibody, wherein the heavy chain variable region has the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFX1FX2X3X4AMSWVRQAPGKGLEWVSAISX5X6GGX7TX8YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPLDSSX9WADFX 10 X 11 X is T or A; X is G, S, or E; X is Q, T, P, or R; X is Y or F; X is A or G; X is A, V, or S; X is T or S; X is K, A, or Y; X is Q, F, or L; 10 is D or Q; and X 11 is L or A.

[0022] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, and SEQ ID NO:87. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, and SEQ ID NO:87.

[0023] In some embodiments, the disclosure provides an isolated anti-CD161 antibody, wherein the light chain variable region has the amino acid sequence: DIQX a TQSPSSVSASVGDRVTITCRASQX 12 IX 13 SWLAWYQQKPGKAPKX b LIYX 14 ASX 15 LQX 16 GVPSRFSGSGSGTDFTLTIX c SLQPEDFATYYCQQX 17 X 18 X 19 LPITFGGGTKVEIK (SEQ ID NO: 189), 12 is G, D, or T; X 13 is D, S, or Y; X 14 is A, Y, or F; X 15 is S, A, G, or F; X 16 is D or S; X 17 is A, H, or Q; X 18 is S, D, W, or L; X 19 is V, D, Y, or K; X a is M or L; X b is L or F; and X c is S or N.

[0024] In some embodiments, the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, and SEQ ID NO: 91. In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, and SEQ ID NO: 91.

[0025] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 11. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 7, and the light chain variable region comprises the sequence of SEQ ID NO: 11.

[0026] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 15, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 19. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 15, and the light chain variable region comprises the sequence of SEQ ID NO: 19.

[0027] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 23, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 27. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 23, and the light chain variable region comprises the sequence of SEQ ID NO: 27.

[0028] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 31, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 35. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 31, and the light chain variable region comprises the sequence of SEQ ID NO: 35.

[0029] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 39, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 43. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 39, and the light chain variable region comprises the sequence of SEQ ID NO: 43.

[0030] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 47, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 51. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 47, and the light chain variable region comprises the sequence of SEQ ID NO: 51.

[0031] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 55, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 59. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 55, and the light chain variable region comprises the sequence of SEQ ID NO: 59.

[0032] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 63, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 67. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 63, and the light chain variable region comprises the sequence of SEQ ID NO: 67.

[0033] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 71, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 75. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 71, and the light chain variable region comprises the sequence of SEQ ID NO: 75.

[0034] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 79, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 83. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 79, and the light chain variable region comprises the sequence of SEQ ID NO: 83.

[0035] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 87, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 91. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 87, and the light chain variable region comprises the sequence of SEQ ID NO: 91.

[0036] In another aspect, the disclosure provides an isolated anti-CD161 antibody thereof comprising a light chain variable region; and a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences, wherein (a) the CDR-H1 sequence is FTFX1X2YYMS (SEQ ID NO: 95); (b) the CDR-H2 sequence is YISPSGX3TIX4YADSVKG (SEQ ID NO: 96); and (c) the CDR-H3 sequence is ARSLMX5TGTHLYFDL (SEQ ID NO: 97); X1 is G, A, P, or S; X2 is N, Q, or D; X3 is A or S; X4 is Y or A; and X5 is A or S. In certain embodiments, the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3 sequences, wherein (a) the CDR-L1 sequence is RASX6X7ISX8WLA (SEQ ID NO: 98); (b) the CDR-L2 sequence is AAX9X 10 LQS (SEQ ID NO: 99); and (c) the CDR-L3 sequence is QQX 11 TSX 12 X 13 X6 is Q or S; X7 is D or G; X8 is D or S; X9 is E or S; X 10 is S, A, G, V, or E; X 11 is A, S, or V; X 12 is F, T, V, Q, or A; and X 13 is L or P.

[0037] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 102; (b) the CDR-H2 sequence is SEQ ID NO: 103; and (c) the CDR-H3 sequence is SEQ ID NO: 104. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 106; (e) the CDR-L2 sequence is SEQ ID NO: 107; and (f) the CDR-L3 sequence is SEQ ID NO: 108.

[0038] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 110; (b) the CDR-H2 sequence is SEQ ID NO: 111; and (c) the CDR-H3 sequence is SEQ ID NO: 112. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 114; (e) the CDR-L2 sequence is SEQ ID NO: 115; and (f) the CDR-L3 sequence is SEQ ID NO: 116.

[0039] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 118; (b) the CDR-H2 sequence is SEQ ID NO: 119; and (c) the CDR-H3 sequence is SEQ ID NO: 120. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 122; (e) the CDR-L2 sequence is SEQ ID NO: 123; and (f) the CDR-L3 sequence is SEQ ID NO: 124.

[0040] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 126; (b) the CDR-H2 sequence is SEQ ID NO: 127; and (c) the CDR-H3 sequence is SEQ ID NO: 128. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 130; (e) the CDR-L2 sequence is SEQ ID NO: 131; and (f) the CDR-L3 sequence is SEQ ID NO: 132.

[0041] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 134; (b) the CDR-H2 sequence is SEQ ID NO: 135; and (c) the CDR-H3 sequence is SEQ ID NO: 136. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 138; (e) the CDR-L2 sequence is SEQ ID NO: 139; and (f) the CDR-L3 sequence is SEQ ID NO: 140.

[0042] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 142; (b) the CDR-H2 sequence is SEQ ID NO: 143; and (c) the CDR-H3 sequence is SEQ ID NO: 144. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 146; (e) the CDR-L2 sequence is SEQ ID NO: 147; and (f) the CDR-L3 sequence is SEQ ID NO: 148.

[0043] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 150; (b) the CDR-H2 sequence is SEQ ID NO: 151; and (c) the CDR-H3 sequence is SEQ ID NO: 152. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 154; (e) the CDR-L2 sequence is SEQ ID NO: 155; and (f) the CDR-L3 sequence is SEQ ID NO: 156.

[0044] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 158; (b) the CDR-H2 sequence is SEQ ID NO: 159; and (c) the CDR-H3 sequence is SEQ ID NO: 160. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 162; (e) the CDR-L2 sequence is SEQ ID NO: 163; and (f) the CDR-L3 sequence is SEQ ID NO: 164.

[0045] In some embodiments, (a) the CDR-H1 sequence is SEQ ID NO: 166; (b) the CDR-H2 sequence is SEQ ID NO: 167; and (c) the CDR-H3 sequence is SEQ ID NO: 168. In some embodiments, alternatively or additionally, (d) the CDR-L1 sequence is SEQ ID NO: 170; (e) the CDR-L2 sequence is SEQ ID NO: 171; and (f) the CDR-L3 sequence is SEQ ID NO: 172.

[0046] In some embodiments, the heavy chain variable region has the amino acid sequence: QVQLVESGGGLVX aPGGSLRLSCAASGFTFX1X2YYMSWIRQAPGKGLEWVSYISPSGX3TIX4YADSVKGRFTISRDNX b KNX c X1 is G, A, P, or S; X2 is N, Q, or D; X3 is A or S; X4 is Y or A; X5 is A or S; a is K or Q; X b is A or S; and X c is S or T.

[0047] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to a sequence selected from the group consisting of SEQ ID NO: 101, SEQ ID NO: 109, SEQ ID NO: 117, SEQ ID NO: 125, SEQ ID NO: 133, SEQ ID NO: 141, SEQ ID NO: 149, SEQ ID NO: 157, and SEQ ID NO: 165. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 101, SEQ ID NO: 109, SEQ ID NO: 117, SEQ ID NO: 125, SEQ ID NO: 133, SEQ ID NO: 141, SEQ ID NO: 149, SEQ ID NO: 157, and SEQ ID NO: 165.

[0048] In some embodiments, the light chain variable region has the amino acid sequence: DIQLTQSPSSVSASVGDRVTITC RASX6X7ISX8WLAWYQQKPGKAPKLLIYAAX9X 10 LQS GVPSRFSGSGSGTFTLTISSLQPEDFATYYC QQX 11 TSX 12 X 13 X6 is Q or S; X7 is D or G; X8 is D or S; X9 is E or S; 10 is S, A, G, V, or E; X 11 is A, S, or V; X 12 is F, T, V, Q, or A; and X 13is L or P.

[0049] In some embodiments, the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to a sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 121, SEQ ID NO: 129, SEQ ID NO: 137, SEQ ID NO: 145, SEQ ID NO: 153, SEQ ID NO: 161, and SEQ ID NO: 169. In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 113, SEQ ID NO: 121, SEQ ID NO: 129, SEQ ID NO: 137, SEQ ID NO: 145, SEQ ID NO: 153, SEQ ID NO: 161, and SEQ ID NO: 169.

[0050] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 101, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 105. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 101, and the light chain variable region comprises the sequence of SEQ ID NO: 105.

[0051] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 109, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 113. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 109, and the light chain variable region comprises the sequence of SEQ ID NO: 113.

[0052] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 117, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 121. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 117, and the light chain variable region comprises the sequence of SEQ ID NO: 121.

[0053] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 125, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 129. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 125, and the light chain variable region comprises the sequence of SEQ ID NO: 129.

[0054] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 133, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 137. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 133, and the light chain variable region comprises the sequence of SEQ ID NO: 137.

[0055] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 141, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 145. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 141, and the light chain variable region comprises the sequence of SEQ ID NO: 145.

[0056] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 149, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 153. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 149, and the light chain variable region comprises the sequence of SEQ ID NO: 153.

[0057] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 157, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 161. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 157, and the light chain variable region comprises the sequence of SEQ ID NO: 161.

[0058] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 165, and the light chain variable region comprises an amino acid sequence at least 90% identical (e.g., at least 95% identical) to the sequence of SEQ ID NO: 169. In some embodiments, the heavy chain variable region comprises the sequence of SEQ ID NO: 165, and the light chain variable region comprises the sequence of SEQ ID NO: 169.

[0059] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is multispecific. In some embodiments, the antibody is a Fab, Fab', F(Ab')2, Fv, scFv, (scFv)2, single-chain antibody molecule, dual variable domain antibody, single variable domain antibody, linear antibody, or V-domain antibody. In some embodiments, the antibody comprises a scaffold. In some embodiments, the scaffold is Fc. In some embodiments, the scaffold is human Fc. In some embodiments, the antibody comprises a heavy chain constant region of a class selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antibody comprises a heavy chain constant region of the class IgG and a subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0060] In certain embodiments, the monoclonal antibody is an aglycosylated human IgG1 antibody. In certain embodiments, the monoclonal antibody comprises an IgG1 Fc region having an alteration at amino acid position N297 (e.g., N297A) according to EU numbering.

[0061] In another aspect, the disclosure provides an isolated antibody that competes with an anti-CD161 antibody disclosed herein for binding to CD161.

[0062] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to the CD161 epitope bound by an anti-CD161 antibody disclosed herein. In some embodiments, the antibody competes with CLEC2D for binding to CD161.

[0063] In some embodiments, the antibody reduces CD161 inhibitory signaling due to CLEC2D binding to CD161. In some embodiments, CD161 is expressed on the surface of a cell. In some embodiments, the cell is a T cell or an NK cell. In some embodiments, the cell is a human cell or a cynomolgus monkey cell. In some embodiments, the antibody reduces suppression of T cell or NK cell activity due to CLEC2D binding to CD161.

[0064] In some embodiments, the antibodies of the present disclosure increase T cell or NK cell activity in the presence of CLEC2D compared to T cell or NK cell activity in the absence of the antibody. In some embodiments, the T cells or NK cells are disposed in a microenvironment comprising cells expressing CLEC2D. In some embodiments, the antibody increases T cell or NK cell activity in a tumor microenvironment containing tumor cells expressing CLEC2D. In some embodiments, the increase in T cell activity is determined by an increase in NFAT signaling. In some embodiments, the antibody has an EC for T cell activation of between 1.5 nM and 4.1 nM. 50 For example, the antibodies have an EC value for T cell activation of about 1.5 nM, about 1.7 nM, about 2.0 nM, about 2.3 nM, about 2.5 nM, about 2.8 nM, about 3.1 nM, about 3.3 nM, about 3.5 nM, about 3.8 nM, about 4.1 nM. 50In some embodiments, the antibodies have therapeutic value. In some embodiments, the antibodies reverse CLEC2D-mediated inhibition and restore T cell activity. In some embodiments, the antibodies enhance the polyfunctionality of primary antigen-specific human T cells. In some embodiments, the antibodies of the disclosure (i) enhance or restore direct T cell-mediated cytotoxicity affected by CD161-CLEC2D interaction, (ii) increase TNFα secretion, (iii) increase IL-2 secretion, (iv) increase IFNγ secretion, or (v) exhibit any combination of characteristics (i), (ii), (iii), or (iv).

[0065] In some embodiments, the increase in NK cell activity is determined by an increase in CD107a expression. In some embodiments, the antibody has an EC50 for NK cell activation of between 0.04 nM and 0.38 nM. 50 For example, the antibodies have an EC50 value for NK cell activation of about 0.04 nM, about 0.06 nM, about 0.08 nM, about 0.1 nM, about 0.12 nM, about 0.14 nM, about 0.16 nM, about 0.18 nM, about 0.20 nM, about 0.22 nM, about 0.24 nM, about 0.28 nM, about 0.30 nM, about 0.32 nM, about 0.34 nM, about 0.36 nM, or about 0.38 nM. 50 In some embodiments, the increase in NK cell activity is determined by an increase in IFNγ expression. In some embodiments, the antibody has an EC value for NK cell activation of between 0.1 nM and 0.5 nM. 50 For example, the antibodies have an EC value for NK cell activation of about 0.1 nM, about 0.15 nM, about 0.2 nM, about 0.25 nM, about 0.3 nM, about 0.35 nM, about 0.4 nM, about 0.45 nM, about 0.5 nM. 50 It has a value.

[0066] In some embodiments, the antibody has a K of 10 nM or less, 0.5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less as measured by an Octet QK384 assay. d In some embodiments, the antibody binds to human CD161 at a concentration of 1 x 10 as measured by Octet QK384 assay.-8 ~1×10 -10 K in the M range d In some embodiments, the antibody binds to human CD161 with an EC50 for HEK293 cells expressing CD161 of less than 5 nM, less than 1 nM, or less than 0.5 nM. 50 In some embodiments, the antibody has an EC 0.1-0.5 nM range for HEK293 cells expressing CD161. 50 In some embodiments, the antibody has an IC in the range of 0.1 to 10 nM. 50 In some embodiments, the antibody reduces CLEC2D binding to CD161 expressed on the surface of a cell with an IC of less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM. 50 In some embodiments, the antibody has a K of greater than 1,000 nM for hKLRF1, hKLRF2, hCLEC12B, hCLEC2D, or any combination thereof. d In some embodiments, the antibody binds to less than 2%, less than 1.5%, less than 1.2%, less than 1.1%, or less than 1% of the population of CD161 homologs compared to the antibody that binds to CD161 under saturating antibody conditions. In some embodiments, the population of CD161 homologs comprises CLEC2D, KLRF1, KLRF2, CLEC12B, or any combination thereof.

[0067] In some embodiments, the antibodies enhance NK cell killing of CLEC2D-expressing cells, for example, under certain circumstances, the antibodies enhance NK cell killing of CLEC2D-expressing cells by blocking the interaction of CD161 with CLEC2D.

[0068] In some embodiments, the antibody enhances the reactivation of antigen-specific effector memory CD4 T cells. In some embodiments, the antibody enhances the reactivation of antigen-specific effector memory CD4 T cells by blocking CD161 interaction with CLEC2D. In some embodiments, the antibody enhances cytokine production of MART-1-specific T cells. In some embodiments, the antibody enhances the cytotoxic function of MART-1-specific T cells. In some embodiments, the antibody does not cause cytokine release syndrome.

[0069] (c) binds to the CD161 epitope bound by an anti-CD161 antibody or antigen-binding fragment thereof; (d) binds to human CD161 at or near the CLEC2D binding site; (e) competes with CLEC2D for binding to CD161; (f) reduces CD161 inhibitory signaling due to CLEC2D binding to CD161; (g) reduces the suppression of T cell activity due to CLEC2D binding to CD161; (h) reduces the suppression of NK cell activity due to CLEC2D binding to CD161; (i) increases T cell activity in the presence of CLEC2D compared to T cell activity in the absence of the antibody or antigen-binding fragment thereof; (j) increases NK cell activity in the presence of CLEC2D compared to NK cell activity in the absence of the antibody or antigen-binding fragment thereof. (k) increasing the activity of T cells disposed in a microenvironment comprising cells expressing CLEC2D; (l) increasing the activity of NK cells disposed in a microenvironment comprising cells expressing CLEC2D; (m) increasing T cell activity in a tumor microenvironment comprising tumor cells expressing CLEC2D; (n) increasing NK cell activity in a tumor microenvironment comprising tumor cells expressing CLEC2D; (o) inhibiting human T cell exhaustion; (p) inducing or increasing activation of CD161-expressing human T cells in response to antigen-expressing target cells; (q) inducing or increasing cytokine production by CD161-expressing human T cells in response to antigen-expressing target cells; (r) inducing or increasing granzyme B expression by CD161-expressing human T cells in response to antigen-expressing target cells; (s) reducing exhaustion of CD161-expressing human T cells in response to antigen-expressing target cells; and (t) any combination of (a)-(s).

[0070] In some embodiments, the antibody binds to human CD161 and cynomolgus CD161, and the binding affinity of the antibody for human CD161 and cynomolgus CD161 differs by no more than 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold. In some embodiments, the antibody binds to human CD161 and cynomolgus CD161, and the binding avidity of the antibody for human CD161 and cynomolgus CD161 differs by no more than 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold.

[0071] In another aspect, the present disclosure also provides a pharmaceutical composition comprising an antibody disclosed herein and a pharmaceutically acceptable carrier.

[0072] In another aspect, the present disclosure also provides an antibody disclosed herein, the V H , that V L The present invention provides an isolated polynucleotide or polynucleotides encoding the antibody, its light chain, its heavy chain, or an antigen-binding portion thereof.

[0073] In another aspect, the present disclosure also provides a vector or vectors comprising the polynucleotide or polynucleotides disclosed herein.

[0074] In another aspect, the present disclosure also provides a host cell comprising a polynucleotide, multiple polynucleotides, or multiple vectors disclosed herein.

[0075] In another aspect, the present disclosure also provides a method of producing an antibody comprising expressing the antibody in a host cell and isolating the expressed antibody.

[0076] In another aspect, the present disclosure also provides a kit comprising an antibody disclosed herein, or a pharmaceutical composition disclosed herein, and instructions for use.

[0077] In another aspect, the present disclosure also provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an immunotherapy (e.g., an anti-CD161 antibody) disclosed herein or an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the cancer is characterized by expression of CLEC2D by cancer cells or other cells in the tumor microenvironment. In some embodiments, the cancer is characterized by increased expression of CLEC2D by cancer cells or other cells in the tumor microenvironment. In some embodiments, the cancer is selected from the group consisting of melanoma, lung cancer, glioma, colorectal cancer, and liver cancer.

[0078] In another aspect, the present disclosure also provides a method for reducing or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein.

[0079] In another aspect, the present disclosure also provides a method for inhibiting or blocking the interaction between human CD161 and CLEC2D in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein.

[0080] In another aspect, the present disclosure also provides a method of inducing or enhancing immune cell activation in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the immune cell activation occurs in the tumor microenvironment. In some embodiments, the immune cells are T cells or NK cells.

[0081] In some embodiments, the present disclosure provides methods of inducing or enhancing a cytotoxic T cell effector response in a subject in need thereof, comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein. Optionally, the T cell effector response is (i) in the tumor microenvironment, (ii) cytokine production (e.g., IL-2, TNFα, IFNγ, or a combination thereof), (iii) granzyme B secretion, or (iv) a combination of two or more of (i), (ii), and (iii).

[0082] These and other aspects and features of the present invention are set forth in the following detailed description and claims.

[0083] The present invention can be more fully understood with reference to the following drawings. [Brief explanation of the drawings]

[0084] [Figure 1A] 1A-1D are graphs showing binding of exemplary human anti-CD161 antibodies to HEK293 cells exogenously expressing human CD161. Iso Ctrl indicates an isotype control antibody. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 2A] 2A-2D are graphs showing binding of exemplary human anti-CD161 antibodies to HEK293 cells exogenously expressing cynomolgus monkey CD161. Iso Ctrl indicates the isotype control antibody. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 3]Figure 3 shows the alignment of human CD161 (KLRB1_human) (SEQ ID NO: 179) with its homologues (killer cell lectin-like receptor subfamily F member 1_human (KLRF1_human) (SEQ ID NO: 183), killer cell lectin-like receptor subfamily F member 2_human (KLRF2_human) (SEQ ID NO: 184), and C-type lectin domain family 12 member B_human (CL12B_human) (SEQ ID NO: 185)). [Figure 4] FIG. 4 shows the alignment of human CD161 (KLRB1_human) (SEQ ID NO: 179) with C-type lectin domain family 2 member D (CLC2D_human) (SEQ ID NO: 186). [Figure 5A] 5A-5B are graphs showing binding of exemplary human anti-CD161 antibodies to HEK293 cells exogenously expressing CD161 or CD161 homologs (CLEC2D, KLRF1, KLRF2, and CLEC12B). Iso Ctrl indicates isotype control antibody, and null represents control HEK293 cells. [Figure 5B] Same as above. [Figure 6A] 6A-6D are graphs showing human anti-CD161 antibody-mediated blockade of the interaction between CD161 and CLEC2D using HEK293 cells overexpressing human CD161 protein and soluble biotinylated Fc-human CLEC2D (H176C) protein. Iso Ctrl indicates the isotype control antibody. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 7A] 7A-7D are graphs showing dose-dependent anti-CD161 antibody-mediated activation of human NK cells as measured by CD107a expression. HP-3G10 represents a mouse anti-human CD161 antibody. Iso Ctrl represents an isotype control antibody. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 8A] Figures 8A-8D are graphs showing anti-CD161 antibody-mediated activation of T cells in a co-culture assay with exogenous CLEC2D expression, as measured by nuclear factor of activated T cells (NFAT) signaling. HP-3G10 represents a mouse anti-human CD161 antibody. An isotype control antibody (Iso Ctrl) was used as a control. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 9] Figure 9 is a graph showing the dose-dependent restoration of NFAT signaling in the presence of human anti-CD161 antibodies Ab8, Ab9, Ab13, and Ab15. An isotype control antibody (Iso Ctrl) was used as a control. [Figure 10A] 10A-10B are graphs showing binding of human anti-CD161 antibodies Ab8, Ab9, Ab13, and Ab15 to human PBMCs (FIG. 10A) and cynomolgus monkey PBMCs (FIG. 10B). Iso Ctrl indicates the isotype control antibody. [Figure 10B] Same as above. [Figure 11] FIG. 11 is a bar graph showing binding of human anti-CD161 antibodies Ab8, Ab9, Ab13, and Ab15 to immune cell populations (MAIT T cells, B cells, monocytes, and dendritic cells) in human PBMCs. [Figure 12A-1] Figures 12A-12B show alignments of antibodies from the first family used to generate consensus sequences for VH and VL, respectively. Figure 12A discloses SEQ ID NOS: 188, 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, and 87 (in order of appearance), respectively, which correspond to the consensus sequence (Cons) to Ab11. Figure 12B discloses SEQ ID NOS: 189, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, and 91 (in order of appearance), respectively, which correspond to the consensus sequence (Cons) to Ab11. [Figure 12A-2] This is a continuation of Figure 12A-1. [Figure 12B-1] As shown in Figure 12A-1. [Figure 12B-2] This is a continuation of Figure 12B-1. [Figure 13A-1] Figures 13A-13B show alignments of antibodies from the second family used to generate consensus sequences for VH and VL, respectively. Figure 13A discloses SEQ ID NOS: 190, 101, 109, 117, 125, 133, 141, 149, 157, and 165 (in order of appearance), which correspond to the consensus sequence (Cons) and Ab12 through Ab20, respectively. Figure 13B discloses SEQ ID NOS: 191, 105, 113, 121, 129, 137, 145, 153, 161, and 169 (in order of appearance), which correspond to the consensus sequence (Cons) and Ab12 through Ab20, respectively. [Figure 13A-2] This is a continuation of Figure 13A-1. [Figure 13B-1] As shown in Figure 13A-1. [Figure 13B-2] This is a continuation of Figure 13B-1. [Figure 14A] Figures 14A-14B show the ELISA assay format, which demonstrates soluble receptor binding to FcγRI (Figure 14A) and FcγRII and FcγRIII (Figure 14B). Biotinylated FcγRI, FcγRII, and FcγRIII are indicated as "FcγRI-bio," "FcγRII-bio," and "FcγRIII-bio," respectively. A mouse antibody binding to the kappa light chain is indicated as "mouse anti-kappa." An HRP-conjugated Fab-anti-human Fab fragment is indicated as "F(ab')2 anti-F(ab')2-HRP." [Figure 14B] Same as above. [Figure 15A]Figures 15A-15E are ELISA binding curves showing the binding kinetics of Ab9 or wild-type human IgG1 antibody ("WT") to the Fcγ receptors FcγRI (Figure 15A), FcγRIIA (Figure 15B), FcγRIIB / C (Figure 15C), FcγRIIIA (Figure 15D), and FcγRIIIB (Figure 15E). H167 and R167 represent two allelic variants of FcγRIIA that differ in their ability to ligate human IgG2. F176 and V176 represent two FcγRIIIA alleles that differ in their ability to bind human IgG1 and IgG3; F176 is a low-binding allele, and V176 is a high-binding allele. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 16A] Figure 16A shows FcRn binding by Octet. Figures 16B-16C are exemplary binding curves for Ab9 (Figure 16B) and wild-type human IgG1 antibody ("WT") (Figure 16C) as determined by Octet. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 17A]Figures 17A-17B are graphs showing anti-CD161 antibody-mediated activation of human NK cells isolated from donor RTD165, as measured by CD107a expression, in a dose-dependent manner on target K562 cells expressing CLEC2D (Figure 17A) or GFP (Figure 17B). Figures 17C-17D are graphs showing anti-CD161 antibody-mediated activation of human NK cells isolated from donor RTD165, as measured by IFN-γ secretion, in a dose-dependent manner on target K562 cells expressing CLEC2D (Figure 17C) or GFP (Figure 17D). Figures 17E-17F are graphs showing anti-CD161 antibody-mediated activation of human NK cells isolated from donor MCB117, as measured by CD107a expression, in a dose-dependent manner on target K562 cells expressing CLEC2D (Figure 17E) or GFP (Figure 17F). Figures 17G-17H are graphs showing anti-CD161 antibody-mediated activation of human NK cells isolated from donor MCB117, as measured by IFN-γ secretion, in a dose-dependent manner on target K562 cells expressing CLEC2D (Figure 17G) or GFP (Figure 17H). [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Same as above. [Figure 17F] Same as above. [Figure 17G] Same as above. [Figure 17H] Same as above. [Figure 18A] 18A-18B are graphs showing the killing of Raji target cells by human NK cells isolated from donor #1 (FIG. 18A) or donor #2 (FIG. 18B) in the presence of anti-CD161 antibody (Ab9). [Figure 18B] Same as above. [Figure 19A]Figures 19A-19B are graphs showing that blockade of the CD161-CLEC2D interaction with anti-CD161 antibody (Ab9) results in enhanced cytokine (measured by IFNγ, Figure 19A) and proliferation (measured by Ki67, Figure 19B) responses in memory CD4 T cells. T cells were exposed to CEFX peptide in the presence of anti-CD161 antibody (Ab9) at a concentration of 6.25 nM, an isotype control (iso.control), or left untreated (no antibody). Proliferation of CD4 effector memory cells (EM) was determined by gating on CD161-expressing CD4 T cells (CD161 Pos) versus CD4 T cells lacking CD161 expression (CD161 Neg). *P ≤ 0.05 **P ≤ 0.01 (two-tailed Student's t-test). [Figure 19B] Same as above. [Figure 20A] Figures 20A-20B are graphs showing that anti-CD161 antibody (Ab9) enhanced IFNγ production in CD161+ MART-1-specific T cells upon reactivation (as the percentage of cytokine-positive cells (Figure 20A) or as geometric mean fluorescence intensity (MFI) (Figure 20B)). **P≦0.01, ***P≦0.001 (two-tailed Student's t-test). Figures 20C-20D are graphs showing that anti-CD161 antibody (Ab9) enhanced IL-2 (Figure 20C) and TNFα (Figure 20D) production in MART-1-specific T cells upon reactivation. *P≦0.05 **P≦0.01, ***P≦0.001 (two-tailed Student's t-test). [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above. [Figure 21A]Figures 21A-21B are graphs showing that anti-CD161 antibody (Ab9) enhances granzyme B production in CD161+ MART-1-specific T cells from two donors upon reactivation. Figures 21C-21D are graphs showing that Ab9 enhanced antigen-specific T cell cytotoxicity of MART-1-specific T cells from two donors. Viable target cells were measured as CD19+, NEAR-IR-negative. *P≦0.05, **P≦0.01 (two-tailed Student's t-test). [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 21D] Same as above. [Figure 22A] Figures 22A-22F are graphs showing cytokine induction from unstimulated healthy human PBMCs after incubation with plate-bound Ab9. Treatment of six donor human PBMCs with plate-bound Ab9, isotype control, anti-CD3 muromonab (anti-CD3), and rituximab treatment (***p<0.0001). The Y-axis is shown on a logarithmic scale to depict the low levels of cytokines induced by the addition of Ab9 and the high levels released by muromonab treatment. Different points in the plots represent individual PBMC donors. [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 22D] Same as above. [Figure 22E] Same as above. [Figure 22F] Same as above. [Figure 23A]Figures 23A-23F are graphs showing cytokine induction from unstimulated healthy human PBMCs after incubation with soluble Ab9. Treatment of six donor human PBMCs with soluble Ab9, isotype control, anti-CD3 muromonab (anti-CD3), and rituximab treatment (***p<0.0001). The Y-axis is shown on a logarithmic scale to depict the low levels of cytokines induced by Ab9 addition and the high levels released by muromonab treatment. Different points in the plots represent individual PBMC donors. The "#" in the IL-6 plot (Figure 23F) represents a high value observed for a single donor at low concentrations of Ab9, suggesting it is an outlier. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 23E] Same as above. [Figure 23F] Same as above. [Figure 24] Figure 24 is a plot of the mean Ab9 serum concentration versus time profile for each dose group using a semi-logarithmic scale. [Figure 25A] Figures 25A-25B are plots of mean Ab9 serum concentration versus time profiles by treatment and sex using a semi-logarithmic scale. [Figure 25B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0085] The present invention is based, in part, on the discovery of high-affinity anti-CD161 antibodies that disrupt the CLEC2D-CD161 axis, which can be used, inter alia, to prevent certain cancers from evading a subject's immune system. The present application further relates to pharmaceutical compositions and therapeutic methods using such antibodies and / or pharmaceutical compositions for the treatment of indications such as cancer. Various features and aspects of the present invention, including the antibodies described in this application, are discussed in detail below.

[0086] definition To facilitate understanding of this application, several terms and phrases are defined below.

[0087] The terms "a" and "an" are used herein to mean "one or more" and include plurals, unless the context is inappropriate.

[0088] As used herein, the term "immunoglobulin" refers to a class of structurally related antibody proteins that typically contain two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an "intact immunoglobulin," all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Paul (2013) FUNDAMENTAL IMMUNOLOGY 7TH ED., Ch. 5 Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically contains a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region typically comprises C H1 , C H2 , and C H3 Each light chain typically contains three domains, abbreviated as V L ) and a light chain constant region. The light chain constant region typically comprises C L It contains one domain, abbreviated as .

[0089] As used herein, unless otherwise indicated, the term "antibody" is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including intact antibodies, antigen-binding fragments, or Fc fragments that have been modified, engineered, or chemically conjugated. Examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0090] As used herein, the term "anti-CD161 antibody" refers to an antibody that specifically binds to CD161 (e.g., human CD161). In certain embodiments, the anti-CD161 antibody can reduce or prevent the interaction of CD161 with CLEC2D.

[0091] The term "antigen-binding fragment" refers to a portion of an antibody capable of specifically binding to an antigen or epitope. Exemplary antigen-binding fragments include Fab, Fab', F(ab')2 (in fragments or recombinant polypeptides), single-chain antibody binding sites (e.g., scFv) (heavy chain variable domains (V)), and the like. H ) in a single polypeptide, connected by a linker (e.g., a polypeptide linker) to a light chain variable domain (V L )), minibody, or nanobody (V HH Antigen-binding fragments can be found in a variety of contexts, including antibodies and chimeric antigen receptors (CARs), e.g., CARs derived from antibodies or antibody fragments such as scFvs.

[0092] The terms "intact antibody," "full length antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain that includes an Fc region. For example, when used to refer to an IgG molecule, a "full length antibody" is an antibody that includes two heavy chains and two light chains.

[0093] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini (2010) J. ALLERGY CLIN. IMMUNOL., 125: S41-52, incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region or may be an altered Fc region as described in the art or elsewhere in this disclosure.

[0094] V H Area and V L The regions can be further subdivided into regions of hypervariability ("hypervariable regions (HVRs)"; also called "complementarity determining regions" (CDRs)) interspersed with more conserved regions, which are called framework regions (FRs). H and V L generally contain three CDRs and four FRs arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and affect the antigen specificity and binding affinity of the antibody. See Kabat et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST 5TH ED., Public Health Service, National Institutes of Health, Bethesda, MD (incorporated by reference in its entirety).

[0095] "Complementarity-determining region (CDR)" refers to the region of an immunoglobulin (Ig or antibody) V H one of the three hypervariable regions (e.g., CDR-H1, CDR-H2, or CDR-H3) within the non-framework regions of the β-sheet framework, or the antibody V L CDR refers to one of the three hypervariable regions (e.g., CDR-L1, CDR-L2, or CDR-L3) within the non-framework regions of the β-sheet framework. CDRs are variable region sequences interspersed within framework region sequences. CDRs are well recognized in the art and are defined, for example, by Kabat as the most hypervariable regions within antibody variable (V) domains. See, for example, Kabat et al. (1977) J. BIOL. CHEM., 252: 6609-6616 and Kabat (1978) ADV. PROTEIN CHEM., 32: 1-75 (each of which is incorporated by reference in its entirety). CDRs have also been structurally defined by Chothia as residues that are not part of the conserved β-sheet framework and are therefore capable of adopting various conformations. See, for example, Chothia and Lesk (1987) J. MOL. BIOL., 196: 901-917 (incorporated by reference in its entirety). Both the Kabat and Chothia nomenclature systems are well known in the art. AbM, Contact, and IMGT have also defined CDRs. The CDR positions within standard antibody variable domains have been determined by comparison of multiple structures. See Morea et al. (2000) METHODS, 20: 267-279 and Al-Lazikani et al. (1997) J. MOL. BIOL., 273: 927-48 (each of which is incorporated by reference in its entirety).

[0096] Several hypervariable region boundaries are used and are included herein. Kabat CDRs are based on sequence variability and are the most commonly used. See Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, DIANE Publishing: 2719 (incorporated by reference in its entirety). Chothia instead refers to the location of structural loops (Chothia and Lesk, supra). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. Contact hypervariable regions are based on the analysis of available complex crystal structures.

[0097] More recently, a universal numbering system, the ImMunoGeneTics (IMGT) Information System™, has been developed and widely adopted. See Lefranc et al. (2003) DEV. COMP. IMMUNOL., 27: 55-77 (incorporated by reference in its entirety). IMGT is an integrated information system specialized for immunoglobulins (IGs), T cell receptors (TRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. IMGT CDRs are referenced both by amino acid sequence and by their location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and occurs in structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. The correspondence between Kabat, Chothia, and IMGT numbering is also well known in the art (Lefranc et al., supra).

[0098] Light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of their constant domain.

[0099] Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also called α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0100] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding the antibody to an antigen but exhibit various effector functions, such as interaction with Fc receptors. This term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the variable domain, the other part of the immunoglobulin that contains the antigen-binding site. The constant domain is the C of the heavy chain. H1 , C H2 and C H3 domain, as well as the C of the light chain L Contains a domain.

[0101] The "EU numbering scheme" is commonly used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra). Unless otherwise specified, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0102] An "Fv" fragment comprises a non-covalent dimer of one heavy- and one light-chain variable domain.

[0103] A "Fab" fragment contains the heavy and light chain variable domains as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 Fab fragments may be produced, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0104] An "F(ab')2" fragment contains two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments may be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0105] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain V in a single polypeptide chain. H Domain and V L Includes domain. V H and V L are generally linked by a peptide linker. Any suitable linker can be used. In some embodiments, the linker is (GGGGS) n (SEQ ID NO: 187), and in certain embodiments, n=1, 2, 3, 4, 5, or 6.

[0106] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain may be linked to the C-terminus of the scFv. The Fc domain may be linked to the orientation of the variable domains in the scFv (i.e., V H -V L or V L -V H ) depending on V H or V L Any suitable Fc domain known in the art or described herein can be used.

[0107] The term "single domain antibody" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of other variable domains. Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al. (1998) FEBS LETTERS, 414: 521-526 and Muyldermans et al. (2001) TRENDS IN BIOCHEM. SCI., 26: 230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies.

[0108] A "multispecific antibody" is an antibody comprising two or more different antigen-binding domains that collectively specifically bind to two or more different epitopes. The two or more different epitopes can be epitopes on the same antigen (e.g., a single CD161 molecule expressed by a cell) or on different antigens (e.g., a CD161 molecule and a non-CD161 molecule). In some embodiments, a multispecific antibody binds to two different epitopes (i.e., a "bispecific antibody"). In some embodiments, a multispecific antibody binds to three different epitopes (i.e., a "trispecific antibody"). In some embodiments, a multispecific antibody binds to four different epitopes (i.e., a "tetraspecific antibody"). In some embodiments, a multispecific antibody binds to five different epitopes (i.e., a "pentaspecific antibody"). In some embodiments, a multispecific antibody binds to six, seven, eight, or more different epitopes. Each binding specificity can be present in any suitable valency. Examples of multispecific antibodies are provided elsewhere in this disclosure.

[0109] A "monospecific antibody" is an antibody that contains one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is a naturally occurring IgG molecule that is bivalent (i.e., has two antigen-binding domains) but recognizes the same epitope in each of the two antigen-binding domains. The binding specificity can be in any suitable valency.

[0110] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies. A substantially homogeneous population of antibodies contains antibodies that are substantially similar and bind to the same epitope(s), except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present in only minor amounts. Monoclonal antibodies are typically obtained by a process that includes selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve its affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0111] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0112] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody, having the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al. (1986) NATURE, 321: 522-525; Riechmann et al. (1988) NATURE, 332: 323-329; and Presta (1992) CURR. OP. STRUCT. BIOL., 2: 593-596, each of which is incorporated by reference in its entirety.

[0113] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or that corresponds to that of an antibody derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies do not include humanized antibodies.

[0114] An "isolated antibody" or "isolated nucleic acid" is an antibody or nucleic acid that has been separated and / or recovered from a component of its natural environment. Components of natural environment may include enzymes, hormones, and other proteinaceous or non-proteinaceous materials. In some embodiments, an isolated antibody is purified sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, by use of a spinning cup sequenator. In some embodiments, an isolated antibody is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions with detection by Coomassie blue or silver staining. In some embodiments, an isolated antibody may include an antibody in situ within recombinant cells, since at least one component of the antibody's natural environment is not present. In some aspects, an isolated antibody or isolated nucleic acid is prepared by at least one purification step. In some embodiments, an isolated antibody or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the isolated antibody or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, the isolated antibody or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% antibody or nucleic acid by volume. In some embodiments, the isolated antibody or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% antibody or nucleic acid by volume.

[0115] "Affinity" refers to the strength of the sum of non-covalent interactions between a binding site (e.g., a single binding site) of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope). The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D ) The kinetic component that contributes to the dissociation equilibrium constant is described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®, e.g., using an Octet QK384 system).

[0116] With respect to antibody binding to a target molecule, the terms "bind," "specific binding," "specifically binds," "specific," "selectively binds," and "selective" to a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from a nonspecific or nonselective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to the non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. Specific binding is then indicated if binding of the antibody to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the CD161 antibody for the non-target molecule is less than about 40% of its affinity for CD161. In some embodiments, the affinity of the CD161 antibody for the non-target molecule is less than about 30% of its affinity for CD161. In some embodiments, the affinity of the CD161 antibody for the non-target molecule is less than about 20% of its affinity for CD161. In some embodiments, the affinity of the CD161 antibody for the non-target molecule is less than about 10% of its affinity for CD161. In some embodiments, the affinity of the CD161 antibody for the non-target molecule is less than about 1% of its affinity for CD161. In some embodiments, the affinity of the CD161 antibody for the non-target molecule is less than about 0.1% of its affinity for CD161.

[0117] The term "k" d " (sec -1 ) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is known as k off Also called value.

[0118] The term "k" a " (M -1 ×sec -1 ) as used herein refers to the association rate constant of a particular antibody-antigen interaction. This value is known as k on Also called value.

[0119] The term “K D" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. K D =k d / k a In some embodiments, the affinity of an antibody is determined by the K D For clarity, as is known in the art, the smaller K D values ​​indicate a higher affinity interaction, while a larger K D Values ​​indicate lower affinity interactions.

[0120] The term “K A " (M -1 ) as used herein refers to the association equilibrium constant of a particular antibody-antigen interaction. A =k a / k d .

[0121] An "affinity matured" antibody is an antibody with one or more alterations (e.g., in one or more CDRs or FRs) relative to a parent antibody (i.e., the antibody from which the altered antibody is derived or engineered) that result in an improvement in the affinity of the antibody for its antigen compared to the parent antibody not possessing the alteration(s). In some embodiments, the affinity matured antibody has nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies can be generated using a variety of methods known in the art. For example, Marks et al. (1992) BIO / TECHNOLOGY, 10: 779-783 (incorporated by reference in its entirety) describe affinity matured antibodies. H and V LAffinity maturation by domain shuffling is described. Random mutagenesis of CDR and / or framework residues is described, for example, by Barbas et al. (1994) PROC. NAT. ACAD. SCI. USA, 91: 3809-3813; Schier et al. (1995) GENE, 169: 147-155; Yelton et al. (1995) J. IMMUNOL., 155: 1994-2004; Jackson et al. (1995) J. IMMUNOL., 154: 3310-33199; and Hawkins et al. (1992) J. MOL. BIOL., 226: 889-896 (each of which is incorporated by reference in its entirety).

[0122] "Fc effector function" refers to a biological activity mediated by the Fc region of an antibody, which may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding, which activates complement-dependent cytotoxicity (CDC), Fc receptor binding, which activates antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0123] As used herein in the context of two or more antibodies, the terms "compete with" or "cross-compete with" indicate that two or more antibodies compete for binding to an antigen (e.g., CD161). In one exemplary assay, CD161 is coated on a surface and contacted with a first CD161 antibody, followed by the addition of a second CD161 antibody. In another exemplary assay, a first CD161 antibody is coated on a surface and contacted with CD161, followed by the addition of a second CD161 antibody. In either assay, antibodies compete with each other if the presence of the first CD161 antibody reduces binding of the second CD161 antibody. The term "compete with" also includes antibody combinations in which one antibody reduces binding of another antibody, but no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first antibody and the second antibody inhibit each other's binding, regardless of the order in which they are added. In some embodiments, one antibody reduces binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. One of skill in the art can select the concentration of antibody to use in a competition assay based on the affinity of the antibody for CD161 and the valency of the antibody. The assays described in this definition are exemplary, and one of skill in the art can utilize any suitable assay to determine whether antibodies compete with each other. Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in ASSAY GUIDANCE MANUAL [INTERNET], Updated December 24, 2014 (www.ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al. (2001) CYTOMETRY, 44: 30-37; and Finco et al. (2011) J. PHARM. BIOMED. ANAL., 54: 351-358 (each of which is incorporated by reference in its entirety).

[0124] As used herein, K D If K values ​​can be measured with the ForteBio Octet using mouse antigens, then an antibody that specifically binds to a human antigen is considered to specifically bind the same antigen of mouse origin. In general, K values ​​for non-human species are D (as measured by ForteBio Octet) D less than 10 times weaker than (i.e., K D An antibody can be considered cross-reactive with non-human species if its K for mouse antigen is greater than 10-fold higher. D The values ​​are the corresponding K D An antibody that specifically binds to a human antigen is considered to be "cross-reactive" with the same antigen of murine origin if the binding affinity is 20-fold or less.

[0125] As used herein, K for cynomolgus monkey antigens D The values ​​are the corresponding K D An antibody that specifically binds to a human antigen is considered to be "cross-reactive" with the same antigen of cynomolgus monkey origin if the binding affinity is 20-fold or less.

[0126] The term "epitope" refers to the portion of an antigen that is specifically bound by an antibody. Epitopes often contain surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, may be lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for determining epitopes, such as testing antibody binding to CD161 variants with various point mutations or to chimeric CD161 variants.

[0127] The percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to those in the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. "Conservative substitution" or "conservative amino acid substitution" refers to the substitution of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. By way of example, the groups of amino acids provided in Tables 1-3 are, in some embodiments, considered conservative substitutions for one another.

[0128] Selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments, are shown in Table 1.

[0129] [Table 1]

[0130] Additional selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments, are shown in Table 2.

[0131] [Table 2]

[0132] Further selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments, are shown in Table 3.

[0133] [Table 3]

[0134] Additional conservative substitutions can be found, for example, in Creighton (1993) PROTEINS: STRUCTURES AND MOLECULAR PROPERTIES 2ND ED. W. H. Freeman & Co., New York, N.Y. Antibodies generated by making one or more conservative substitutions of amino acid residues in a parent antibody are referred to as "conservatively modified variants."

[0135] The term "amino acid" refers to the 20 common naturally occurring amino acids, including alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0136] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0137] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and to the progeny of such cells. Host cells include "transformants" (or "transformed cells") and "transfectants" (or "transfected cells"), which include the original transformed or transfected cell, respectively, and its derived progeny. Such progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations.

[0138] The term "treating" (and variations thereof, such as "treat" or "treatment") refers to a clinical intervention that seeks to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both prophylactically and during the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of the disease, ameliorating one or more symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliation of the disease state, and remission or improved prognosis.

[0139] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an antibody or pharmaceutical composition provided herein that is sufficient to produce a beneficial or desired result when administered to a subject, e.g., the antibody or pharmaceutical composition is effective to treat a disease or disorder or ameliorate one or more symptoms of a disease or disorder when administered to a subject. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0140] As used herein, the term "subject" means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, pigs, and sheep. In certain embodiments, the subject is a human subject. In some embodiments, the subject has a disease or condition that can be treated with an antibody provided herein. For example, the disease or condition is cancer.

[0141] The term "package insert" is used to refer to instructions typically included in a commercial package (e.g., a kit) of a therapeutic or diagnostic product that contains information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings for the use of the therapeutic or diagnostic product.

[0142] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include "antihormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth.

[0143] The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredients contained therein to be effective in treating a subject, and that does not contain additional ingredients that are unacceptably toxic to a subject in the amounts provided in the pharmaceutical composition.

[0144] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or alternatively activating or increasing, the recited variable.

[0145] The terms "increase" and "activate" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater increase in the recited variable.

[0146] The terms "reduce" and "inhibit" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater decrease in the recited variable.

[0147] The term "agonize" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to and agonizes a receptor.

[0148] The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor. As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably include humans.

[0149] The phrase "at least one of" should be understood to include each of the listed items following the phrase individually, and various combinations of two or more of the listed items, unless otherwise understood from context and usage. The phrase "and / or" in connection with more than two listed items should be understood to have the same meaning, unless otherwise understood from context.

[0150] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing" (including their grammatical equivalents) should generally be understood as open-ended and non-limiting, and for example, does not exclude additional, unrecited elements or steps, unless otherwise specified or understood from context.

[0151] The term "about" refers to and encompasses the indicated value, as well as a range above and below that value. In certain embodiments, the term "about" refers to the indicated value ±10%, ±5%, or ±1%. In certain embodiments, where applicable, the term "about" refers to the indicated value(s) ±1 standard deviation of that value(s).

[0152] At various places in this specification, components, or features thereof, are disclosed in groups or ranges. The description is specifically intended to include any and all individual subcombinations of the members of such groups and ranges. As another example, an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0153] Throughout the description, when compositions are described as having, including, or comprising certain components, or when processes and methods are described as having, including, or comprising certain steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and processes and methods of the invention that consist essentially of or consist of the recited processing steps.

[0154] In this application, when an element or component is said to be included in and / or selected from a list of recited elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0155] Furthermore, it should be understood that elements and / or features of the compositions or methods described herein, whether expressly or implicitly stated herein, can be combined in various ways without departing from the spirit and scope of the invention. For example, when reference is made to a particular compound, that compound can be used in various embodiments of the compositions of the invention and / or in the methods of the invention, unless otherwise understood from the context. In other words, within this application, embodiments are described and depicted so as to enable a clear and concise application to be written and depicted, but it is intended and understood that the embodiments can be combined or separated in various ways without departing from the present teachings and invention(s). For example, it is understood that all features described and depicted herein are applicable to all aspects of the invention(s) described and depicted herein.

[0156] I. Anti-CD161 antibody Human CD161 (also known as NK receptor-P1A (NKR-P1A), killer cell lectin-like receptor subfamily B member 1 (KLRB1), or C-type lectin domain family 5 member B (CLEC5B)) is expressed on Th17 cells, natural killer (NK) cells, and NKT cells. CD161 belongs to the killer cell lectin-like receptor (KLR) family, whose members contain a single C-type lectin-like domain in the extracellular region responsible for ligand recognition. Mice have several CD161 molecules, which are both activating and inhibitory receptors, known as the NKR-P1 family, whereas humans have only one CD161 molecule, which is an inhibitory receptor. The CD161 receptor binds to a ligand known as C-type lectin domain family 2 member D (CLEC2D) (see Aldemir et al. (2005) J. IMMUNOL., 175(12): 7791-5; Rosen et al. (2005) J. IMMUNOL., 175(12): 7796-9). CLEC2D is also known as human lectin-like transcript 1 molecule (LLT1). CD161 family molecules are type II transmembrane glycoproteins that form disulfide-linked homodimers. Inhibiting the interaction between CD161 and CLEC2D promotes T cell activation in response to tumor cells. All orthologs and isoforms of CLEC2D and CD161 are considered within the scope of the present disclosure.

[0157] The amino acid sequence of human CD161 is represented by SEQ ID NO: 179. Amino acid residues 1 to 45, 46 to 66, and 67 to 225 of SEQ ID NO: 179 comprise the cytoplasmic domain, the transmembrane domain, and the extracellular domain, respectively. The amino acid sequences of cynomolgus monkey CD161, mouse CD161, and rat CD161 are represented by SEQ ID NOs: 180, 181, and 182, respectively.

[0158] The amino acid sequence of human CLEC2D is represented by SEQ ID NO: 186. Amino acid residues 1 to 38, 39 to 59, and 60 to 191 of SEQ ID NO: 186 comprise the cytoplasmic domain, the transmembrane domain, and the extracellular domain, respectively.

[0159] 1. Anti-CD161 antibody sequence In certain embodiments, the present disclosure provides antigen-binding fragments that bind to CD161 (eg, human CD161) derived from the antibodies listed in Table 4.

[0160] The sequences of exemplary antibodies that bind to CD161 are shown in Table 4.

[0161] [Table 4] TIFF2024534265000005.tif241160TIFF2024534265000006.tif239160TIFF2024534265000007.tif24216 0TIFF2024534265000008.tif244160TIFF2024534265000009.tif239160TIFF2024534265000010.tif61160

[0162] 1.1 V H domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. H Contains arrays.

[0163] In some embodiments, the antibodies provided herein comprise a V H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 15. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 23. HIn some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 31. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 39. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 47. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 55. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 63. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 71. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 79. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 87. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 101. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 109. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 117. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 125. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 133. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 141. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 149. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 157. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 165. H Contains arrays.

[0164] In some embodiments, the antibodies provided herein comprise an exemplary VVL sequence provided in SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. H V having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the sequence H In some embodiments, the antibodies provided herein comprise a VLK sequence as provided in SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. H In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0165] 1.2 V L domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. L Contains arrays.

[0166] In some embodiments, the antibodies provided herein comprise a V LIn some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 19. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 27. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 35. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 43. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 51. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 59. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 67. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 75. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 83. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 91. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 105. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 113. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 121. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 129. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 137. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 145. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 153. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 161. LIn some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 169. L Contains arrays.

[0167] In some embodiments, the antibodies provided herein comprise an exemplary VVL sequence provided in SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. L V having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the sequence L In some embodiments, the antibodies provided herein comprise a V sequence as provided in SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. L In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0168] 1.3 V H -V L combination of In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165.H and V selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. L Contains arrays.

[0169] In some embodiments, the antibodies provided herein comprise a V H Sequence and V of SEQ ID NO: 11 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 15. H Sequence and V of SEQ ID NO: 19 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 23. H Sequence and V of SEQ ID NO: 27 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 31. H Sequence and V of SEQ ID NO: 35 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 39. H Sequence and V of SEQ ID NO: 43 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 47. H Sequence and V of SEQ ID NO: 51 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 55. H Sequence and V of SEQ ID NO: 59 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 63. H Sequence and V of SEQ ID NO: 67 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 71. H Sequence and V of SEQ ID NO: 75 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 79. H Sequence and V of SEQ ID NO: 83 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 87. H Sequence and V of SEQ ID NO: 91L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 101. H Sequence and V of SEQ ID NO: 105 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 109. H Sequence and V of SEQ ID NO: 113 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 117. H Sequence and V of SEQ ID NO: 121 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 125. H Sequence and V of SEQ ID NO: 129 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 133. H Sequence and V of SEQ ID NO: 137 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 141. H Sequence and V of SEQ ID NO: 145 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 149. H Sequence and V of SEQ ID NO: 153 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 157. H Sequence and V of SEQ ID NO: 161 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 165. H Sequence and V of SEQ ID NO: 169 L Contains arrays.

[0170] In some embodiments, the antibodies provided herein comprise an exemplary VVL sequence provided in SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. H V having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the sequence HExemplary V sequences are provided in SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. L V having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the sequence L In some embodiments, the antibodies provided herein comprise a VLK sequence as provided in SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. H and V sequences provided in SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. L In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0171] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11.

[0172] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:15, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19.

[0173] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:27.

[0174] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:35.

[0175] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:39, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:43.

[0176] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:51.

[0177] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:55, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:59.

[0178] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:63, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:67.

[0179] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:71, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:75.

[0180] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:79, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:83.

[0181] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:87, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:91.

[0182] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 105.

[0183] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 109, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 113.

[0184] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 117, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 121.

[0185] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 125, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 129.

[0186] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 133, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 137.

[0187] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 141, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 145.

[0188] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 149, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153.

[0189] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 161.

[0190] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 165, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169.

[0191] In certain embodiments, the heavy chain variable region sequence, e.g., the V of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, or 165, H It is contemplated that the sequences, or amino acid variants thereof, can be covalently linked to various heavy chain constant region sequences known in the art. Similarly, light chain variable region sequences, such as the V sequences of SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, or 169, can be covalently linked to various heavy chain constant region sequences known in the art. LIt is contemplated that the , or amino acid variants thereof, may be covalently linked to various light chain constant region sequences known in the art.

[0192] For example, an antibody molecule may have a heavy chain constant region selected from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE heavy chain constant regions, particularly, e.g., IgG1, IgG2, IgG3, and IgG4 (e.g., human) heavy chain constant regions. In another embodiment, an antibody molecule has a light chain constant region selected from, e.g., kappa or lambda (e.g., human) light chain constant regions. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In another embodiment, the antibody does not recruit effector cells or fix complement. In another embodiment, the antibody has reduced ability to bind to Fc receptors or does not have the ability to bind to Fc receptors. For example, the antibody may be an isotype or subtype, fragment or other variant that does not support binding to an Fc receptor, e.g., the antibody has a mutated or deleted Fc receptor binding region.

[0193] 1.4 CDR In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. H In some embodiments, the antibodies provided herein comprise one to three CDRs of a V domain selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. HIn some embodiments, the antibodies provided herein comprise two to three CDRs of a V domain selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. H The domain comprises three CDRs. In some embodiments, the CDRs are annotated using the Kabat numbering system. In some embodiments, the CDRs are annotated using the Chothia numbering system. In some embodiments, the CDRs are annotated using the AbM numbering system. In some embodiments, the CDRs are annotated using the Contact numbering system. In some embodiments, the CDRs are annotated using the IMGT numbering system. In some embodiments, the CDRs are annotated using a typical numbering system, where amino acid residues at positions 27-35, 50-65, and 93-102 represent CDR-H1, CDR-H2, and CDR-H3, respectively.

[0194] In some embodiments, the CDRs are CDRs that have at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. In some embodiments, the CDR-H1 is selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165 with up to 1, 2, 3, 4, or 5 amino acid substitutions. H In some embodiments, the CDR-H2 is a CDR-H1 of the V domain selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. HIn some embodiments, the CDR-H3 is a CDR-H2 of the V domain selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. H The CDR-H3 of the domain. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0195] In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. L In some embodiments, the antibodies provided herein comprise one to three CDRs of a V domain selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. L In some embodiments, the antibodies provided herein comprise two to three CDRs of a V domain selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. LThe CDRs comprise three CDRs of the domain. In some embodiments, the CDRs are annotated using the Kabat numbering system. In some embodiments, the CDRs are annotated using the Chothia numbering system. In some embodiments, the CDRs are annotated using the AbM numbering system. In some embodiments, the CDRs are annotated using the Contact numbering system. In some embodiments, the CDRs are annotated using the IMGT numbering system. In some embodiments, the CDRs are annotated using a typical numbering system, where amino acid residues at positions 24-34, 50-56, and 89-97 designate CDR-L1, CDR-L2, and CDR-L3, respectively.

[0196] In some embodiments, the CDRs are CDRs that have at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. In some embodiments, the CDR-L1 is selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169 with up to 1, 2, 3, 4, or 5 amino acid substitutions. L In some embodiments, the CDR-L2 is a CDR-L1 of the V domain selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. LIn some embodiments, the CDR-L3 is a CDR-L2 of the V domain selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169, and 760, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. L The CDR-L3 of the domain. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0197] In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. H 1 to 3 CDRs of the domain and a V selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. L In some embodiments, the antibodies provided herein comprise one to three CDRs of a V domain selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. H 2 to 3 CDRs of the domain and a V selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169 LIn some embodiments, the antibodies provided herein comprise two to three CDRs of a V domain selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. H the three CDRs of the domain and a V selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169; L The CDR comprises three CDRs of the domain. In some embodiments, the CDR is a Kabat CDR. In some embodiments, the CDR is a Chothia CDR. In some embodiments, the CDR is an AbM CDR. In some embodiments, the CDR is a Contact CDR. In some embodiments, the CDR is an IMGT CDR. In some embodiments, the CDR is a typical CDR.

[0198] In some embodiments, the CDRs are at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165. and CDRs having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169. In some embodiments, the CDR-H1 is selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165 with up to 1, 2, 3, 4, or 5 amino acid substitutions. HCDR-H1 of the domain; and CDR-H2 is selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. H CDR-H3 is a CDR-H2 of the V domain selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 101, 109, 117, 125, 133, 141, 149, 157, and 165 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. H CDR-L1 is a CDR-H3 of the V domain selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. L CDR-L1 of the domain; and CDR-L2 is selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169 with up to 1, 2, 3, or 4 amino acid substitutions. L and CDR-L3 is selected from SEQ ID NOs: 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 105, 113, 121, 129, 137, 145, 153, 161, and 169, with up to 1, 2, 3, 4, or 5 amino acid substitutions. LThe CDR-L3 of the domain. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0199] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168, as determined by a typical numbering system, in which amino acid residues at positions 93-102 represent CDR-H3. In some embodiments, the CDR-H3 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-H3 of SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168. In some embodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0200] In some embodiments, the antibodies provided herein comprise a CDR-H2 selected from SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 159, and 167, as determined by a typical numbering system, in which amino acid residues at positions 50-65 represent CDR-H2. In some embodiments, the CDR-H2 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-H2 of SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 159, and 167. In some embodiments, the CDR-H2 is selected from SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 159, and 167, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0201] In some embodiments, the antibodies provided herein comprise a CDR-H1 selected from SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166, as determined by a typical numbering system, in which amino acid residues at positions 27-35 represent CDR-H1. In some embodiments, the CDR-H1 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-H1 of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166. In some embodiments, the CDR-H1 is a CDR-H1 selected from SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0202] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168, and a CDR-H2 selected from SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 159, and 167. In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168; a CDR-H2 selected from SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 159, and 167; and a CDR-H1 selected from SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166.In some embodiments, the CDR-H3 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-H3 of SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168; and the CDR-H2 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-H3 of SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 159, and 167, and the CDR-H1 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-H1 of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166. In some embodiments, the CDR-H3 is selected from SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H2 is selected from SEQ ID NOs: 9, 17, 25, 33, 44, 45, 46, 47, 48, 49, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. and CDR-H1 is CDR-H1 selected from SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166, with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants."In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, hi some embodiments, such variants are not obtained from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0203] In some embodiments, the antibodies provided herein comprise a CDR-L3 selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172, as determined by a typical numbering system, in which amino acid residues at positions 89-97 represent CDR-L3. In some embodiments, the CDR-L3 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-L3 of SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172. In some embodiments, the CDR-L3 is a CDR-L3 selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0204] In some embodiments, the antibodies provided herein comprise a CDR-L2 selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 107, 115, 123, 131, 139, 147, 155, 163, and 171, as determined by a typical numbering system, in which amino acid residues at positions 50-56 represent CDR-L2. In some embodiments, the CDR-L2 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-L2 of SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 107, 115, 123, 131, 139, 147, 155, 163, and 171. In some embodiments, the CDR-L2 is selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 107, 115, 123, 131, 139, 147, 155, 163, and 171, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0205] In some embodiments, the antibodies provided herein comprise a CDR-L1 selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170, as determined by a typical numbering system, in which amino acid residues at positions 24-34 represent CDR-L1. In some embodiments, the CDR-L1 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-L1 of SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170. In some embodiments, the CDR-L1 is a CDR-L1 selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170, with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not obtained from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0206] In some embodiments, the antibodies provided herein comprise a CDR-L3 selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172, and a CDR-L2 selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 107, 115, 123, 131, 139, 147, 155, 163, and 171. In some embodiments, the antibodies provided herein comprise a CDR-L3 selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172; a CDR-L2 selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 107, 115, 123, 131, 139, 147, 155, 163, and 171; and a CDR-L1 selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170.In some embodiments, the CDR-L3 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-L3 of SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172; and the CDR-L2 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-L3 of SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 107, 115, 123, 131, 139, 147, 155, 163, and 171, and the CDR-L1 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the CDR-L1 of SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170. In some embodiments, the CDR-L3 is selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172 with up to 1, 2, 3, 4, or 5 amino acid substitutions; and the CDR-L2 is selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172 with up to 1, 2, 3, or 4 amino acid substitutions. and CDR-L1 is CDR-L1 selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170, with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants."In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, hi some embodiments, such variants are not obtained from the sequences provided herein, but may be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0207] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168; a CDR-H2 selected from SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 159, and 167; a CDR-H3 selected from SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166; CDR-H1 selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172; CDR-L2 selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 107, 115, 123, 131, 139, 147, 155, 163, and 171; and CDR-L1 selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170.In some embodiments, the CDR-H3 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-H3 of SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168; and the CDR-H2 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-H3 of SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 160, and 162. and CDR-H1 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to CDR-H2 of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166. and CDR-L3 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to CDR-L3 of SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172; and CDR-L2 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to CDR-L3 of SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 107, 115, 123, 131, 139, 147, 155, 163, and 17 and the CDR-L1 has at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CDR-L1 of SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170.In some embodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NOs: 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H2 is a CDR-H3 selected from SEQ ID NOs: 9, 17, 28, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 66, 74, 82, 90, 104, 112, 120, 128, 136, 144, 152, 160, and 168 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. 5, 33, 41, 49, 57, 65, 73, 81, 89, 103, 111, 119, 127, 135, 143, 151, 159, and 167; and CDR-H1 is selected from SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 102, 110, 118, 126, 134, 142, 150, 158, and 166 with up to 1, 2, 3, 4, or 5 amino acid substitutions. CDR-H1 is CDR-H1; CDR-L3 is CDR-L3 selected from SEQ ID NOs: 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172 with up to 1, 2, 3, or 4 amino acid substitutions; CDR-L2 is CDR-L3 selected from SEQ ID NOs: 13, 21, 29, 37, 45, 53, 61, 62, 70, 78, 86, 94, 108, 116, 124, 132, 140, 148, 156, 164, and 172 with up to 1, 2, 3, or 4 amino acid substitutions. and CDR-L1 is selected from SEQ ID NOs: 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 106, 114, 122, 130, 138, 146, 154, 162, and 170, with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are obtained from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein.In some embodiments, such variants are not derived from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0208] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 8, a CDR-H2 of SEQ ID NO: 9, a CDR-H3 of SEQ ID NO: 10, a CDR-L1 of SEQ ID NO: 12, a CDR-L2 of SEQ ID NO: 13, and a CDR-L3 of SEQ ID NO: 14, as determined by a typical numbering system.

[0209] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 16, a CDR-H2 of SEQ ID NO: 17, a CDR-H3 of SEQ ID NO: 18, a CDR-L1 of SEQ ID NO: 20, a CDR-L2 of SEQ ID NO: 21, and a CDR-L3 of SEQ ID NO: 22, as determined by a typical numbering system.

[0210] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 24, a CDR-H2 of SEQ ID NO: 25, a CDR-H3 of SEQ ID NO: 26, a CDR-L1 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 29, and a CDR-L3 of SEQ ID NO: 30, as determined by a typical numbering system.

[0211] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 32, a CDR-H2 of SEQ ID NO: 33, a CDR-H3 of SEQ ID NO: 34, a CDR-L1 of SEQ ID NO: 36, a CDR-L2 of SEQ ID NO: 37, and a CDR-L3 of SEQ ID NO: 38, as determined by a typical numbering system.

[0212] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 40, a CDR-H2 of SEQ ID NO: 41, a CDR-H3 of SEQ ID NO: 42, a CDR-L1 of SEQ ID NO: 44, a CDR-L2 of SEQ ID NO: 45, and a CDR-L3 of SEQ ID NO: 46, as determined by a typical numbering system.

[0213] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 48, a CDR-H2 of SEQ ID NO: 49, a CDR-H3 of SEQ ID NO: 50, a CDR-L1 of SEQ ID NO: 52, a CDR-L2 of SEQ ID NO: 53, and a CDR-L3 of SEQ ID NO: 54, as determined by a typical numbering system.

[0214] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 56, a CDR-H2 of SEQ ID NO: 57, a CDR-H3 of SEQ ID NO: 58, a CDR-L1 of SEQ ID NO: 60, a CDR-L2 of SEQ ID NO: 61, and a CDR-L3 of SEQ ID NO: 62, as determined by a typical numbering system.

[0215] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 64, a CDR-H2 of SEQ ID NO: 65, a CDR-H3 of SEQ ID NO: 66, a CDR-L1 of SEQ ID NO: 68, a CDR-L2 of SEQ ID NO: 69, and a CDR-L3 of SEQ ID NO: 70, as determined by a typical numbering system.

[0216] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 72, a CDR-H2 of SEQ ID NO: 73, a CDR-H3 of SEQ ID NO: 74, a CDR-L1 of SEQ ID NO: 76, a CDR-L2 of SEQ ID NO: 77, and a CDR-L3 of SEQ ID NO: 78, as determined by a typical numbering system.

[0217] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 80, a CDR-H2 of SEQ ID NO: 81, a CDR-H3 of SEQ ID NO: 82, a CDR-L1 of SEQ ID NO: 84, a CDR-L2 of SEQ ID NO: 85, and a CDR-L3 of SEQ ID NO: 86, as determined by a typical numbering system.

[0218] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 88, a CDR-H2 of SEQ ID NO: 89, a CDR-H3 of SEQ ID NO: 90, a CDR-L1 of SEQ ID NO: 92, a CDR-L2 of SEQ ID NO: 93, and a CDR-L3 of SEQ ID NO: 94, as determined by a typical numbering system.

[0219] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 102, a CDR-H2 of SEQ ID NO: 103, a CDR-H3 of SEQ ID NO: 104, a CDR-L1 of SEQ ID NO: 106, a CDR-L2 of SEQ ID NO: 107, and a CDR-L3 of SEQ ID NO: 108, as determined by a typical numbering system.

[0220] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 110, a CDR-H2 of SEQ ID NO: 111, a CDR-H3 of SEQ ID NO: 112, a CDR-L1 of SEQ ID NO: 114, a CDR-L2 of SEQ ID NO: 115, and a CDR-L3 of SEQ ID NO: 116, as determined by a typical numbering system.

[0221] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 118, a CDR-H2 of SEQ ID NO: 119, a CDR-H3 of SEQ ID NO: 120, a CDR-L1 of SEQ ID NO: 122, a CDR-L2 of SEQ ID NO: 123, and a CDR-L3 of SEQ ID NO: 124, as determined by a typical numbering system.

[0222] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 126, a CDR-H2 of SEQ ID NO: 127, a CDR-H3 of SEQ ID NO: 128, a CDR-L1 of SEQ ID NO: 130, a CDR-L2 of SEQ ID NO: 131, and a CDR-L3 of SEQ ID NO: 132, as determined by a typical numbering system.

[0223] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 134, a CDR-H2 of SEQ ID NO: 135, a CDR-H3 of SEQ ID NO: 136, a CDR-L1 of SEQ ID NO: 138, a CDR-L2 of SEQ ID NO: 139, and a CDR-L3 of SEQ ID NO: 140, as determined by a typical numbering system.

[0224] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 142, a CDR-H2 of SEQ ID NO: 143, a CDR-H3 of SEQ ID NO: 144, a CDR-L1 of SEQ ID NO: 146, a CDR-L2 of SEQ ID NO: 147, and a CDR-L3 of SEQ ID NO: 148, as determined by a typical numbering system.

[0225] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 150, a CDR-H2 of SEQ ID NO: 151, a CDR-H3 of SEQ ID NO: 152, a CDR-L1 of SEQ ID NO: 154, a CDR-L2 of SEQ ID NO: 155, and a CDR-L3 of SEQ ID NO: 156, as determined by a typical numbering system.

[0226] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 158, a CDR-H2 of SEQ ID NO: 159, a CDR-H3 of SEQ ID NO: 160, a CDR-L1 of SEQ ID NO: 162, a CDR-L2 of SEQ ID NO: 163, and a CDR-L3 of SEQ ID NO: 164, as determined by a typical numbering system.

[0227] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 166, a CDR-H2 of SEQ ID NO: 167, a CDR-H3 of SEQ ID NO: 168, a CDR-L1 of SEQ ID NO: 170, a CDR-L2 of SEQ ID NO: 171, and a CDR-L3 of SEQ ID NO: 172, as determined by a typical numbering system.

[0228] 1.5 Consensus Sequence V of representative antibodies from each family H Sequence and V LConsensus sequences were developed that represent the sequences of the V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, V14, V15, V16, V17, V18, V19, V20, V21, V22, V23, V24, V25, V26, V27, V28, V29, V30, V31 H Sequence and V L The positions where amino acids differ within the framework regions of the sequence are 、 X a , X b , X c ...The amino acids that may represent the consensus sequence at each X position are identified herein.

[0229] In some embodiments, there is provided a first family of antibodies (see Figures 12A-12B), wherein the antibodies of the family have the following six CDR sequences: (a) a CDR-H1 having the sequence FX1FX2X3X4AMS (SEQ ID NO: 1) (X1 is T or A, X2 is G, S, or E, X3 is Q, T, P, or R, and X4 is Y or F); (b) a CDR-H2 having the sequence AISX5X6GGX7TX8YADSVKG (SEQ ID NO: 2) (X5 is A or G, X6 is A, V, or S, X7 is T or S, and X8 is K, A, or Y); (c) a CDR-H2 having the sequence AKPLDSSX9WADFX 10 X 11 (SEQ ID NO: 3) (X9 is Q, F, or L, and X 10 is D or Q, and X 11 is L or A); (d) a CDR-H3 having the sequence RASQX 12 IX 13 SWLA (SEQ ID NO: 4)(X 12 is G, D, or T, and X 13 (e) a CDR-L1 having the sequence X 14 ASX 15 LQX 16 (SEQ ID NO: 5)(X 14 is A, Y, or F, and X 15 is S, A, G, or F, and X 16 is D or S); and (f) a CDR-L2 having the sequence QQX 17 X 18 X 19LPIT (SEQ ID NO: 6) (X 17 is A, H, or Q; X 18 is S, D, W, or L; and X 19 A first family of antibodies is provided herein, comprising a CDR-L3 having a CDR-L3 with a CDR-L3 of V, D, Y, or K.

[0230] In some embodiments, the antibody of the family comprises the V of SEQ ID NO: 188. H Sequence and V of SEQ ID NO: 189 L In some embodiments, provided herein are antibodies within the first family. Figures 12A-12B each show a sequence of each V H and V L 1 shows an alignment of antibodies from the first family used to generate the consensus sequence of

[0231] In some embodiments, there is provided a second family of antibodies (see Figures 13A-13B), wherein the antibodies of the family have the following six CDR sequences: (a) CDR-H1 having the sequence FTFX1X2YYMS (SEQ ID NO: 95) (X1 is G, A, P, or S, and X2 is N, Q, or D); (b) CDR-H2 having the sequence YISPSGX3TIX4YADSVKG (SEQ ID NO: 96) (X3 is A or S, and X4 is Y or A); (c) CDR-H3 having the sequence ARSLMX5TGTHLYFDL (SEQ ID NO: 97) (X5 is A or S); (d) CDR-L1 having the sequence RASX6X7ISX8WLA (SEQ ID NO: 98) (X6 is Q or S, X7 is D or G, and X8 is D or S); (e) CDR-L1 having the sequence AAX9X 10 LQS (SEQ ID NO: 99) (X9 is E or S and X 10 is S, A, G, V, or E); and (f) a CDR-L2 having the sequence QQX 11 TSX 12 X 13 PYT (SEQ ID NO: 100) (X 11 is A, S, or V, and X 12 is F, T, V, Q, or A, and X 13A second family of antibodies is provided herein, comprising a CDR-L3 having a CDR-L3 where L is L or P.

[0232] In some embodiments, the antibody of the family comprises the V of SEQ ID NO: 190. H Sequence and V of SEQ ID NO: 191 L In some embodiments, provided herein are antibodies within the second family. Figures 13A-13B each show a sequence of each V H and V L 1 shows an alignment of antibodies from a second family used to generate the consensus sequence of

[0233] 2. Functional properties of anti-CD161 antibodies In some embodiments, the CD161 antibody binds (e.g., specifically binds) to CD161. In some embodiments, human CD161 is represented by SEQ ID NO: 179. In some embodiments, the CD161 antibody binds to human CD161 and induces or promotes activation of human NK cells. In some embodiments, the CD161 antibody binds to human CD161 and induces or promotes activation of human T cells.

[0234] In some embodiments, the CD161 antibody has a K of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM or less when measured using a standard binding assay, e.g., surface plasmon resonance, biolayer interferometry, or Octet QK384 assay. D binds to human CD161 at these K DFor example, in certain embodiments, the antibody has a binding affinity of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, about 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, or about 0.1 nM to about 7 nM. nM, approximately 0.1nM to approximately 6nM, approximately 0.1nM to approximately 5nM, approximately 0.1nM to approximately 4nM, approximately 0.1nM to approximately 3nM, approximately 0.1nM to approximately 2nM, approximately 0.1nM to approximately 1nM, approximately 0.1nM to approximately 0.5nM, approximately 0.5nM to approximately 10nM, approximately K in the range of 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, about 8nM to about 10nM, or about 9nM to about 10nM D It binds to human CD161.

[0235] In certain embodiments, in addition to binding to human CD161, the disclosed antibodies also bind to Macaca fascicularis (cynomolgus monkey) CD161. In some embodiments, the cynomolgus monkey CD161 is represented by SEQ ID NO: 180. In some embodiments, the CD161 antibodies bind to cynomolgus monkey CD161 and induce or promote activation of cynomolgus monkey NK cells. In some embodiments, the CD161 antibodies bind to cynomolgus monkey CD161 and induce or promote activation of cynomolgus monkey T cells. For example, the antibody may have a K of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM or less when measured using a standard binding assay, e.g., surface plasmon resonance, biolayer interferometry, or Octet QK384 assay. Dbinds to cynomolgus CD161 at 100 kJ / s (i.e., these K D In certain embodiments, the antibody has a binding affinity of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, or about 0.1 nM to about 7 nM, as measured using a standard binding assay, for example, surface plasmon resonance, biolayer interferometry, or Octet QK384 assay. nM, approximately 0.1nM to approximately 6nM, approximately 0.1nM to approximately 5nM, approximately 0.1nM to approximately 4nM, approximately 0.1nM to approximately 3nM, approximately 0.1nM to approximately 2nM, approximately 0.1nM to approximately 1nM, approximately 0.1nM to approximately 0.5nM, approximately 0.5nM to approximately 10nM, approximately K in the range of 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, about 8nM to about 10nM, or about 9nM to about 10nM D It binds to cynomolgus monkey CD161.

[0236] In certain embodiments, EC measured in CD161-expressing HEK293 cells 50 The affinity of the antibodies disclosed herein for human CD161, as indicated by the EC20 saturation index (EC20 saturation index), is 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or less. In certain embodiments, the EC20 saturation index (EC20 saturation index) ... 50The affinity of the antibodies disclosed herein for human CD161 when represented by the formula (I) is about 20 nM to about 0.05 nM, about 20 nM to about 0.075 nM, about 20 nM to about 0.1 nM, about 20 nM to about 0.5 nM, about 20 nM to about 1 nM, about 10 nM to about 0.05 nM, about 10 nM to about 0.075 nM, about 10 nM to about 0.1 nM, about 10 nM to about 0.5 nM, about 10 nM to about 1 nM, about 5 nM to about 0.05 nM, about 5 nM to about 0.075 nM, about 5 nM to about 0.1 nM, about 5 nM to about 0.5 nM, about 5 nM to about 1 nM, about 3 nM to about 0.05 nM, about 3 nM to about 0.075 nM, about 3nM to about 0.1nM, about 3nM to about 0.5nM, about 3nM to about 1nM, about 3nM to about 2nM, about 2nM to about 0.05nM, about 2nM ~about 0.075nM, about 2nM to about 0.1nM, about 2nM to about 0.5nM, about 2nM to about 1nM, about 1nM to about 0.05nM, about 1nM to about 0 In certain embodiments, the EC200 / ... 50 The affinity of the antibodies disclosed herein for human CD161, as indicated by the formula (I), is about 0.5 nM to about 0.1 nM (e.g., about 0.45 nM, about 0.4 nM, about 0.35 nM, about 0.3 nM, about 0.25 nM, about 0.2 nM, about 0.15 nM, or about 0.05 nM).

[0237] In certain embodiments, the present disclosure provides antibodies that bind to the same epitope present in CD161 as the epitope bound by the disclosed antibodies, hi certain embodiments, the present disclosure provides antibodies that compete with the disclosed antibodies for binding to CD161.

[0238] Competitive assays for determining whether an antibody binds to the same epitope as a disclosed antibody or competes for binding with a disclosed antibody are known in the art. Exemplary competitive assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance (e.g., BIAcore analysis), biolayer interferometry, and flow cytometry. Typically, competitive assays involve the use of an antigen (e.g., human CD161 protein or a fragment thereof) bound to a solid surface or expressed on a cell surface, a test CD161-binding antibody, and a reference antibody. The reference antibody is labeled, and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess (e.g., 1x, 5x, 10x, 20x, or 100x). Antibodies identified by competition assays (i.e., competing antibodies) include antibodies that bind to the same epitope as the reference antibody or a similar (e.g., overlapping) epitope, as well as antibodies that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antibody so that steric hindrance occurs.

[0239] Competitive assays can be performed in both directions to ensure that the presence of label does not interfere with or otherwise inhibit binding. For example, in one direction, the reference antibody is labeled and the test antibody is unlabeled, and in the second direction, the test antibody is labeled and the reference antibody is unlabeled. A test antibody competes with the reference antibody for specific binding to an antigen if an excess of one antibody (e.g., 1×, 5×, 10×, 20×, or 100×) inhibits binding of the other antibody by at least 50%, 75%, 90%, 95%, or 99%, as measured, for example, in a competitive binding assay.

[0240] Two antibodies can be determined to bind the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies can be determined to bind overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody.

[0241] Several reports have shown that activation of CD161 by CLEC2D on tumor cells and immunosuppressive cells attenuates T cell responses against tumor cells. Accordingly, the present application relates to CD161 antibodies capable of binding to CD161 and inhibiting the interaction of CD161 with CLEC2D. In some embodiments, the CD161 antibodies of the present disclosure block, inhibit, or antagonize CLEC2D binding. In some embodiments, the CD161 antibodies of the present disclosure compete with CLEC2D for binding to CD161. In some embodiments, the CD161 antibodies of the present disclosure reduce CD161 inhibitory signaling due to CLEC2D binding to CD161. In some embodiments, CD161 is expressed on the surface of a cell. In certain embodiments, the present disclosure provides antibodies that compete with CLEC2D for binding to CD161.

[0242] In some embodiments, the CD161 antibody binds to a region of CD161 that overlaps with the ligand-binding region of CD161. In some embodiments, the CD161 antibody binds to or near the ligand-binding region of CD161. In some embodiments, the ligand-binding region of CD161 is a CLEC2D-binding region. In some embodiments, binding of the CD161 antibody, or antigen-binding fragment thereof, to CD161 prevents CLEC2D binding to CD161. In some embodiments, blockage of CD161 is measured by determining the concentration of cytokines produced by CD161-expressing immune cells. In some embodiments, the immune cells are NK cells or T cells. In some embodiments, blockage of CD161 is measured by determining the concentration of cytokines produced by CD161-expressing T cells in response to target cells (e.g., tumor cells). In some embodiments, increased cytokine production by the immune cells indicates blockage of CD161. In some embodiments, blockage of CD161 is measured by analyzing proliferation of CD161-expressing immune cells. In some embodiments, increased immune cell proliferation is indicative of blockade of CD161. In some embodiments, blockade of CD161 is measured by measuring the level of cell signaling by quantifying phosphorylation or expression of a gene reporter induced by an associated transcription factor. In some embodiments, increased cell signaling is indicative of blockade of CD161.

[0243] In certain embodiments, the CD161 antibodies of the disclosure have an IC of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or lower. 50This reduces CLEC2D binding to CD161 expressed on the surface of cells. In certain embodiments, the antibody may be present in a concentration range of about 20 nM to about 0.05 nM, about 20 nM to about 0.075 nM, about 20 nM to about 0.1 nM, about 20 nM to about 0.5 nM, about 20 nM to about 1 nM, about 10 nM to about 0.05 nM, about 10 nM to about 0.075 nM, about 10 nM to about 0.1 nM, about 10 nM to about 0.5 nM, about 10 nM to about 1 nM, about 5 nM to about 0.05 nM, about 5 nM to about 0.075 nM, about 5 nM to about 0.1 nM, about 5 nM to about 0.5 nM, about 5 nM to about 1 nM, about 3 nM to about 0.05 nM, about 3 nM to about 0.075 nM, or about 3 nM to about 0. 1nM, about 3nM to about 0.5nM, about 3nM to about 1nM, about 3nM to about 2nM, about 2nM to about 0.05nM, about 2nM to about 0.075n M, about 2nM to about 0.1nM, about 2nM to about 0.5nM, about 2nM to about 1nM, about 1nM to about 0.05nM, about 1nM to about 0.075n M, about 1nM to about 0.1nM, about 1nM to about 0.5nM, about 0.5nM to about 0.05nM, about 0.5nM to about 0.075nM, about 0.5 IC of nM to about 0.1nM, about 0.1nM to about 0.05nM, about 0.1nM to about 0.075nM, or about 0.075nM to about 0.05nM 50 In certain embodiments, the antibody reduces CLEC2D binding at a concentration of about 0.1 nM to about 10 nM (e.g., about 0.15 nM, about 0.2 nM, about 0.25 nM, about 0.3 nM, about 0.35 nM, about 0.4 nM, about 0.45 nM, about 0.5 nM, about 0.55 nM, about 0.6 nM, about 0.65 nM, about 0.7 nM, about 0.75 nM, about 0.8 nM, , about 0.85nM, about 0.9nM, about 0.95nM, about 1nM, about 1.5nM, about 2nM, about 2.5nM, about 3nM, about 3.5nM, about 4nM, about 4. IC of 5nM, about 5nM, about 5.5nM, about 6nM, about 6.5nM, about 7nM, about 7.5nM, about 8nM, about 8.5nM, about 9nM, and about 9.5nM) 50 reduces CLEC2D binding.

[0244] In certain embodiments, the CD161 antibody has a K of 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1,000 nM, 1,500 nM, 2,000 nM, 3,000 nM, 4,000 nM, 5,000 nM, 10,000 nM, or 20,000 nM or greater. D and binds to hKLRF1, hKLRF2, hCLEC12B, hCLEC2D, or any combination thereof. In some embodiments, the CD161 antibody does not bind to human KLRF1. In some embodiments, the CD161 antibody does not bind to human KLRF2. In some embodiments, the CD161 antibody does not bind to human CLEC12B. In some embodiments, the CD161 antibody does not bind to human CLEC2D.

[0245] In certain embodiments, the CD161 antibody binds to human CD161 and cynomolgus monkey CD161. In certain embodiments, the binding affinity K D (as measured by a monovalent affinity assay) is within about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold of the affinity for cynomolgus CD161.

[0246] In some embodiments, the CD161 antibodies of the disclosure reduce the inhibition of T cell or NK cell activity due to CLEC2D binding to CD161. In some embodiments, the inhibition of T cell or NK cell activity due to CLEC2D binding to CD161 is reduced by at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold compared to a control antibody (e.g., an antibody that does not bind to CD161).

[0247] In some embodiments, the CD161 antibody increases T cell or NK cell activity in the presence of CLEC2D compared to T cell or NK cell activity in the absence of the antibody or antigen-binding fragment. In certain embodiments, T cell or NK cell activity is increased by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold compared to a control antibody (e.g., an antibody that does not bind to CD161).

[0248] In some embodiments, T cells or NK cells are located in a microenvironment containing cells that express CLEC2D. In some embodiments, the CD161 antibody increases T cell or NK cell activity in a tumor microenvironment containing tumor cells that express CLEC2D. In some embodiments, the increase in T cell activity is determined by an increase in NFAT signaling. In some embodiments, the increase in NK cell activity is determined by an increase in CD107a expression. In certain embodiments, T cell or NK cell activity in the tumor microenvironment is increased by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold compared to a control antibody (e.g., an antibody that does not bind to CD161).

[0249] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein reduce CD161 inhibitory signaling as measured by one or more of the assays or biological effects described herein. In some embodiments, the CD161 antibodies of the present disclosure reduce CD161 inhibitory signaling due to CLEC2D binding to CD161. In certain embodiments, CD161 inhibitory signaling is reduced by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold compared to a control antibody (e.g., an antibody that does not bind to CD161).

[0250] In some embodiments, the CD161 antibodies of the present disclosure promote CD8+ T cell activation and cytokine production within the tumor microenvironment. In certain embodiments, the antibodies or antigen-binding fragments thereof activate CD8+ T cells and increase cytokine production within the tumor microenvironment by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold compared to a control antibody (e.g., an antibody that does not bind to CD161).

[0251] In certain embodiments, the antibodies provided herein comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:11.

[0252] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 19.

[0253] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:27, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO:27.

[0254] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 31, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 35, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 35.

[0255] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 39, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 43, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 43.

[0256] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 47, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 51.

[0257] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 55, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 59, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 59.

[0258] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 63, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 67, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 67.

[0259] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 71, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 75, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 71 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 75.

[0260] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 79, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 83, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 83.

[0261] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 87, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 91, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 91.

[0262] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 105, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 101 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 105.

[0263] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 109, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 113, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 109 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 113.

[0264] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 117, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 121, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 117 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 121.

[0265] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 125, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 129, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 125 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 129.

[0266] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 133, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 137, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 137.

[0267] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 141, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 145, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 145.

[0268] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 149, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 153, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 153.

[0269] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 157, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 161, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 157 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 161.

[0270] In certain embodiments, the antibodies provided herein (i) comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 165, and an immunoglobulin light chain variable region comprising an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 169, and (ii) compete for binding to human CD161 with, and / or bind to the same epitope on human CD161 as, an antibody comprising an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 165 and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 169.

[0271] The antibodies disclosed herein may be further optimized (e.g., affinity matured) to improve biochemical properties, including affinity and / or specificity; improve biophysical properties, including aggregation, stability, precipitation, and / or nonspecific interactions; and / or reduce immunogenicity. For example, diversity can be introduced into the immunoglobulin heavy chains and / or immunoglobulin light chains by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis, and / or site-directed mutagenesis.

[0272] In certain embodiments, the isolated human antibody contains one or more somatic mutations. In these cases, the antibody can be modified to human germline sequences to optimize the antibody (i.e., a process called germlining).

[0273] Generally, an optimized antibody has at least the same, or substantially the same, affinity for the antigen as the non-optimized (or parent) antibody from which it is derived. Preferably, the optimized antibody has a higher affinity for the antigen when compared to the parent antibody.

[0274] The antibodies disclosed herein can be conjugated to effector agents such as small molecule toxins or radionuclides using standard in vitro conjugation chemistry. If the effector agent is a polypeptide, the antibody may be chemically conjugated to the effector or may be bound to the effector as a fusion protein. Construction of fusion proteins is within the ordinary skill in the art.

[0275] (c) binds to the CD161 epitope bound by an anti-CD161 antibody or antigen-binding fragment thereof; (d) binds to human CD161 at or near the CLEC2D binding site; (e) competes with CLEC2D for binding to CD161; (f) reduces CD161 inhibitory signaling due to CLEC2D binding to CD161; (g) reduces suppression of T cell activity due to CLEC2D binding to CD161; (h) reduces suppression of NK cell activity due to CLEC2D binding to CD161; (i) increases T cell activity in the presence of CLEC2D compared to T cell activity in the absence of the antibody or antigen-binding fragment thereof; (j) increases NK cell activity in the absence of the antibody or antigen-binding fragment thereof. (k) increasing the activity of T cells disposed in a microenvironment comprising cells expressing CLEC2D; (l) increasing the activity of NK cells disposed in a microenvironment comprising cells expressing CLEC2D; (m) increasing T cell activity in a tumor microenvironment comprising tumor cells expressing CLEC2D; (n) increasing NK cell activity in a tumor microenvironment comprising tumor cells expressing CLEC2D; (o) inhibiting human T cell exhaustion; (p) inducing or increasing activation of CD161-expressing human T cells in response to antigen-expressing target cells; (q) inducing or increasing cytokine production by CD161-expressing human T cells in response to antigen-expressing target cells; (r) inducing or increasing granzyme B expression by CD161-expressing human T cells in response to antigen-expressing target cells; (s) reducing exhaustion of CD161-expressing human T cells in response to antigen-expressing target cells; and (t) any combination of (a)-(s).

[0276] 3. Monospecific and polyspecific anti-CD161 antibodies In certain embodiments, the antibodies provided herein are monospecific antibodies. However, in certain embodiments, the antibodies provided herein are multispecific antibodies. For example, a multispecific antibody can bind to two or more antigens, e.g., two antigens, three antigens, four antigens, or five antigens. In some embodiments, a multispecific antibody can bind to two or more epitopes on the CD161 antigen, e.g., two epitopes on the CD161 antigen or three epitopes on the CD161 antigen.

[0277] Many multispecific antibody constructs are known in the art, and the antibodies provided herein can be provided in the form of any suitable multispecific construct. In some embodiments, the multispecific antibody comprises an immunoglobulin comprising at least two different heavy chain variable regions, each paired with a common light chain variable region (i.e., a "common light chain antibody"). The common light chain variable region forms a unique antigen-binding domain with each of the two different heavy chain variable regions (see Merchant et al. (1998) NATURE BIOTECHNOL., 16: 677-681, incorporated by reference in its entirety).

[0278] In some embodiments, multispecific antibodies include immunoglobulins comprising antibodies linked to one or more of the N-terminus or C-terminus of the heavy or light chains of the immunoglobulin (see Coloma and Morrison (1997) NATURE BIOTECHNOL., 15: 159-163, incorporated by reference in their entireties). In some aspects, such antibodies comprise tetravalent bispecific antibodies. In some embodiments, multispecific antibodies comprise hybrid immunoglobulins comprising at least two different heavy chain variable regions and at least two different light chain variable regions (see Milstein and Cuello (1983) NATURE, 305: 537-540; and Staerz and Bevan (1986) PROC. NATL. ACAD. SCI. USA, 83: 1453-1457, each of which is incorporated by reference in its entirety).

[0279] In some embodiments, multispecific antibodies comprise immunoglobulin chains with modifications that reduce the formation of non-multispecific by-products. In some aspects, the antibodies comprise one or more "knobs-into-holes" modifications, as described in U.S. Pat. No. 5,731,168 (incorporated by reference in its entirety). In some embodiments, multispecific antibodies comprise immunoglobulin chains with one or more electrostatic modifications to promote Fc heteromultimer assembly (see WO 2009 / 089004 (incorporated by reference in its entirety)). In some embodiments, multispecific antibodies comprise bispecific single-chain molecules (see Traunecker et al. (1991) EMBO J., 10: 3655-3659; and Gruber et al. (1994) J. IMMUNOL., 152: 5368-5374 (each of which is incorporated by reference in its entirety)).

[0280] In some embodiments, multispecific antibodies comprise heavy and light chain variable domains connected by a polypeptide linker, the length of which is selected to facilitate assembly of the multispecific antibody with the desired multispecificity. For example, monospecific scFvs generally form when heavy and light chain variable domains are connected by a polypeptide linker of more than 12 amino acid residues (see U.S. Pat. Nos. 4,946,778 and 5,132,405, each of which is incorporated by reference in its entirety). In some embodiments, shortening the polypeptide linker length to less than 12 amino acid residues prevents pairing of heavy and light chain variable domains on the same polypeptide chain, thereby allowing pairing of heavy and light chain variable domains from one chain with complementary domains on another chain. Thus, the resulting antibody is multispecific, with the specificity of each binding site provided by more than one polypeptide chain. Polypeptide chains comprising heavy and light chain variable domains joined by a linker of between 3 and 12 amino acid residues form predominantly dimers (called diabodies). With linkers of between 0 and 2 amino acid residues, trimers (called triabodies) and tetramers (called tetrabodies) may be preferred. However, the exact type of oligomerization depends on the composition of amino acid residues in each polypeptide chain and the order of the variable domains (e.g., V, V). H -Linker-V L Against V L -Linker-V H ) The skilled artisan can select an appropriate linker length based on the desired level of multispecificity.

[0281] In some embodiments, the multispecific antibody comprises a diabody (see Hollinger et al. (1993) PROC. NATL. ACAD. SCI. USA, 90: 6444-6448, incorporated by reference in its entirety), or a triabody (see Todorovska et al. (2001) J. IMMUNOL. METHODS, 248: 47-66, incorporated by reference in its entirety), or a tetrabody (see ibid., incorporated by reference in its entirety).

[0282] In some embodiments, multispecific antibodies include trispecific F(ab')3 derivatives (see Tutt et al. (1991) J. IMMUNOL., 147: 60-69, incorporated by reference in its entirety).

[0283] In some embodiments, multispecific antibodies comprise cross-linked antibodies (see U.S. Pat. No. 4,676,980; Brennan et al. (1985) SCIENCE, 229: 81-83; Staerz et al. (1985) NATURE, 314: 628-631; and EP 0453082, each of which is incorporated by reference in its entirety). In some embodiments, multispecific antibodies comprise antigen-binding domains associated by a leucine zipper (see Kostelny et al. (1992) J. IMMUNOL., 148: 1547-1553, incorporated by reference in its entirety).

[0284] In some embodiments, the multispecific antibody comprises a DuoBody®, e.g., as in Labrijn et al. (2013) PROC. NATL. ACAD. SCI. USA, 110: 5145-5150; Gramer et al. (2013) MABS, 5: 962-972; and Labrijn et al. (2014) NATURE PROTOCOLS, 9: 2450-2463, each of which is incorporated by reference in its entirety.

[0285] 4. Glycosylation variants In certain embodiments, the antibodies provided herein may be altered to increase, decrease, or eliminate the extent to which they are glycosylated. Glycosylation of polypeptides is typically either "N-linked" or "O-linked." "N-linked" glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. "O-linked" glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used).

[0286] Addition of N-linked glycosylation sites to or deletion of N-linked glycosylation sites from the antibodies provided herein can be accomplished by altering the amino acid sequence so as to create or remove one or more of the above tripeptide sequences. Addition or deletion of O-linked glycosylation sites can be accomplished by adding, deleting, or substituting one or more serine or threonine residues in, or (as the case may be) to, the sequence of the antibody.

[0287] In some embodiments, the antibodies provided herein comprise a glycosylation motif that differs from naturally occurring antibodies. Any suitable naturally occurring glycosylation motif can be engineered into the antibodies provided herein. For example, the structure and glycosylation characteristics of immunoglobulins are known in the art and are summarized, for example, in Schroeder and Cavacini (2010) J. ALLERGY CLIN. IMMUNOL., 125: S41-52, which is incorporated by reference in its entirety.

[0288] In some embodiments, the antibodies provided herein comprise an IgG1 Fc region with modifications to the oligosaccharide attached to asparagine 297 (Asn297). Naturally occurring IgG1 antibodies produced by mammalian cells typically have a C nucleotide sequence in the Fc region. H2 The oligosaccharides attached to Asn297 may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure.

[0289] In some embodiments, the oligosaccharide attached to Asn297 is modified to generate an antibody with altered ADCC. In some embodiments, the oligosaccharide is modified to improve ADCC. In some embodiments, the oligosaccharide is modified to decrease ADCC.

[0290] In some embodiments, the antibodies provided herein comprise an IgG1 domain having a reduced fucose content at position Asn297 compared to a naturally occurring IgG1 domain. Such Fc domains are known to have improved ADCC (see Shields et al. (2002) J. BIOL. CHEM., 277: 26733-26740, incorporated by reference in its entirety). In some embodiments, such antibodies do not comprise fucose at position Asn297. The amount of fucose may be determined using any suitable method, for example, as described in WO2008 / 077546, incorporated by reference in its entirety.

[0291] In some embodiments, the antibodies provided herein comprise bisected oligosaccharides, such as biantennary oligosaccharides, attached to the Fc region of the antibody, bisected by a GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; and U.S. Patent No. 6,602,684 (each of which is incorporated by reference in its entirety).

[0292] In some embodiments, the antibodies provided herein comprise an Fc region having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764 (each of which is incorporated by reference in its entirety).

[0293] Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (see Ripka et al. (1986) ARCH. BIOCHEM. BIOPHYS., 249: 533-545; U.S. Patent Publication No. 2003 / 0157108; WO2004 / 056312, each of which is incorporated by reference in its entirety), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene or FUT8 knockout CHO cells (Yamane-Ohnuki et al. (2004) BIOTECH. BIOENG., 87: 614-622; Kanda et al. (2006) BIOTECHNOL. BIOENG., 94: 680-688; and WO2003 / 085107 (each of which is incorporated by reference in its entirety).

[0294] In some embodiments, the antibodies provided herein are aglycosylated antibodies. Aglycosylated antibodies can be produced using any method known in the art or described herein. In some aspects, aglycosylated antibodies are produced by modifying an antibody to remove all glycosylation sites. In some aspects, glycosylation sites are removed only from the Fc region of the antibody. In some aspects, aglycosylated antibodies are produced by expressing the antibody in an organism incapable of glycosylation, such as E. coli, or by expressing the antibody in a cell-free reaction mixture.

[0295] In some embodiments, the antibodies provided herein have a constant region that has reduced effector function compared to a native IgG1 antibody, hi some embodiments, the affinity of the constant region of the Fc region of the antibodies provided herein for an Fc receptor is less than the affinity of the native IgG1 constant region for the Fc receptor.

[0296] 5. Fc region The anti-CD161 antibodies of the present disclosure are IgG-type antibodies. For example, each of the anti-CD161 antibodies disclosed in Table 4 is fused to an immunoglobulin Fc domain (e.g., an immunoglobulin Fc domain derived from human IgG1, human IgG2, human IgG3, human IgG4, human IgA1, human IgA2, human IgD, human IgE, or human IgM Fc domain). In an exemplary embodiment, each of the anti-CD161 antibodies disclosed in Table 4 is fused to an immunoglobulin Fc domain derived from human IgG1.

[0297] In certain embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions, insertions, or deletions compared to a naturally occurring Fc region. In some aspects, such substitutions, insertions, or deletions result in an antibody with altered stability, glycosylation, or other properties. In some aspects, such substitutions, insertions, or deletions result in an aglycosylated antibody.

[0298] In certain embodiments, the immunoglobulin Fc domain is derived from a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM Fc domain. A single amino acid substitution (S228P according to Kabat numbering; referred to as IgG4Pro) may be introduced to eliminate the heterogeneity observed in recombinant IgG4 antibodies (see Angal, S. et al. (1993) MOL. IMMUNOL., 30:105-108).

[0299] In some aspects, the Fc region of the antibodies provided herein is modified to produce antibodies with altered affinity for Fc receptors or to produce antibodies that are more immunologically inert. In some embodiments, the antibody variants provided herein retain some, but not all, effector functions. Such antibodies may be useful, for example, when antibody half-life is important in vivo, but certain effector functions (e.g., complement activation and ADCC) are unnecessary or deleterious.

[0300] In some embodiments, the Fc region of an antibody provided herein is a human IgG4 Fc region comprising one or more of the hinge-stabilizing mutations S228P and L235E (see Aalberse et al. (2002) IMMUNOLOGY, 105: 9-19, incorporated by reference in its entirety). In some embodiments, the IgG4 Fc region comprises one or more of the following mutations: E233P, F234V, and L235A (see Armour et al. (2003) MOL. IMMUNOL., 40: 585-593, incorporated by reference in its entirety). In some embodiments, the IgG4 Fc region comprises a deletion at position G236.

[0301] In some embodiments, the Fc region of an antibody provided herein is a human IgG1 Fc region comprising one or more mutations to reduce Fc receptor binding. In some embodiments, the Fc domain comprises one or more mutations, such as those described in U.S. Patent No. 8,394,925 (incorporated herein by reference in its entirety). In some embodiments, the Fc region is a variant Fc region comprising amino acid substitutions at positions 428 and 434, where the amino acid substitutions are a non-wild-type amino acid leucine at position 428 and a non-wild-type amino acid serine at position 434, and the polypeptide is an antibody, where the numbering is according to the EU index in Kabat et al. In some embodiments, the Fc region comprises an S228P, L235E, M428L, or N434S substitution. In some embodiments, the Fc region comprises an M428L substitution. In some embodiments, the Fc region comprises an N434S substitution. In some embodiments, the Fc region comprises an M428L and an N434S substitution. In some embodiments, the Fc region comprises M252Y, S254T, and / or T256E substitutions. In some aspects, the antibody comprises a PVA236 mutation. PVA236 refers to the substitution of the amino acid sequence ELLG (SEQ ID NO: 195) from amino acid positions 233-236 of IgG1 or EFLG (SEQ ID NO: 196) of IgG4 with PVA (see U.S. Patent No. 9,150,641, incorporated by reference in its entirety).

[0302] In some embodiments, the Fc region of an antibody provided herein is a human IgG2 Fc region comprising one or more of the mutations A330S and P331S.

[0303] In some embodiments, the Fc region of an antibody provided herein comprises an amino acid substitution at one or more positions selected from 238, 265, 269, 270, 297, 327, and 329 (see U.S. Pat. No. 6,737,056, incorporated by reference in its entirety). Such Fc variants include Fc variants comprising substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including so-called "DANA" Fc variants comprising substitutions of residues 265 and 297 with alanine (see U.S. Pat. No. 7,332,581, incorporated by reference in its entirety). In some embodiments, the antibody comprises an alanine at amino acid position 265. In some embodiments, the antibody comprises an alanine at amino acid position 297. In certain embodiments, the human IgG1 constant region is modified at amino acid Asn297 (e.g., Asn297Ala (N297A)) to prevent glycosylation of the antibody. In certain embodiments, the mutations are effective to eliminate binding of the anti-CD161 antibodies disclosed herein to Fcγ receptors (e.g., RI, RIIA, RIIB / C, RIIA, and RIIIB). In certain embodiments, the mutations are effective to significantly reduce binding of the anti-CD161 antibodies disclosed herein to Fcγ receptors (e.g., RI, RIIA, RIIB / C, RIIA, and RIIIB). In exemplary embodiments, each of the anti-CD161 antibodies disclosed in Table 4 is fused to a human IgG1 constant region modified at amino acid Asn297.

[0304] In certain embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at one or more of Fc region positions 298, 333, and 334. In some embodiments, the antibodies provided herein comprise an Fc region with one or more amino acid substitutions at positions 239, 332, and 330, as described in Lazar et al. (2006) PROC. NATL. ACAD. SCI. USA, 103: 4005-4010, which is incorporated by reference in its entirety.

[0305] In some embodiments, the antibodies provided herein comprise one or more alterations to increase half-life. In some embodiments, the Fc domain comprises a mutation, such as those described in U.S. Pat. No. 7,670,600 (incorporated herein by reference in its entirety). In some embodiments, the constant region comprises a mutation at amino acid residue 428, as compared to a wild-type human IgG constant domain, numbered according to the EU numbering index of Kabat. Without being bound by any particular theory, antibodies comprising a mutation corresponding to residue 428 may have an increased half-life compared to the half-life of an IgG having a wild-type human IgG constant domain. In some embodiments, the mutation is a substitution of the native residue with threonine, leucine, phenylalanine, or serine. In some embodiments, the antibody further comprises one or more amino acid substitutions, as compared to the corresponding wild-type human IgG constant domain, at one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 429-436, as numbered according to the EU numbering index of Kabat. Specific mutations or substitutions at these positions are described in US Pat. No. 7,670,600, which is incorporated herein by reference in its entirety.

[0306] 6. Antibodies with reduced immunogenicity If the antibodies are to be administered to humans, they are preferably human antibodies or engineered to reduce or eliminate antigenicity in humans. Preferably, each humanized antibody has the same, or substantially the same, affinity for the antigen as the non-humanized murine antibody from which it is derived.

[0307] One humanization approach creates chimeric proteins in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions (see, e.g., Morrison et al., 1984, PROC. NAT. ACAD. SCI. 81:6851-6855; Neuberger et al., 1984, NATURE 312:604-608; U.S. Patent Nos. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).

[0308] In an approach known as CDR grafting, the CDRs of the light and heavy chain variable regions are grafted onto a framework from another species. For example, murine CDRs can be grafted onto human framework regions (FRs). In some embodiments, the CDRs of the light and heavy chain variable regions of an antibody are grafted onto human FRs or consensus human FRs. To create consensus human FRs, FRs from several human heavy or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR porting is commonly used in U.S. Patent Nos. 7,022,500 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332 (Hoogenboom); 5,585,089 (Queen); 5,530,101 (Queen); Jones et al. (1986) NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332: 323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter (1998) FEBS LETT 430: 92-94.

[0309] In an approach called "SUPERHUMANIZATION™," human CDR sequences are selected from human germline genes based on the structural similarity of the human CDRs to the CDRs of the mouse antibody to be humanized. See, e.g., U.S. Patent No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL. 169:1119-1125.

[0310] Other methods for reducing immunogenicity include "reshaping," "hyperchimerization," and "veneering / resurfacing." See, e.g., Vaswami et al., 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81:105; Roguska et al., 1996, PROT. ENGINEER 9:895-904; and U.S. Pat. No. 6,072,035 (Hardman). In the veneering / resurfacing approach, surface-accessible amino acid residues in a mouse antibody are replaced with amino acid residues more frequently found at the same positions in human antibodies. This type of antibody resurfacing is described, for example, in U.S. Pat. No. 5,639,641 (Pedersen).

[0311] Another approach for converting murine antibodies into a form suitable for human clinical use is known as ACTIVMAB™ technology (Vaccinex, Inc., Rochester, NY), which involves the use of vaccinia virus-based vectors for expressing antibodies in mammalian cells. High levels of combinatorial diversity of IgG heavy and light chains can be generated. See, e.g., U.S. Patent Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer). Another approach for converting murine antibodies into a form suitable for human use is the technology commercially implemented by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology involves the use of a proprietary human "acceptor" library to generate an "epitope-focused" library for antibody selection. Another approach for modifying murine antibodies into a form suitable for medical use in humans is the HUMAN ENGINEERING™ technology, which is commercially practiced by XOMA (US) LLC. See, e.g., International (PCT) Publication No. WO 93 / 11794 and U.S. Patent Nos. 5,766,886 (Studnicka); 5,770,196 (Studnicka); 5,821,123 (Studnicka); and 5,869,619 (Studnicka), each of which is incorporated by reference in its entirety.

[0312] Any suitable approach, including any of the approaches described above, can be used to reduce or eliminate the human immunogenicity of an antibody.

[0313] Furthermore, it is possible to generate fully human antibodies in mice. Fully human mAbs lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice by techniques referenced, for example, in Lonberg et al., NATURE 368:856-859, 1994; Fishwild et al., NATURE BIOTECHNOLOGY 14:845-851, 1996; and Mendez et al., NATURE GENETICS 15:146-156, 1997. Fully human monoclonal antibodies can also be prepared and optimized from phage display libraries by techniques referenced, for example, in Knappik et al., J. MOL. BIOL. 296:57-86, 2000; and Krebs et al., J. IMMUNOL. METH. 254:67-84, 2001.

[0314] 7. Anti-CD161 antibody-drug conjugates The present disclosure further provides antibody conjugates containing one or more of the antibodies disclosed herein. As used herein, unless otherwise indicated, the term "antibody conjugate" is understood to refer to an antibody or functional fragment thereof comprising antigen-binding activity (e.g., anti-CD161 antigen-binding activity) and / or Fc receptor-binding activity conjugated (e.g., covalently linked) to an additional functional moiety.

[0315] In certain embodiments, the antibody conjugate is an antibody-drug conjugate (ADC) comprising an antibody that specifically binds to CD161 and a cytotoxic agent. The cytotoxic agent can be directly or indirectly linked to the anti-CD161 antibody. In some embodiments, the ADC further comprises a linker that covalently attaches the cytotoxic agent to the anti-CD161 antibody.

[0316] Exemplary cytotoxic agents useful in the generated ADCs include, for example, certain anti-tumor or anti-cancer agents known in the art. In some embodiments, the cytotoxic agent causes destruction of cancer cells. In some embodiments, the cytotoxic agent inhibits the growth or proliferation of cancer cells. Exemplary cytotoxic agents include anti-angiogenic agents, pro-apoptotic agents, anti-mitotic agents, anti-kinase agents, alkylating agents, hormones, hormone agonists, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes, antimetabolites, antibiotics, alkaloids, and radioisotopes.

[0317] In certain embodiments, the cytotoxic agent is attached to the anti-CD161 antibody via a linker. In some embodiments, the linker used to generate the ADC comprises two reactive ends: an antibody conjugation reactive end and a cytotoxic agent conjugation reactive end. The antibody conjugation reactive end of the linker can be conjugated to the antibody via a cysteine ​​thiol or lysine amine group on the antibody, for example, via a thiol-reactive group, such as a double bond, a leaving group, such as a chloro, bromo, or iodo, an R-sulfanyl or sulfonyl group, or an amine-reactive group, such as a carboxyl group. The cytotoxic agent conjugation reactive end of the linker can be conjugated to the cytotoxic agent, for example, by forming an amide bond with a basic amine or carboxyl group, typically a carboxyl or basic amine group, on the cytotoxin.

[0318] Depending on the intended purpose of the ADC, the linker may be a non-cleavable or a cleavable linker.

[0319] 8. How to generate CD161 antibodies 8.1 CD161 antigen preparation The CD161 antigen used to isolate the antibodies provided herein may be intact CD161 or a fragment of CD161. The CD161 antigen may be, for example, in the form of an isolated protein or in the form of a protein expressed on the surface of a cell. In some embodiments, the CD161 antigen is a non-naturally occurring variant of CD161, e.g., a CD161 protein having an amino acid sequence or post-translational modification that does not occur in nature.

[0320] In some embodiments, the CD161 antigen is truncated, for example, by removal of the intracellular or transmembrane sequence, or the signal sequence. In some embodiments, the CD161 antigen is fused at its C-terminus to a human IgG1 Fc domain or a polyhistidine tag.

[0321] 8.2 Methods for producing monoclonal antibodies Monoclonal antibodies may be obtained, for example, using the hybridoma method first described by Kohler et al. (1975) NATURE, 256: 495-497, incorporated by reference in its entirety, and / or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567, incorporated by reference in its entirety). Monoclonal antibodies may also be obtained using, for example, phage display libraries (see, e.g., U.S. Pat. No. 8,258,082, incorporated by reference in its entirety) or yeast-based libraries (see, e.g., U.S. Pat. Nos. 8,691,730 and 9,354,228, incorporated by reference in their entireties).

[0322] In the hybridoma method, a mouse or other suitable host animal is immunized to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding JW (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 3 RD ed., Academic Press, San Diego, CA (incorporated by reference in its entirety).

[0323] The hybridoma cells are seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0324] Useful myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to culture conditions, such as the presence or absence of HAT medium. Among these, preferred myeloma cell lines are mouse myeloma lines, such as those derived from MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, CA), and SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, MD). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (see, e.g., Kozbor (1984) J. IMMUNOL., 133:3001, incorporated by reference in its entirety).

[0325] After identification of hybridoma cells producing antibodies of the desired specificity, affinity, and / or biological activity, selected clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0326] DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells can therefore serve as a useful source of DNA encoding antibodies with the desired characteristics. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells that would not otherwise produce the antibody, such as bacteria (e.g., E. coli), yeast (e.g., Saccharomyces or Pichia species), COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to produce the monoclonal antibody.

[0327] 8.3 Methods for Producing Antibodies with Reduced Immunogenicity Humanized antibodies may be produced by replacing most or all of the structural portions of a non-human monoclonal antibody with corresponding human antibody sequences, resulting in a hybrid molecule in which only the antigen-specific variable elements, i.e., the CDRs, are composed of non-human sequences. Methods for obtaining humanized antibodies include, for example, those described in Winter and Milstein (1991) NATURE, 349: 293-299; Rader et al. (1998) PROC. NAT. ACAD. SCI. USA, 95: 8910-8915; Steinberger et al. (2000) J. BIOL. CHEM., 275: 36073-36078; Queen et al. (1989) PROC. NATL. ACAD. SCI. USA, 86: 10029-10033; and U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370 (each of which is incorporated by reference in its entirety).

[0328] Human antibodies can be produced by various techniques known in the art, such as by using transgenic animals (e.g., humanized mice). See, e.g., Jakobovits et al. (1993) PROC. NATL. ACAD. SCI. USA, 90: 2551; Jakobovits et al. (1993) NATURE, 362: 255-258; Bruggermann et al. (1993) YEAR IN IMMUNO., 7: 33; and U.S. Patent Nos. 5,591,669, 5,589,369, and 5,545,807 (each of which is incorporated by reference in its entirety). Human antibodies can also be obtained from phage display libraries (see, e.g., Hoogenboom et al. (1991) J. MOL. BIOL., 227: 381-388; Marks et al. (1991) J. MOL. BIOL., 222: 581-597; and U.S. Patent Nos. 5,565,332 and 5,573,905, each of which is incorporated by reference in its entirety). Human antibodies can also be generated by in vitro activated B cells (see, e.g., U.S. Patent Nos. 5,567,610 and 5,229,275, each of which is incorporated by reference in its entirety). Human antibodies can also be obtained from yeast-based libraries (see, e.g., U.S. Patent Nos. 8,691,730 and 9,354,228, each of which is incorporated by reference in its entirety).

[0329] Exemplary methods for producing chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al. (1984) PROC. NATL. ACAD. SCI. USA, 81: 6851-6855, each of which is incorporated by reference in its entirety. In some embodiments, chimeric antibodies are produced by using recombinant techniques to combine non-human variable regions (e.g., variable regions derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) with human constant regions.

[0330] 8.4 Methods for Producing Antibody Fragments The antibody fragments provided herein can be produced by any suitable method, including exemplary methods described herein or known in the art. Suitable methods include recombinant techniques and proteolytic digestion of whole antibodies. Exemplary methods for producing antibody fragments are described, for example, in Hudson et al. (2003) NAT. MED., 9: 129-134 (incorporated by reference in its entirety). Methods for producing scFv antibodies are described, for example, in Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, VOL. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315; WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458 (each of which is incorporated by reference in its entirety).

[0331] 8.5 Methods for generating polyspecific antibodies The multispecific antibodies provided herein can be produced by any suitable method, including exemplary methods described herein or known in the art. Methods for producing common light chain antibodies are described in Merchant et al. (1998) NATURE BIOTECHNOL., 16: 677-681 (incorporated by reference in their entirety). Methods for producing tetravalent bispecific antibodies are described in Coloma and Morrison (1997) NATURE BIOTECHNOL., 15: 159-163 (incorporated by reference in their entirety). Methods for producing hybrid immunoglobulins are described in Milstein and Cuello (1983) NATURE, 305: 537-540; and Staerz and Bevan (1986) PROC. NATL. ACAD. SCI. USA, 83: 1453-1457 (each of which is incorporated by reference in its entirety). Methods for producing immunoglobulins with knob-into-hole modifications are described in U.S. Patent No. 5,731,168 (incorporated by reference in its entirety). Methods for producing immunoglobulins with electrostatic modifications are provided in WO 2009 / 089004 (incorporated by reference in its entirety). Methods for producing bispecific single-chain antibodies are described in Traunecker et al. (1991) EMBO J., 10: 3655-3659; and Gruber et al. (1994) J. IMMUNOL., 152: 5368-5374 (each of which is incorporated by reference in its entirety). Methods for producing single-chain antibodies with variable linker lengths are described in U.S. Patent Nos. 4,946,778 and 5,132,405 (each of which is incorporated by reference in its entirety). Methods for producing diabodies are described in Hollinger et al. (1993) PROC. NATL. ACAD. SCI. USA, 90: 6444-6448, which is incorporated by reference in its entirety.Methods for making triabodies and tetrabodies are described in Todorovska et al. (2001) J. IMMUNOL. METHODS, 248: 47-66 (incorporated by reference in its entirety). Methods for making trispecific F(ab')3 derivatives are described in Tutt et al. (1991) J. IMMUNOL., 147: 60-69 (incorporated by reference in its entirety). Methods for making cross-linked antibodies are described in U.S. Pat. No. 4,676,980; Brennan et al. (1985) SCIENCE, 229: 81-83; Staerz et al. (1985) NATURE, 314: 628-631; and EP 0453082 (each of which is incorporated by reference in its entirety).

[0332] 8.6 How to create variants In some embodiments, the antibodies provided herein are affinity matured variants of a parent antibody, which may be generated, for example, using phage display-based affinity maturation techniques. Briefly, one or more CDR residues may be mutated, and the variant antibodies, or portions thereof, may be displayed on phage and screened for affinity. Such alterations may be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during somatic maturation (see Chowdhury (2008) METHODS MOL. BIOL., 207: 179-196, incorporated by reference in its entirety), and / or in residues that contact the antigen.

[0333] Variability can be introduced into the polynucleotide sequence(s) encoding an antibody using any suitable method, including error-prone PCR, chain shuffling, and oligonucleotide-directed mutagenesis, e.g., trinucleotide-directed mutagenesis (TRIM). In some embodiments, several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted for mutation.

[0334] The introduction of diversity into the variable regions and / or CDRs can be used to generate a secondary library. The secondary library is then screened to identify antibody variants with improved affinity. Affinity maturation by constructing and reselecting from a secondary library is described, for example, in Hoogenboom et al. (2001) METHODS MOL. BIOL., 178: 1-37 (incorporated by reference in its entirety).

[0335] 8.7 Vectors, host cells, and recombinant methods Also provided are isolated nucleic acids encoding CD161 antibodies, vectors containing the nucleic acids, and host cells containing the vectors and nucleic acids, as well as recombinant techniques for the production of the antibodies.

[0336] For recombinant production of an antibody, the nucleic acid(s) encoding the antibody can be isolated and inserted into a replicable vector for further cloning (i.e., amplification of the DNA) or expression. In some embodiments, the nucleic acid can be produced by homologous recombination, as described, for example, in U.S. Patent No. 5,204,244, which is incorporated by reference in its entirety.

[0337] Many different vectors are known in the art. The vector components generally include one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, as described, for example, in U.S. Patent No. 5,534,615, which is incorporated by reference in its entirety.

[0338] Examples of suitable host cells include any suitable prokaryotic cell (eg, a bacterial cell), lower eukaryotic cell (eg, a yeast cell), or higher eukaryotic cell (eg, a mammalian cell). Suitable prokaryotes include eubacteria, for example, gram-negative or gram-positive organisms, such as Enterobacteriaceae, for example, Escherichia (E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia (S. marcescans), Shigella, Bacilli (B. subtilis and B. licheniformis), Pseudomonas (P. aeruginosa), aeruginosa, and Streptomyces. One useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli X1776, and E. coli W3110 are also suitable.

[0339] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for CD161 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is a commonly used lower eukaryotic host microorganism. However, Schizosaccharomyces pombe, Kluyveromyces species (K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K. thermotolerans, and K. marxianus), Yarrowia species, Pichia pastoris, Candida species (C. albicans), Trichoderma reesei, Neurospora crassa, and others have been reported to be pathogenic. Several other genera, species, and strains are available and useful, such as S. crassa, Schwanniomyces (S. occidentalis), and filamentous fungi, e.g., Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A. niger).

[0340] Useful mammalian host cells include COS-7 cells, HEK293 cells, baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO) cells, mouse Sertoli cells, African green monkey kidney cells (VERO-76), and the like.

[0341] The host cells used to produce the CD161 antibody of the present invention can be cultured in a variety of media. For example, commercially available media such as Ham's F10, minimal essential medium (MEM), RPMI-1640, and Dulbecco's modified Eagle's medium (DMEM) are suitable for culturing host cells. In addition, any of the media described in Ham et al. (1979) METH. ENZ., 58: 44; Barnes et al. (1980) ANAL. BIOCHEM., 102: 255; and U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, and 5,122,469; or WO1990 / 03430 and WO1987 / 00195 (each of which is incorporated by reference in its entirety) can be used.

[0342] Any of these media may be supplemented as necessary with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

[0343] The culture conditions, such as temperature, pH, etc., are those previously used with the host cell selected for expression and will be apparent to one skilled in the art.

[0344] When using recombinant techniques, antibodies may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If antibodies are produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. For example, Carter et al. (1992) BIO / TECHNOLOGY, 10: 163-167 (incorporated by reference in its entirety) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation.

[0345] In some embodiments, the antibody is produced in a cell-free system. In some aspects, the cell-free system is an in vitro transcription and translation system as described in Yin et al. (2012) MABS, 4: 217-225 (incorporated by reference in its entirety). In some aspects, the cell-free system utilizes a cell-free extract from a eukaryotic or prokaryotic cell. In some aspects, the prokaryotic cell is Escherichia coli. Cell-free expression of an antibody can be useful, for example, when the antibody accumulates in the cell as insoluble aggregates or when yields from periplasmic expression are low.

[0346] Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, e.g., an Amicon® or Millipore® Pellcon® ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0347] Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a particularly useful purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies containing human γ1, γ2, or γ4 heavy chains (Lindmark et al. (1983) J. IMMUNOL. METH., 62: 1-13, incorporated by reference in its entirety). Protein G is useful for all mouse isotypes and for human γ3 (Guss et al. (1986) EMBO J., 5: 1567-1575, incorporated by reference in its entirety).

[0348] The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. H3 If the domain is involved, BakerBond ABX® resin is useful for purification.

[0349] Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin Sepharose®, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available and can be applied by one of skill in the art.

[0350] After any preliminary purification step(s), the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, generally performed at a low salt concentration (e.g., about 0 to about 0.25 M salt) and using an elution buffer at a pH between about 2.5 and about 4.5.

[0351] 9. Functional Assay Various assays known in the art may be used to identify and characterize the anti-CD161 antibodies and anti-CD161 ADCs provided herein.

[0352] 9.1 Binding, Competition, and Epitope Mapping Assays 9.1.1 Antigen Binding Assays In some embodiments, antigen binding activity is determined using an antigen binding assay. In some embodiments, the antigen binding assay measures the binding affinity (K D In some embodiments, the antigen binding assay determines the kinetic rate constant (e.g., k) for the binding interaction of an anti-CD161 antibody to a CD161 polypeptide. on , k off In some embodiments, the kinetic characteristics of the binding interaction of a CD161 antibody to a target molecule are determined using an Octet QK384 assay.

[0353] Although examples of antigen-binding activity assays are provided herein, the specific antigen-binding activity of the CD161 antibodies provided herein may also be assessed by any suitable method, including using surface plasmon resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium exclusion binding assay (KinExA), gel shift assay, pull-down assay, quantitative immunoblot, equilibrium dialysis, analytical ultracentrifugation, fluorescence anisotropy, solution equilibrium titration, equilibrium exclusion binding assay, and isothermal titration calorimetry. These methods are well known in the art.

[0354] In some embodiments, the antigen binding assay comprises measuring the binding affinity of a labeled anti-CD161 antibody to a CD161 polypeptide expressed on the cell surface. In some embodiments, the anti-CD161 antibody is labeled with a fluorescent molecule (e.g., a fluorescent dye). In some embodiments, binding is detected using a fluorescent detection method (e.g., flow cytometry). In some embodiments, binding of an anti-CD161 antibody disclosed herein to an antigen-expressing cell is compared to a reference cell lacking expression of the antigen.

[0355] In some embodiments, the antigen binding assay is surface plasmon resonance. "Surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biomolecular specific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U., et al. (1993) ANN. BIOL. CLIN., 51: 19-26; Jonsson, U., et al. (1991) BIOTECHNIQUES, 11: 620-627; Johnsson, B., et al. (1995) J. MOL. RECOGNIT., 8: 125-131; and Johnson, B., et al. (1991) ANAL. BIOCHEM., 198: 268-277.

[0356] In some embodiments, the antigen binding assay is biolayer interferometry (BLI). The phrases "biolayer interferometry" or "BLI" refer to an optical phenomenon that allows for the measurement of sub-nanometer changes in the thickness of the optical layer of the sensing surface. In some embodiments, biomolecules bind to the sensor surface, causing a change in the thickness of the optical layer. The magnitude of the change in the thickness of the optical layer is proportional to the mass or molecular weight of the bound molecule. In some embodiments, CD161 is immobilized on the sensor surface to measure binding by the antibody, and binding causes a change in molecular weight, resulting in a corresponding change in the thickness of the optical layer. In some embodiments where CD161 is immobilized on the sensor surface, samples of anti-CD161 antibodies are prepared and injected by serial dilution, and the K is determined from modeling of the binding curve versus antibody concentration. D In some embodiments, the BLI is performed on an Octet QK384 system (ForteBio), i.e., the antigen binding assay is an Octet QK384 assay.

[0357] 9.1.2 Ligand Binding Assays In some embodiments, the anti-CD161 antibodies described herein bind to human CD161 and block or inhibit binding by CLEC2D as determined by a ligand binding assay. A ligand binding assay is an assay that provides a measure of the degree of interaction and / or affinity between a receptor and a ligand. For example, in some embodiments, a ligand binding assay is used to determine the degree of binding of a ligand molecule (e.g., CLEC2D) to a receptor (e.g., CD161). In some embodiments, the ligand binding assay comprises detecting the formation of a complex between the ligand and the receptor. In some embodiments, to determine the degree of ligand binding to the receptor, the ligand binding assay comprises determining the dissociation of the ligand:receptor complex. In some embodiments, the formation and / or dissociation of the ligand:receptor complex is determined by detecting a fluorescently labeled ligand in complex with the receptor. In some embodiments, the formation and / or dissociation of the ligand:receptor complex is determined by detecting and / or quantifying the amount of fluorescently labeled receptor in complex with the ligand. In some embodiments, the formation and / or dissociation of the ligand:receptor complex is determined by detecting and / or quantifying the amount of fluorescently labeled antibody that specifically binds to the ligand:receptor complex. Methods for detecting and quantifying fluorescence are known in the art and include, but are not limited to, fluorescence polarization (FP), fluorescence anisotropy (FA), flow cytometry, and microscopy. In some embodiments, the formation and / or dissociation of a ligand:receptor complex is determined by detecting and / or quantifying the amount of radiolabeled ligand in complex with the receptor. In some embodiments, the formation and / or dissociation of a ligand:receptor complex is determined by detecting and / or quantifying the amount of radiolabeled receptor in complex with the ligand. In some embodiments, the formation and / or dissociation of a ligand:receptor complex is determined by detecting and / or quantifying the amount of radiolabeled antibody that specifically binds to the ligand:receptor complex.

[0358] In some embodiments, an antibody of the present disclosure (e.g., an anti-CD161 antibody) binds to a receptor (e.g., CD161) and disrupts, inhibits, or blocks formation of a ligand:receptor complex (e.g., a CD161:CLEC2D complex).

[0359] 9.1.3 Competitive Assays Assays for measuring competition between two antibodies, or between an antibody and another molecule (e.g., one or more ligands of CD161), are well known in the art, e.g., Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CH.14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (incorporated by reference in its entirety).

[0360] 9.1.4 Epitope Mapping Assays The CD161 antibodies described herein are characterized by epitope binding on CD161. In some embodiments, the binding epitope of each CD161 antibody on a CD161 ligand is determined by surface plasmon resonance, for example, using a Biacore 8K instrument. Assays for mapping the epitopes to which the antibodies provided herein bind are described, for example, in Morris (1996) "Epitope Mapping Protocols," in METHODS IN MOLECULAR BIOLOGY, vol. 66, Humana Press, Totowa, NJ (incorporated by reference in its entirety). In some embodiments, the epitope is determined by peptide competition. In some embodiments, the epitope is determined by mass spectrometry. In some embodiments, the epitope is determined by crystallography (e.g., X-ray crystallography). The crystal structure of a bound antibody-antigen pair allows for the highly accurate determination of important interactions between individual amino acids, both from side chains and main chain atoms, in both the antigen epitope and the antibody paratope. Amino acids within 4 angstroms (Å) of each other are generally considered to be contact residues. This method typically involves purification of the antibody and antigen, complex formation and purification, followed by successive rounds of crystallization screening and optimization to obtain diffraction-quality crystals. Structural solutions are often obtained after X-ray crystallography at synchrotron sources. Thus, anti-CD161 antibodies or antigen-binding portions thereof provided by the present disclosure can be evaluated by X-ray crystallography of a crystal structure comprising the antibody or a fragment or portion thereof bound to human CD161. In some embodiments, the epitope bound by an antibody provided by the present disclosure is identified by determining residues on the human CD161 antigen that reside or are located within 4 angstroms (Å) of the antibody paratope residues. Other structural methods for epitope mapping include, but are not limited to, hydrogen-deuterium exchange coupled to mass spectrometry, cross-linked mass spectrometry, and nuclear magnetic resonance (NMR) (e.g., Morris (1996), supra; Abbott et al. (2014) IMMUNOLOGY, 142(4): 5 26-535).

[0361] Functional methods for epitope mapping are well known in the art and typically involve assessing or quantifying antibody binding to whole proteins, protein fragments, or peptides. Functional methods for epitope mapping can be used, for example, to identify linear or conformational epitopes and / or to infer when two or more specific antibodies bind to the same or similar epitopes. Functional methods for epitope mapping include, for example, immunoblotting assays, immunoprecipitation assays, and fluorescence-based labeling assays, in which overlapping or consecutive peptides from CD161 are tested for reactivity with anti-CD161 antibodies (e.g., HP-3G10). Other functional methods for epitope mapping include array-based oligopeptide scanning (also known as "overlapping peptide scanning" or "pepscan analysis"), site-directed mutagenesis (e.g., alanine scanning mutagenesis), and high-throughput mutagenesis mapping (e.g., shotgun mutagenesis mapping).

[0362] In some embodiments, the epitope bound by the anti-CD161 antibodies described herein is determined using site-directed mutagenesis or alanine scanning mutagenesis. Site-directed mutagenesis involves targeted site-specific mutagenesis, in which critical amino acids are identified by systematically introducing substitutions along a protein sequence and then determining the effect of each substitution on antibody binding. This may be performed by "alanine scanning mutagenesis" (Cunningham and Wells (1989) SCIENCE, 244: 1081-085) or some other form of point mutagenesis of amino acid residues in CD161. As described herein, alanine scanning is a technique that involves substituting alanine residues for wild-type residues in a polypeptide, followed by evaluation of the stability or function(s) (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparison with the wild-type polypeptide. Without being bound by theory, two or more antibodies (e.g., a test antibody and a reference antibody) have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of a first antibody reduce or eliminate binding of a second or subsequent antibody. In some embodiments, the epitope bound by the anti-CD161 antibodies described herein is determined using shotgun mutagenesis. Shotgun mutagenesis mapping utilizes a comprehensive plasmid mutation library for a target gene, with each clone in the library containing a unique amino acid mutation, and the entire library covers all amino acids in the target protein. The clones comprising the mutation library are individually plated in microplates, expressed in live mammalian cells, and tested for immunoreactivity with the antibody of interest. Amino acids important to the antibody epitope are identified by loss of reactivity and then mapped onto the protein structure to visualize the epitope.Expression of target protein antigens in mammalian cells often results in the native structure of the target protein antigen, allowing both linear and conformational epitope structures to be mapped onto complex proteins (Paes et al. (2009) J. AM. CHEM. SOC., 131(20): 6952-6954; Banik and Doranz (2010) GENETIC ENGINEERING AND BIOTECHNOLOGY NEWS, 3(2): 25-28).

[0363] In some embodiments, the epitope bound by the anti-CD161 antibodies described herein is determined using peptide scanning. In peptide scanning, a library of short peptide sequences from overlapping segments of a target protein (e.g., CD161) is tested for their ability to bind to an antibody of interest. Peptides are synthesized and screened for binding by any of several methods for solid-phase screening, such as in the "pepscan" method (WO1984 / 03564; WO1993 / 09872, each of which is incorporated by reference in its entirety), using ELISA or BIACORE, or on a chip (Reineke et al. (2001) CURR. OPIN. BIOTECHNOL., 12: 59-64). Conformational epitopes may also be identified by chemical conjugation of peptides onto a scaffold (CLIPS). The loose ends of the peptides are attached to a synthetic scaffold, allowing the scaffold peptide to adopt the same spatial structure as the corresponding sequence in the intact protein. Using CLIPS technology, linear peptides are fixed into circular structures ("single loop" format) and different portions of protein binding sites are joined ("double loop", "triple loop", etc. formats) to create conformational epitopes that can be assayed for antibody binding (U.S. Patent No. 7,972,993). The epitopes bound by the antibodies provided by the present disclosure may also be mapped using computational methods. In these methods, for example, libraries of peptide fragments are displayed on the surface of phage or cells. Epitopes are then mapped by screening antibodies against these fragments using selective binding assays. Several computational tools have been developed that allow prediction of conformational epitopes based on linear affinity-selected peptides obtained using phage display (Mayrose et al. (2007) BIOINFORMATICS, 23: 3244-3246). Methods for the detection of conformational epitopes by phage display are also available. Microbial display systems can be used to display peptides on cell surfaces for the identification of conformational epitopes. Properly folded antigenic fragments may be expressed (Cochran et al. (2004) J. IMMUNOL. METH., 287: 147-158; Rockberg et al. (2008) NATURE METHODS, 5: 1039-1045). Antibody epitopes may be determined using methods involving proteolysis and mass spectroscopy (Baerga-Ortiz et al. (2002) PROTEIN SCI., 11(6): 1300-1308). In limited proteolysis, antigens are cleaved by various proteases in the presence and absence of antibodies, and fragments are identified by mass spectrometry. Epitopes are regions of antigens that are protected from proteolysis upon antibody binding (Suckau et al. (1990) PROC. NATL. ACAD. SCI. USA, 87: 9848-9852). Additional proteolysis-based methods include, for example, selective chemical modification (Fiedler et al. (1998) BIOCONJUGATE CHEMISTRY, 9(2): 236-234), epitope excision (Van de Water et al. (1997) CLIN. IMMUNOL. IMMUNOPATHOL., 85(3): 229-235), and the recently developed method of hydrogen-deuterium (H / D) exchange (Flanagan, N. (2010) GENETIC ENGINEERING AND BIOTECHNOLOGY NEWS 3(2): 25-28).

[0364] 9.1.5 Assays for Effector Function In some embodiments, an anti-CD161 antibody as described herein binds to CD161 and induces or promotes activation of immune cell effector function, where the immune cell is any CD161-expressing immune cell, or the immune cell is a CD161-expressing T cell, a CD161-expressing NK cell, or a combination thereof. In some embodiments, an anti-CD161 antibody as described herein binds to CD161 and induces or promotes activation, proliferation, cytokine production, cytolytic function, or any combination thereof, of a CD161-expressing T cell or a CD161-expressing NK cell. In some embodiments, an anti-CD161 antibody as described herein activates mucosal-associated invariant T cells (MAIT cells). In some embodiments, an anti-CD161 antibody of the present disclosure activates MAIT cells by CD161 blockade. In some embodiments, an anti-CD161 antibody as described herein binds to CD161 and induces or promotes T cell activation, a cytotoxic T lymphocyte (CTL) response, T cell proliferation, cytokine production, or a combination thereof.

[0365] In some embodiments, an anti-CD161 antibody as described herein binds to CD161 and competes with or blocks CD161 binding to CLEC2D. In some embodiments, blockade of CD161 is measured by determining ligand (e.g., CLEC2D) binding. In some embodiments, an anti-CD161 antibody as described herein binds to CD161 on CD161-expressing T cells or CD161-expressing NK cells and induces or promotes activation of T cell or NK cell immune cell effector function in the presence of CLEC2D.

[0366] In some embodiments, activation of CD161-expressing NK cells is determined by adding an anti-CD161 antibody to a culture of CD161-expressing NK cells in the presence of CLEC2D. In some embodiments, the CLEC2D ligand is expressed on human cancer cells co-cultured with NK cells. Activation of human NK cells can be measured by CD107a expression on NK cells after exposure to an anti-CD161 antibody, e.g., in a dose-dependent manner. Activation of human NK cells can be measured by IFNγ secretion after exposure to an anti-CD161 antibody, e.g., in a dose-dependent manner. In some embodiments, the efficacy of a CD161 antibody to block inhibitory signaling in human NK cells in the presence of CLEC2D is measured by EC107a expression on NK cells after exposure to an anti-CD161 antibody, e.g., as determined in Example 7. 50 In some embodiments, the EC values ​​for CD161 antibody activation or blockade of human NK cells in the presence of CLEC2D are provided as (nM) values. 50 The values ​​are between 0.04 nM and 0.38 nM.

[0367] In some embodiments, activation of CD161-expressing T cells is determined by adding an anti-CD161 antibody to a culture of CD161-expressing T cells in the presence of CLEC2D. In some embodiments, the CLEC2D ligand is expressed on human cancer cells co-cultured with T cells. In some embodiments, T cell activation is measured using an NFAT-luciferase reporter gene system (InvivoGen) in engineered T cells. In some embodiments, the efficacy of anti-CD161 to block inhibitory signaling in T cells in the presence of CLEC2D is provided, for example, as described in Examples 8 and 9. In some embodiments, EC values ​​for CD161 antibody activation or blockade of human T cells in the presence of CLEC2D are 50 The values ​​are between 1.5 nM and 4.1 nM.

[0368] In some embodiments, the antibodies of the present disclosure reverse CLEC2D-mediated inhibition, restore primary NK cell and T cell function, and further restore enhanced T cell recall responses and direct T cell-mediated cytotoxicity to antigen in a TCR-dependent manner. In some embodiments, the antibodies disclosed herein reverse the inhibition of NK cell killing by blocking the interaction of CD161 on NK cells with CLEC2D on target cells, as described, for example, in Example 13. In certain embodiments, the antibodies restore not only cytotoxic degranulation by NK cells but also IFNγ expression. In certain embodiments, restoration of NK cell activation can be measured, for example, by CD107a expression by NK cells after exposure to an anti-CD161 antibody in a dose-dependent manner. Activation of human NK cells can be measured, for example, by IFNγ secretion after exposure to an anti-CD161 antibody in a dose-dependent manner.

[0369] In some embodiments, the antibodies disclosed herein enhance NK cell killing of CLEC2D-expressing target cells. In certain embodiments, the antibodies disclosed herein enhance NK cell killing of CLEC2D-expressing target cells by blocking the interaction of CD161 on NK cells with CLEC2D on the target cells, as described, for example, in Example 14.

[0370] In some embodiments, the antibodies disclosed herein enhance the reactivation of antigen-specific effector memory CD4 T cells by blocking CD161 (expressed on T cells) interaction with CLEC2D (expressed on monocyte-derived DCs), e.g., as described in Example 15. In some embodiments, the antibodies result in enhanced cytokine production and increased proliferation (e.g., as measured by Ki-67 expression) by antigen-specific effector memory T (EM cells). In some embodiments, the antibodies disclosed herein enhance cytokine production of MART-1-specific T cells, e.g., as described in Example 16. In some embodiments, the antibodies disclosed herein enhance interferon gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNFα) production from CD8+ T cells, e.g., in a concentration-dependent manner. In some embodiments, the antibodies of the present disclosure enhance the cytotoxic function of MART-1-specific T cells, e.g., as described in Example 17. In some embodiments, the antibodies of the present disclosure increase the frequency of granzyme B-producing T cells, e.g., in a concentration-dependent manner.

[0371] In some embodiments, antibodies of the present disclosure did not induce cytokine release (eg, cytokine release syndrome) in unstimulated human PBMCs from healthy donors.

[0372] Effector function assays using a variety of in vitro and in vivo assays are known in the art, see, e.g., Ravetch and Kinet (1991) ANNU. REV. IMMUNOL., 9: 457-492; U.S. Pat. Nos. 5,500,362 and 5,821,337; Hellstrom et al. (1986) PROC. NAT'L ACAD. SCI. USA, 83: 7059-7063; Hellstrom et al. (1985) PROC. NAT'L ACAD. SCI. USA, 82:1499-1502; Bruggemann et al. (1987) J. EXP. MED., 166: 1351-1361; Clynes et al. (1998) PROC. NAT'L ACAD. SCI. USA, 95: 652-656; WO2006 / 029879; WO2005 / 100402; Gazzano-Santoro et al. (1996) J. IMMUNOL. METHODS, 202: 163-171; Cragg et al. (2003) BLOOD, 101: 1045-1052; Cragg et al. (2004) BLOOD, 103: 2738-2743; and Petkova et al. (2006) INT'L. IMMUNOL., 18: 1759-1769 (each of which is incorporated by reference in its entirety).

[0373] 10. Pharmaceutical Compositions For therapeutic uses, the antibody or antigen-binding fragment thereof or antibody conjugate is preferably combined with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0374] The term "pharmaceutically acceptable carrier," as used herein, refers to buffers, carriers, and excipients that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin (1975) REMINGTON'S PHARMACEUTICAL SCIENCES, 15TH ED., Mack Publ. Co., Easton, PA. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art.

[0375] In certain embodiments, pharmaceutical compositions may contain formulatory materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability of the composition.In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides. preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (e.g., REMINGTON'S See PHARMACEUTICAL SCIENCES, 18th ed. (Mack Publishing Company, 1990) and Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23d ed. 2020).

[0376] In certain embodiments, the pharmaceutical composition may contain nanoparticles, such as polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED., 1: 10-29).

[0377] In certain embodiments, the pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. The sustained-release formulation may contain, for example, porous polymer microparticles or semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. The sustained-release matrix may include polyester, hydrogel, polylactide, copolymer of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also contain liposomes, which can be prepared by any of several methods known in the art.

[0378] Pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof, or antibody conjugates disclosed herein can be presented in dosage unit form and can be prepared by any suitable method. Pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. In certain embodiments, the antibodies or antigen-binding fragments thereof, or antibody conjugates disclosed herein are administered by IV infusion. In certain embodiments, the antibodies or antigen-binding fragments thereof, or antibody conjugates disclosed herein are administered by intratumoral injection. Useful formulations can be prepared by methods known in the pharmaceutical arts (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. (Mack Publishing Company, 1990) and Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rd ed. 2020)). Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.

[0379] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0380] In certain embodiments, the pharmaceutical composition may contain a stabilizer. In certain embodiments, the stabilizer is a cation, for example, a divalent cation. In certain embodiments, the cation is calcium or magnesium. The cation may be in the form of a salt, such as calcium chloride (CaCl) or magnesium chloride (MgCl).

[0381] In certain embodiments, the stabilizer is present in an amount of about 0.05 mM to about 5 mM. For example, the stabilizer may be present in an amount of about 0.05 mM to about 4 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 2 mM, about 0.05 mM to about 1 mM, about 0.05 mM to about 0.5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM.

[0382] Pharmaceutical preparations are preferably sterile.Sterilization can be achieved by any suitable method, for example, by filtration through a sterile filtration membrane.When the composition is lyophilized, sterilization by filtration can be carried out before or after lyophilization and reconstitution.

[0383] The compositions described herein may be administered locally or systemically. Administration is generally parenteral. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in an even more preferred embodiment, it is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0384] Generally, a therapeutically effective amount of an active ingredient, e.g., an antibody, ranges from about 0.1 mg / kg to about 100 mg / kg, e.g., about 1 mg / kg to about 100 mg / kg, e.g., about 1 mg / kg to about 10 mg / kg. The amount administered depends on variables such as the patient's weight, the type and severity of the disease or indication being treated, the patient's overall health, the in vivo efficacy of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased above the upper level to rapidly achieve the desired blood or tissue level. Alternatively, the initial dosage can be lower than the optimal dosage, and the daily dosage can be gradually increased over the course of treatment. Human dosages can be optimized in a conventional Phase I dose-escalation study designed to be, for example, about 0.1 mg / kg to about 20 mg / kg. The frequency of administration can vary depending on factors such as the route of administration, dosage, serum half-life of the antibody, and the disease being treated. Exemplary administration frequencies are once a day, once a week, once every two weeks, or once every three weeks. A preferred administration route is parenteral, for example, intravenous infusion. In certain embodiments, the antibody is lyophilized and then reconstituted in buffered saline at the time of administration. In certain embodiments, a starting dose of 6 mg (0.1 mg / kg based on a patient weight of 60 kg) of anti-CD161 antibody is administered (e.g., by IV) once every three weeks.

[0385] 11. Dosage and Unit Dosage Form In human therapeutics, a physician will determine the posology deemed most appropriate depending on whether prophylactic or curative treatment is desired, and on the age, weight, condition, and other factors specific to the subject being treated. In certain embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein comprise a therapeutically effective amount of one or more therapeutic antibodies.

[0386] The amount of antibody effective for preventing or treating a disorder or one or more symptoms thereof can vary depending on the nature and severity of the disease or condition, and the route by which the antibody is administered. The frequency and dosage can also vary based on factors specific to each subject, depending on the specific therapy (e.g., therapeutic or prophylactic) administered, the severity of the disorder, disease, or condition, the route of administration, and the age, weight, response, and prior medical history of the subject. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0387] As will be readily apparent to those skilled in the art, various therapeutically effective amounts may be applicable to various diseases and conditions. Similarly, amounts sufficient to prevent, manage, treat, or ameliorate such disorders, but insufficient to cause adverse effects associated with the antibodies or ADCs provided herein, or amounts sufficient to reduce such adverse effects, are also encompassed by the dosage and administration frequency schedules provided herein. Furthermore, when a subject is administered multiple doses of the compositions provided herein, not all of the doses need to be the same. For example, the dosage administered to a subject may be increased to improve the prophylactic or therapeutic effect of the composition, or decreased to reduce one or more side effects experienced by a particular subject.

[0388] As discussed in more detail elsewhere in this disclosure, the antibodies provided herein may optionally be administered with one or more additional agents useful for preventing or treating a disease or disorder. The effective amount of such additional agents may depend on the amount of agent present in the formulation, the type of disorder or treatment, and other factors known in the art or described herein.

[0389] 12. Therapeutic applications It is contemplated that the antibodies disclosed herein can be administered to a mammal, generally a human, in a pharmaceutically acceptable dosage form via one or more of the administration approaches described herein.

[0390] Any of the antibodies described herein can be used to treat any disease or condition associated with CD161. It is contemplated that the agents described herein can be used to treat cancer, which can be achieved by administering an effective amount of an immunotherapy (e.g., an anti-CD161 antibody) disclosed herein to a subject. It is also contemplated that the agents described herein can be used to treat autoimmune disorders, which can be achieved by administering an effective amount of an anti-CD161 antibody disclosed herein to a subject.

[0391] The present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an immunotherapy (e.g., an anti-CD161 antibody) disclosed herein or an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the cancer is characterized by expression of CLEC2D by cancer cells or other cells in the tumor microenvironment. In some embodiments, the cancer is characterized by increased expression of CLEC2D by cancer cells or other cells in the tumor microenvironment.

[0392] Any suitable cancer can be treated with the agents disclosed herein. Examples of cancer include solid tumors, soft tissue tumors, hematopoietic tumors, and metastatic lesions. Examples of hematopoietic tumors include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), such as transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Richter's syndrome (Richter's transformation). Examples of solid tumors include malignant tumors of various organ systems, e.g., sarcomas, adenocarcinomas, and carcinomas, such as those affecting the head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung cancer (NSCLC)), breast, lymphatic, gastrointestinal (e.g., mouth, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive and genitourinary tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testes), CNS (e.g., neuronal or glial cells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma and metastatic Merkel cell carcinoma (MCC)).

[0393] In certain embodiments, the cancer is lymphoma. In certain embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In certain embodiments, the cancer is non-small cell lung cancer (NSCLC). In certain embodiments, the cancer is hepatocellular carcinoma (HCC). In certain embodiments, the cancer is triple-negative breast cancer (TNBC). In certain embodiments, the cancer is melanoma. In certain embodiments, the cancer is glioblastoma. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is liver cancer. In some embodiments, the cancer is cervical cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is renal cancer. In certain embodiments, the cancer is lung adenocarcinoma. In certain embodiments, the cancer is glioma. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is bladder cancer. In certain embodiments, the cancer is colon adenocarcinoma. In certain embodiments, the cancer is kidney cancer.

[0394] In certain embodiments, cancers that may benefit from CD161 blockade immunotherapy have high densities of CLEC2D, as identified using immunofluorescence data. + and CD161 + In certain embodiments, the cancer has a high density of CLEC2D + and CD161 + In certain embodiments, the cancer is a cancer having B-cell cells. In certain embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin's lymphoma, and T-cell lymphoma, such as peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), NK / T-cell lymphoma, or anaplastic large cell lymphoma (ALCL). In certain embodiments, the cancer is non-small cell lung cancer (NSCLC), such as NSCLC-squamous cell carcinoma or NSCLC-adenocarcinoma, head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), or cutaneous squamous cell carcinoma.

[0395] In certain circumstances, the cancer is selected from the group consisting of melanoma, lung cancer, glioma, colon cancer, and liver cancer.

[0396] Furthermore, the present disclosure provides a method for reducing or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein.

[0397] The present disclosure also provides a method for inhibiting or blocking the interaction between human CD161 and CLEC2D in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein.

[0398] The present disclosure also provides methods for inducing or enhancing immune cell activation in a subject in need thereof, the method comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the immune cell activation occurs in the tumor microenvironment. In some embodiments, the immune cells are T cells or NK cells.

[0399] In some embodiments, the present disclosure provides methods of inducing or enhancing a cytotoxic T cell effector response in a subject in need thereof, comprising administering to the subject an effective amount of an anti-CD161 antibody disclosed herein or an effective amount of a pharmaceutical composition disclosed herein. Optionally, the T cell effector response is (i) in the tumor microenvironment, (ii) cytokine production (e.g., IL-2, TNFα, IFNγ, or a combination thereof), (iii) granzyme B secretion, or (iv) a combination of two or more of (i), (ii), and (iii).

[0400] In some embodiments, the present disclosure provides methods for reducing CD161 inhibitory signaling caused by CLEC2D binding to CD161. In some embodiments, the present disclosure provides methods for reducing the suppression of T cell activity caused by CLEC2D binding to CD161. In some embodiments, the present disclosure provides methods for reducing the suppression of NK cell activity caused by CLEC2D binding to CD161. In some embodiments, the present disclosure provides methods for increasing T cell activity in the presence of CLEC2D compared to T cell activity in the absence of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the present disclosure provides methods for increasing NK cell activity in the presence of CLEC2D compared to NK cell activity in the absence of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the present disclosure provides methods for increasing T cell activity disposed in a microenvironment comprising cells expressing CLEC2D. In some embodiments, the present disclosure provides methods for increasing NK cell activity disposed in a microenvironment comprising cells expressing CLEC2D. In some embodiments, the present disclosure provides methods for increasing T cell activity in a tumor microenvironment comprising tumor cells expressing CLEC2D. In some embodiments, the present disclosure provides methods for increasing NK cell activity in a tumor microenvironment comprising tumor cells expressing CLEC2D. In some embodiments, the present disclosure provides methods for inhibiting human T cell exhaustion. In some embodiments, the present disclosure provides methods for inducing or increasing activation of CD161-expressing human T cells in response to antigen-expressing target cells. In some embodiments, the present disclosure provides methods for inducing or increasing cytokine production by CD161-expressing human T cells in response to antigen-expressing target cells. In some embodiments, the present disclosure provides methods for inducing or increasing granzyme B expression by CD161-expressing human T cells in response to antigen-expressing target cells. In some embodiments, the present disclosure provides methods for reducing exhaustion of CD161-expressing human T cells in response to antigen-expressing target cells.

[0401] In certain embodiments, provided herein are methods of delaying the onset of cancer in a subject in need thereof by administering to the subject an effective amount of an immunotherapy (e.g., an immunotherapeutic agent) disclosed herein (e.g., an anti-CD161 antibody or antigen-binding fragment or antibody conjugate thereof). In certain embodiments, provided herein are methods of preventing the onset of cancer in a subject in need thereof by administering to the subject an effective amount of an agent disclosed herein. In some embodiments, provided herein are methods of reducing tumor size in a subject in need thereof by administering to the subject an effective amount of an agent disclosed herein. In certain embodiments, provided herein are methods of reducing the number of metastases in a subject in need thereof by administering to the subject an effective amount of an agent disclosed herein. In certain embodiments, provided herein are methods of extending overall survival, median survival, or progression-free survival in a subject in need thereof by administering to the subject an agent disclosed herein. In certain embodiments, provided herein are methods of treating a subject that has become resistant to standard of care by administering to the subject an effective amount of an agent disclosed herein.

[0402] 13. Combination Therapy The methods and compositions described herein may be used alone or in combination with other therapeutic agents and / or modalities. The term "administered in combination," as used herein, is understood to mean that two (or more) different therapies are delivered to a subject over the course of the subject's illness, such that the effects of the therapies on the patient overlap at some point. In certain embodiments, the delivery of one treatment is still occurring when the delivery of a second treatment begins, and thus there is an overlap in administration. This is sometimes referred to herein as "simultaneous" or "co-delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments in either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective than would be seen if the second treatment were administered in the absence of the first treatment; e.g., a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, or a similar situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that which would be observed with one treatment delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Delivery may be such that the effect of the first treatment delivered remains detectable when the second treatment is delivered.

[0403] In certain embodiments, the methods or compositions described herein are administered in combination with one or more additional therapies, such as surgery, radiation therapy, or administration of another therapeutic agent. In certain embodiments, the additional therapy may include chemotherapy, such as a cytotoxic agent. In certain embodiments, the additional therapy may include a targeted therapy, such as a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In certain embodiments, the additional therapy may include an anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compound, such as a steroid, a biological immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodilator, a statin, an anti-inflammatory agent (e.g., methotrexate), or an NSAID. In certain embodiments, the additional therapy may include a combination of different classes of therapeutic agents.

[0404] In certain embodiments, the methods or compositions described herein are administered in combination with a second checkpoint inhibitor, which may be chosen from, for example, a PD-1 antagonist, a second PD-L1 antagonist, a CTLA-4 antagonist, an adenosine A2A receptor antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a KIR antagonist, a LAG3 antagonist, a TIM-3 antagonist, a VISTA antagonist, or a TIGIT antagonist.

[0405] In certain embodiments, the checkpoint inhibitor is a PD-1 or second PD-L1 inhibitor. PD-1 is a receptor present on the surface of T cells and functions as an immune system checkpoint that inhibits or otherwise modulates T cell activity in a timely manner to prevent an overactive immune response. However, cancer cells can exploit this checkpoint to silence or modulate T cell activity by expressing a ligand, e.g., PD-L1, that interacts with PD-1 on the surface of T cells. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapeutics. Exemplary therapeutic methods using PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Pat. Nos. 8,728,474 and 9,073,994 and European Patent No. 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149.Exemplary anti-PD-L1 antibodies include atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0406] In certain embodiments, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of CTLA-4 on T cells with its ligands (e.g., CD80, also known as B7-1, and CD86) on the surface of antigen-presenting cells (but not cancer cells) leads to T cell inhibition. Exemplary CTLA-4-based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910, 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,144,796, and 8,145,797. 3,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984, International (PCT) Publication Nos. WO 1998 / 42752, WO 2000 / 37504, and WO 2001 / 14424, and European Patent No. EP 1212422 B1 (each of which is incorporated by reference in its entirety). Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0407] In certain embodiments, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor, such as a CTLA-4 inhibitor disclosed herein.

[0408] In certain embodiments, the methods or compositions described herein are administered in combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-tryptophan (also known as indoximod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205.

[0409] Exemplary cytotoxic agents that can be administered in combination with the methods or compositions described herein include, for example, microtubule inhibitors, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinum compounds, inhibitors of nucleic acid synthesis, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethylenimines, alkylsulfonates, triazenes, folate analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, drugs capable of interfering with signal transduction pathways, drugs that promote apoptosis and radiation, or antibody molecule conjugates that bind to surface proteins and deliver toxic drugs. In one embodiment, cytotoxic agents that can be administered with the methods or compositions described herein include platinum-based agents (e.g., cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacitidine, vorinostat, ixabepilone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine , mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin), daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansinoids.

[0410] The present invention also provides methods for increasing expression of HLA-DR, CD86, CD83, IFNγ, IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6 in a cell, tissue, or subject. The methods include contacting the cell, tissue, or subject with an effective amount of an antibody, fusion protein, and / or antibody conjugate, such as an antibody, fusion protein, or antibody conjugate disclosed herein. In certain embodiments, the cell is selected from a dendritic cell and a peripheral blood mononuclear cell (PBMC).

[0411] In certain embodiments, expression of HLA-DR, CD86, CD83, IFNγ, IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6 in a cell, tissue, or subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to a similar or otherwise identical cell or tissue not contacted with the antibody, fusion protein, or antibody conjugate. Gene expression may be measured by any suitable method known in the art, for example, by ELISA or by Luminex multiplex assay.

[0412] The present invention also provides a method for promoting immune cell infiltration into tumors in a subject in need thereof, comprising administering to the subject an effective amount of an antibody disclosed herein. In certain embodiments, the immune cells are T cells, e.g., CD4 + and / or CD8 + T cells, e.g., CD69 + CD8 + and / or GzmB + CD8 +The immune cells are T cells. In certain embodiments, the immune cells are natural killer (NK) cells. In certain embodiments, immune cell infiltration into tumors in a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to a similar or otherwise identical tumor and / or subject not receiving the drug. Immune cell infiltration into tumors may be measured by any suitable method known in the art, for example, by antibody staining.

[0413] The invention also provides a method for increasing expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12, and / or Ccl5 in a cell, tissue, or subject, comprising contacting the cell, tissue, or subject with an effective amount of an antibody disclosed herein, so as to increase expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12, and / or Ccl5, compared to the cell, tissue, or subject prior to contact with such agent. In certain embodiments, expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12, and / or Ccl5 in a cell, tissue, or subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% in a cell, tissue, or subject compared to a similar or otherwise identical cell, tissue, or subject not contacted with such agent. Gene expression may be measured by any suitable method known in the art, for example, by ELISA, Luminex multiplex assay, or Nanostring technology.

[0414] In certain embodiments, the cells affected by treatment are tumor cells, dendritic cells (DCs), or monocytes. In certain embodiments, the cells are monocytes, and the method results in increased expression of MHC-II molecules (e.g., HLA-DR) on the monocytes. In certain embodiments, expression of MHC-II molecules in cells or tissues is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%, compared to similar or otherwise identical cells or tissues not contacted with such agents. Gene expression may be measured by any suitable method known in the art, for example, by ELISA, by Luminex multiplex assay, or by flow cytometry.

[0415] 14. Diagnostic Methods Also provided are methods for detecting the presence of CD161 on cells from a subject. Such methods can be used, for example, to predict and assess responsiveness to treatment with the antibodies disclosed herein.

[0416] In some embodiments, the method can be used to detect CD161 in a subject having or suspected of having a disease or condition. In some embodiments, the method includes (a) receiving a sample from the subject; and (b) detecting the presence or level of CD161 in the sample by contacting the sample with an antibody disclosed herein. In certain embodiments, the disease or condition is cancer. The antibodies provided herein can be labeled with a detectable label, e.g., a fluorescent label, a radioactive label, or an enzymatic label.

[0417] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0418] Any examples or use of exemplary language herein, such as "such as" or "including," are intended solely to better describe the invention and do not impose limitations on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [Example]

[0419] Having now generally described the invention, it will be more readily understood by reference to the following examples, which are...

Claims

1. A light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 sequences; and Heavy chain variable region including CDR-H1, CDR-H2, and CDR-H3 sequences 1. An isolated anti-CD161 antibody comprising: (a) The CDR-H1 sequence is FX 1 FX 2 X 3 X 4 AMS (SEQ ID NO: 1); (b) the CDR-H2 sequence is AISX 5 X 6 GGX 7 TX 8 YADSVKG (SEQ ID NO: 2); and (c) the CDR-H3 sequence is AKPLDSSX 9 WADFX 10 X 11 (SEQ ID NO: 3); X 1 is T or A; X 2 is G, S, or E; X 3 is Q, T, P, or R; X 4 is Y or F; X 5 is A or G; X 6 is A, V, or S; X 7 is T or S; X 8 is K, A, or Y; X 9 is Q, F, or L; X 10 is D or Q; and X 11 is L or A; and (d) the CDR-L1 sequence is RASQX 12 IX 13 SWLA (SEQ ID NO: 4); (e) the CDR-L2 sequence is X 14 ASX 15 LQX 16 (SEQ ID NO: 5); and (f) the CDR-L3 sequence is QQX 17 X 18 X 19 LPIT (SEQ ID NO: 6); X 12 is G, D, or T; X 13 is D, S, or Y; X 14 is A, Y, or F; X 15 is S, A, G, or F; X 16 is D or S; X 17 is A, H, or Q; X 18 is S, D, W, or L; and X 19 is V, D, Y, or K; Isolated anti-CD161 antibody.

2. (a) the CDR-H1 sequence is SEQ ID NO: 72; (b) the CDR-H2 sequence is SEQ ID NO: 73; (c) the CDR-H3 sequence is SEQ ID NO: 74; (d) the CDR-L1 sequence is SEQ ID NO: 76; (e) the CDR-L2 sequence is SEQ ID NO: 77; and (f) the CDR-L3 sequence is SEQ ID NO: 78; The isolated anti-CD161 antibody of claim 1.

3. (a) the CDR-H1 sequence is SEQ ID NO: 16; (b) the CDR-H2 sequence is SEQ ID NO: 17; (c) the CDR-H3 sequence is SEQ ID NO: 18; (d) the CDR-L1 sequence is SEQ ID NO: 20; (e) the CDR-L2 sequence is SEQ ID NO: 21; and (f) the CDR-L3 sequence is SEQ ID NO: 22; The isolated anti-CD161 antibody of claim 1.

4. (a) the CDR-H1 sequence is SEQ ID NO: 24; (b) the CDR-H2 sequence is SEQ ID NO: 25; (c) the CDR-H3 sequence is SEQ ID NO: 26; (d) the CDR-L1 sequence is SEQ ID NO: 28; (e) the CDR-L2 sequence is SEQ ID NO: 29; and (f) the CDR-L3 sequence is SEQ ID NO: 30; The isolated anti-CD161 antibody of claim 1.

5. (a) the CDR-H1 sequence is SEQ ID NO: 32; (b) the CDR-H2 sequence is SEQ ID NO: 33; (c) the CDR-H3 sequence is SEQ ID NO: 34; (d) the CDR-L1 sequence is SEQ ID NO: 36; (e) the CDR-L2 sequence is SEQ ID NO: 37; and (f) the CDR-L3 sequence is SEQ ID NO: 38; The isolated anti-CD161 antibody of claim 1.

6. (a) the CDR-H1 sequence is SEQ ID NO: 40; (b) the CDR-H2 sequence is SEQ ID NO: 41; and (c) the CDR-H3 sequence is SEQ ID NO: 42; (d) the CDR-L1 sequence is SEQ ID NO: 44; (e) the CDR-L2 sequence is SEQ ID NO: 45; and (f) the CDR-L3 sequence is SEQ ID NO: 46; The isolated anti-CD161 antibody of claim 1.

7. (a) the CDR-H1 sequence is SEQ ID NO: 48; (b) the CDR-H2 sequence is SEQ ID NO: 49; (c) the CDR-H3 sequence is SEQ ID NO: 50; (d) the CDR-L1 sequence is SEQ ID NO: 52; (e) the CDR-L2 sequence is SEQ ID NO: 53; and (f) the CDR-L3 sequence is SEQ ID NO: 54; The isolated anti-CD161 antibody of claim 1.

8. (a) the CDR-H1 sequence is SEQ ID NO: 56; (b) the CDR-H2 sequence is SEQ ID NO: 57; (c) the CDR-H3 sequence is SEQ ID NO: 58; (d) the CDR-L1 sequence is SEQ ID NO: 60; (e) the CDR-L2 sequence is SEQ ID NO: 61; and (f) the CDR-L3 sequence is SEQ ID NO: 62; The isolated anti-CD161 antibody of claim 1.

9. (a) the CDR-H1 sequence is SEQ ID NO: 64; (b) the CDR-H2 sequence is SEQ ID NO: 65; (c) the CDR-H3 sequence is SEQ ID NO: 66; (d) the CDR-L1 sequence is SEQ ID NO: 68; (e) the CDR-L2 sequence is SEQ ID NO: 69; and (f) the CDR-L3 sequence is SEQ ID NO: 70; The isolated anti-CD161 antibody of claim 1.

10. (a) the CDR-H1 sequence is SEQ ID NO: 8; (b) the CDR-H2 sequence is SEQ ID NO: 9; (c) the CDR-H3 sequence is SEQ ID NO: 10; (d) the CDR-L1 sequence is SEQ ID NO: 12; (e) the CDR-L2 sequence is SEQ ID NO: 13; and (f) the CDR-L3 sequence is SEQ ID NO: 14; The isolated anti-CD161 antibody of claim 1.

11. (a) the CDR-H1 sequence is SEQ ID NO: 80; (b) the CDR-H2 sequence is SEQ ID NO: 81; (c) the CDR-H3 sequence is SEQ ID NO: 82; (d) the CDR-L1 sequence is SEQ ID NO: 84; (e) the CDR-L2 sequence is SEQ ID NO: 85; and (f) the CDR-L3 sequence is SEQ ID NO: 86; The isolated anti-CD161 antibody of claim 1.

12. (a) the CDR-H1 sequence is SEQ ID NO: 88; (b) the CDR-H2 sequence is SEQ ID NO: 89; (c) the CDR-H3 sequence is SEQ ID NO: 90; and (d) the CDR-L1 sequence is SEQ ID NO: 92; (e) the CDR-L2 sequence is SEQ ID NO: 93; and (f) the CDR-L3 sequence is SEQ ID NO: 94; The isolated anti-CD161 antibody of claim 1.

13. The heavy chain variable region has the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFX 1 FX 2 X 3 X 4 AMSWVRQAPGKGLEWVSAISX 5 X 6 GGX 7 TX 8 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPLDSSX 9 WADFX 10 X 11 WGRGTLVTVSS (SEQ ID NO: 188), X 1 is T or A; X 2 is G, S, or E; X 3 is Q, T, P, or R; X 4 is Y or F; X 5 is A or G; X 6 is A, V, or S; X 7 is T or S; X 8 is K, A, or Y; X 9 is Q, F, or L; X 10 is D or Q; and X 11 is L or A; and the light chain variable region comprises the amino acid sequence: DIQX a TQSPSSVSASVGDRVTITCRASQX 12 IX 13 SWLAWYQQKPGKAPKX b LIYX 14 ASX 15 LQX 16 GVPSRFSGSGSGTDFTLTIX c SLQPEDFATYYCQQX 17 X 18 X 19 LPITFGGGTKVEIK (SEQ ID NO: 189); X 12 is G, D, or T; X 13 is D, S, or Y; X 14 is A, Y, or F; X 15 is S, A, G, or F; X 16 is D or S; X 17 is A, H, or Q; X 18 is S, D, W, or L; X 19 is V, D, Y, or K; X a is M or L; X b is L or F; and X c is S or N; The isolated anti-CD161 antibody of claim 1.

14. The heavy chain variable region of claim 13, wherein the amino acid sequence of the heavy chain variable region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 71; and the amino acid sequence of the light chain variable region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 75; The isolated anti-CD161 antibody of claim 1.

15. 2. The isolated anti-CD161 antibody of claim 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 71 and the light chain variable region comprises the sequence of SEQ ID NO:

75.

16. A light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 sequences; and Heavy chain variable region including CDR-H1, CDR-H2, and CDR-H3 sequences 1. An isolated anti-CD161 antibody comprising: (a) The CDR-H1 sequence is FTFX 1 X 2 YYMS (SEQ ID NO: 95); (b) The CDR-H2 sequence is YISPSGX 3 TIX 4 YADSVKG (SEQ ID NO: 96); and (c) the CDR-H3 sequence is ARSLMX 5 TGTHLYFDL (SEQ ID NO: 97); X 1 is G, A, P, or S; X 2 is N, Q, or D; X 3 is A or S; X 4 is Y or A; and X 5 is A or S; and (a) the CDR-L1 sequence is RASX 6 X 7 ISX 8 WLA (SEQ ID NO: 98); (b) the CDR-L2 sequence is AAX 9 X 10 LQS (SEQ ID NO: 99); and (c) the CDR-L3 sequence is QQX 11 TSX 12 X 13 PYT (SEQ ID NO: 100); X 6 is Q or S; X 7 is D or G; X 8 is D or S; X 9 is E or S; X 10 is S, A, G, V, or E; X 11 is A, S, or V; X 12 is F, T, V, Q, or A; and X 13 is L or P; Isolated anti-CD161 antibody.

17. (a) the CDR-H1 sequence is SEQ ID NO: 102; (b) the CDR-H2 sequence is SEQ ID NO: 103; (c) the CDR-H3 sequence is SEQ ID NO: 104; (d) the CDR-L1 sequence is SEQ ID NO: 106; (e) the CDR-L2 sequence is SEQ ID NO: 107; and (f) the CDR-L3 sequence is SEQ ID NO: 108; 17. The isolated anti-CD161 antibody of claim 16.

18. (a) the CDR-H1 sequence is SEQ ID NO: 110; (b) the CDR-H2 sequence is SEQ ID NO: 111; (c) the CDR-H3 sequence is SEQ ID NO: 112; (d) the CDR-L1 sequence is SEQ ID NO: 114; (e) the CDR-L2 sequence is SEQ ID NO: 115; and (f) the CDR-L3 sequence is SEQ ID NO: 116; 17. The isolated anti-CD161 antibody of claim 16.

19. (a) the CDR-H1 sequence is SEQ ID NO: 118; (b) the CDR-H2 sequence is SEQ ID NO: 119; (c) the CDR-H3 sequence is SEQ ID NO: 120; (d) the CDR-L1 sequence is SEQ ID NO: 122; (e) the CDR-L2 sequence is SEQ ID NO: 123; and (f) the CDR-L3 sequence is SEQ ID NO: 124; 17. The isolated anti-CD161 antibody of claim 16.

20. (a) the CDR-H1 sequence is SEQ ID NO: 126; (b) the CDR-H2 sequence is SEQ ID NO: 127; and (c) the CDR-H3 sequence is SEQ ID NO: 128; and (d) the CDR-L1 sequence is SEQ ID NO: 130; (e) the CDR-L2 sequence is SEQ ID NO: 131; and (f) the CDR-L3 sequence is SEQ ID NO: 132; 17. The isolated anti-CD161 antibody of claim 16.

21. (a) the CDR-H1 sequence is SEQ ID NO: 134; (b) the CDR-H2 sequence is SEQ ID NO: 135; (c) the CDR-H3 sequence is SEQ ID NO: 136; (d) the CDR-L1 sequence is SEQ ID NO: 138; (e) the CDR-L2 sequence is SEQ ID NO: 139; and (f) the CDR-L3 sequence is SEQ ID NO: 140; 17. The isolated anti-CD161 antibody of claim 16.

22. (a) the CDR-H1 sequence is SEQ ID NO: 142; (b) the CDR-H2 sequence is SEQ ID NO: 143; (c) the CDR-H3 sequence is SEQ ID NO: 144; (d) the CDR-L1 sequence is SEQ ID NO: 146; (e) the CDR-L2 sequence is SEQ ID NO: 147; and (f) the CDR-L3 sequence is SEQ ID NO: 148; 17. The isolated anti-CD161 antibody of claim 16.

23. (a) the CDR-H1 sequence is SEQ ID NO: 150; (b) the CDR-H2 sequence is SEQ ID NO: 151; (c) the CDR-H3 sequence is SEQ ID NO: 152; (d) the CDR-L1 sequence is SEQ ID NO: 154; (e) the CDR-L2 sequence is SEQ ID NO: 155; and (f) the CDR-L3 sequence is SEQ ID NO: 156; 17. The isolated anti-CD161 antibody of claim 16.

24. (a) the CDR-H1 sequence is SEQ ID NO: 158; (b) the CDR-H2 sequence is SEQ ID NO: 159; (c) the CDR-H3 sequence is SEQ ID NO: 160; (d) the CDR-L1 sequence is SEQ ID NO: 162; (e) the CDR-L2 sequence is SEQ ID NO: 163; and (f) the CDR-L3 sequence is SEQ ID NO: 164; 17. The isolated anti-CD161 antibody of claim 16.

25. (a) the CDR-H1 sequence is SEQ ID NO: 166; (b) the CDR-H2 sequence is SEQ ID NO: 167; (c) the CDR-H3 sequence is SEQ ID NO: 168; (d) the CDR-L1 sequence is SEQ ID NO: 170; (e) the CDR-L2 sequence is SEQ ID NO: 171; and (f) the CDR-L3 sequence is SEQ ID NO: 172; 17. The isolated anti-CD161 antibody of claim 16.

26. The isolated anti-CD161 antibody of claim 1, wherein the antibody is a monoclonal antibody.

27. The isolated anti-CD161 antibody of claim 1, wherein the antibody is multispecific.

28. The antibody is Fab, Fab', F(Ab') 2 , Fv, scFv, (scFv) 2 2. The isolated anti-CD161 antibody of claim 1, which is a single-chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V-domain antibody.

29. An isolated anti-CD161 antibody described in claim 1, wherein the antibody is a full-length antibody.

30. The isolated anti-CD161 antibody of claim 1 , wherein the antibody comprises a scaffold.

31. The isolated anti-CD161 antibody of claim 30, wherein the scaffold is Fc.

32. 32. The isolated anti-CD161 antibody of claim 31, wherein the scaffold is human Fc.

33. The isolated anti-CD161 antibody of claim 1, wherein the antibody comprises a heavy chain constant region of a class selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

34. The antibody is of class IgG and 1 , IgG 2 , IgG 3 , and IgG 4 34. The isolated anti-CD161 antibody of claim 33, comprising a heavy chain constant region of a subclass selected from the group consisting of:

35. An isolated anti-CD161 antibody described in claim 1, wherein the antibody is a non-glycosylated antibody.

36. 27. The isolated anti-CD161 antibody of claim 26, wherein the monoclonal antibody is a non-glycosylated human IgG1 antibody.

37. 37. The isolated anti-CD161 antibody of claim 36, wherein the monoclonal antibody comprises an IgG1 Fc region having an alteration at amino acid position N297 according to EU numbering.

38. 38. The isolated anti-CD161 antibody of claim 37, wherein the mutation is N297A.

39. A pharmaceutical composition comprising the antibody of any one of claims 1 to 38 and a pharmaceutically acceptable carrier.

40. The antibody according to any one of claims 1 to 38, H , that V L , its light chain, its heavy chain, or an antigen-binding portion thereof.

41. 41. A vector or vectors comprising the polynucleotide or polynucleotides of claim 40.

42. 41. A host cell comprising a polynucleotide or polynucleotides according to claim 40.

43. 43. A method of producing an antibody, comprising expressing the antibody in a host cell of claim 42 and isolating the expressed antibody.

44. A kit comprising an antibody according to any one of claims 1 to 38 and instructions for use.

45. A pharmaceutical composition for the treatment of cancer, comprising an anti-CD161 antibody described in any one of claims 1 to 38.

46. 46. The pharmaceutical composition of claim 45, wherein the cancer is characterized by expression of CLEC2D by cancer cells or other cells in the tumor microenvironment.

47. 46. The pharmaceutical composition of claim 45, wherein the cancer is characterized by increased expression of CLEC2D by cancer cells or other cells in the tumor microenvironment.

48. The pharmaceutical composition described in claim 45, wherein the cancer is a solid tumor or a hematopoietic tumor.

49. The pharmaceutical composition described in claim 45, wherein the cancer is a metastatic lesion.

50. The pharmaceutical composition described in claim 45, wherein the cancer is leukemia, lymphoma, or multiple myeloma.

51. The pharmaceutical composition of claim 45, wherein the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, non-Hodgkin's lymphoma, or Burkitt's lymphoma.

52. The pharmaceutical composition described in claim 45, wherein the cancer is a sarcoma, adenocarcinoma, or carcinoma.

53. The pharmaceutical composition described in claim 45, wherein the cancer is head and neck cancer, thyroid cancer, lung cancer, breast cancer, lymphoma, mouth cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, small intestine cancer, colon cancer, anal canal cancer, kidney cancer, urothelial cancer, bladder cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, prostate cancer, testicular cancer, central nervous system cancer, or skin cancer.

54. A pharmaceutical composition for the treatment of an autoimmune disorder, comprising an anti-CD161 antibody described in any one of claims 1 to 38.

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