Anti-CLEC2d antibodies and methods of use thereof

Abstract

To provide anti-CLEC2D antibodies and methods of use thereof.SOLUTION: The present application disclosure relates to anti-CLEC2D (CLEC2D encodes the gene for the Lectin Like Transcript-1-LLT1- protein, which is a functional ligand for the human NKR-P1A receptor) antibodies and related compositions and methods of use thereof. These antibodies are used as therapeutics, and in prognostic and diagnostic applications in various cancers and other diseases. The present disclosure relates to immunology, particularly to immuno-oncology.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of Indian Provisional Patent Application No. 201941005395, filed February 11, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to immunology, particularly immuno-oncology. In particular, the present disclosure relates to novel antibody molecules against CLEC2D antigen. The present disclosure also relates to multiple formats and amino acid compositions of the disclosed antibody molecules, the variable regions of the heavy and light chains of the antibody molecules, and the composition and length distribution of CDRs against CLEC2D antigen. The compositions of the present disclosure can be used either as single agent therapeutics or in combination with other antibody molecules or any other therapeutics suitable for treating or preventing diseases such as cancer. [Background technology]

[0003] Modulation of immune cell checkpoint receptors by antibody-based / directed therapeutic approaches has attracted constant interest over the past decade. Many of these receptors are involved in regulating checkpoints in T cells. However, modulation of checkpoints in B cells, natural killer (NK) cells, and myeloid cells has received increasing attention.

[0004] NK cells are part of the innate immune system that recognize and induce cytotoxicity against a wide range of target cells, such as tumor cells or virus-infected cells. In addition, NK cells are involved in the induction and progression of adaptive immune responses through the production of various cytokines. In general, these responses are regulated by the interaction of a wide variety of activating and inhibitory receptors with ligands on the surface of target cells and immune cells.

[0005] NK cell receptors are classified into two main structural classes, the immunoglobulin superfamily and the C-type lectin-like (CTL) superfamily. NKR-P1 receptors (e.g., CD161) are a family of C-type lectin-like transmembrane molecules that are important immunoregulatory genes and are expressed on various cell types, including splenic dendritic cells, T cells, and granulocyte subtypes. Lectin-like transcript 1 (LLT1) molecules or C-type lectin domain family 2 member D (CLEC2D) molecules or osteoclast inhibitory lectin (OCIL) molecules are ligands for the CD161 receptor, and this interaction differentially regulates the function of NK cells and T cells. There are six splice variants of CLEC2D, but isoform 1 is the canonical sequence expressed on NK cells, T cells, monocytes / macrophages, activated B cells, and activated dendritic cells, and functions as a human NK cell activating receptor. The polypeptide chain of CLEC2D can be divided into an N-terminal cytoplasmic portion, a transmembrane and stalk region, and a C-terminal CTL ectodomain with two predicted N-glycosylation sites.

[0006] The interaction of CLEC2D and CD161 leads to evasion from host defense in several disease scenarios, including various cancers. Such immune evasion has been reported in human glioblastoma and other diseases. Furthermore, CLEC2D expression on B cells is thought to regulate the crosstalk between NK cells and antigen-presenting cells (APCs). Blocking the CLEC2D-CD161 interaction provides a novel therapeutic option for treating various cancers.

[0007] The downstream signaling of CLEC2D-CD161 interaction is poorly understood. CLEC2D / CD161 interaction inhibits NK cell function and promotes T cell proliferation and cytokine secretion. Therefore, monoclonal antibodies that specifically bind to CLEC2D can be used to reverse the effects of CLEC2D / CD161 interaction by inhibiting the interaction between CLEC2D and its known receptor CD161 or other unknown cellular mechanisms. Summary of the Invention

[0008] The disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:46, SEQ ID NO:65, SEQ ID NO:59, and SEQ ID NO:99; or (b) at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 99% identical to a sequence selected from SEQ ID NO:57, SEQ ID NO:91, SEQ ID NO:98, SEQ ID NO:84, SEQ ID NO:58, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:47, SEQ ID NO:17, and SEQ ID NO:8. (c) a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:93, SEQ ID NO:53, SEQ ID NO:95, SEQ ID NO:23, SEQ ID NO:103, and SEQ ID NO:7.(d) a sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:45, SEQ ID NO:15, SEQ ID NO:51, SEQ ID NO:44, SEQ ID NO:73, SEQ ID NO:36, SEQ ID NO:77, SEQ ID NO:50, and SEQ ID NO:6; (e) a sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:97, SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:9, SEQ ID NO:89, SEQ ID NO:107, SEQ ID NO:68, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical, or 100% identical, (f) SEQ ID NO:105, SEQ ID NO:101, SEQ ID NO:4, SEQ ID NO:72, SEQ ID NO:28, SEQ ID NO:64, SEQ ID NO:25, SEQ ID NO:60, SEQ ID NO:55, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:43, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:14, SEQ ID NO:85, SEQ ID NO:13, SEQ ID NO:61, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:10, SEQ ID NO:49, SEQ ID NO:24, SEQ ID NO: 40, SEQ ID NO:63, SEQ ID NO:78, SEQ ID NO:2, SEQ ID NO:94, and SEQ ID NO:5, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least (g) a sequence selected from SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:86, SEQ ID NO:22, SEQ ID NO:69, SEQ ID NO:41, SEQ ID NO:3, SEQ ID NO:66, SEQ ID NO:37, SEQ ID NO:56, SEQ ID NO:21, SEQ ID NO:38, SEQ ID NO:90, SEQ ID NO:100, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:83, SEQ ID NO:1, and SEQ ID NO:19. For the selected sequence, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.and (h) a sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical, or 100% identical to a sequence selected from SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:87, SEQ ID NO:82, and SEQ ID NO:104, wherein the antibody or antigen-binding fragment thereof binds to C-type lectin domain family 2 member D (CLEC2D).

[0009] The disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the light chain (a) has a sequence identity at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least similar to a sequence selected from SEQ ID NO:218, SEQ ID NO:249, SEQ ID NO:230, SEQ ID NO:279, SEQ ID NO:316, SEQ ID NO:237, SEQ ID NO:322, SEQ ID NO:225, SEQ ID NO:318, SEQ ID NO:233, SEQ ID NO:305, SEQ ID NO:280, SEQ ID NO:283, SEQ ID NO:242, SEQ ID NO:286, SEQ ID NO:297, SEQ ID NO:309, and SEQ ID NO:246. or (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% or 100% identical to a sequence selected from SEQ ID NO:222, SEQ ID NO:258, SEQ ID NO:219, SEQ ID NO:313, SEQ ID NO:294, SEQ ID NO:303, SEQ ID NO:317, SEQ ID NO:273, SEQ ID NO:266, SEQ ID NO:315, SEQ ID NO:257, SEQ ID NO:288, SEQ ID NO:301, SEQ ID NO:221, SEQ ID NO:240, SEQ ID NO:299, SEQ ID NO:247, SEQ ID NO:263, and SEQ ID NO:274.9% or 100% identical to a sequence selected from SEQ ID NO:231, SEQ ID NO:250, SEQ ID NO:260, SEQ ID NO:226, SEQ ID NO:271, SEQ ID NO:256, SEQ ID NO:272, SEQ ID NO:278, SEQ ID NO:302, SEQ ID NO:320, SEQ ID NO:295, SEQ ID NO:292, SEQ ID NO:229, SEQ ID NO:264, SEQ ID NO:252, SEQ ID NO:267, SEQ ID NO:304, SEQ ID NO:300, SEQ ID NO:311, and SEQ ID NO:324. (d) sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to SEQ ID NO:259, SEQ ID NO:239, SEQ ID NO:281, SEQ ID NO:228, SEQ ID NO:217, SEQ ID NO:227, and SEQ ID NO: 251; (e) a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:307, SEQ ID NO:262, SEQ ID NO:253, SEQ ID NO:27 6, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5 ...9%, or 100% identical to a sequence selected from SEQ ID NO:254, SEQ ID NO:289, SEQ ID NO:238, SEQ ID NO:268, SEQ ID NO:248, SEQ ID NO:284, SEQ ID NO:244, SEQ ID NO:310, SEQ ID NO:243, SEQ ID NO:285, SEQ ID NO:220, SEQ ID NO:255, SEQ ID NO:293, SEQ ID NO:298, SEQ ID NO:235, SEQ ID NO:319, SEQ ID NO:245, SEQ ID NO:224, SEQ ID NO:291, SEQ ID NO:277, and SEQ ID NO:232; and (g) a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:282, SEQ ID NO:308, SEQ ID NO:287, SEQ ID NO:321, SEQ ID NO:236, SEQ ID NO:265, SEQ ID NO:270, SEQ ID NO:275, SEQ ID NO:306, SEQ ID NO:296, SEQ ID NO:241, SEQ ID NO:314, and SEQ ID NO:223, wherein the antibody or antigen-binding fragment thereof binds to CLEC2D.

[0010] The present disclosure relates to a method and apparatus for producing a nucleic acid sequence comprising: (a) (i) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:46, SEQ ID NO:65, SEQ ID NO:59, and SEQ ID NO:99; (ii) a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:57, SEQ ID NO:91, SEQ ID NO:98, SEQ ID NO:84, SEQ ID NO:58, SEQ ID NO:8 (iii) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:93, SEQ ID NO:53, SEQ ID NO:95, SEQ ID NO:23, SEQ ID NO: 103, and SEQ ID NO: 7; (iv) a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO: 45, SEQ ID NO: 15, SEQ ID NO: 51, At least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5 ...9% or 100% identical sequences, (v) SEQ ID NO:97, SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:9, SEQ ID NO:89, SEQ ID NO:107, SEQ ID NO:68, SEQ ID NO:29, SEQ ID NO:67, SEQ ID NO:74, SEQ ID NO:32, SEQ ID NO:81, SEQ ID NO:106, SEQ ID NO:31, SEQ ID NO:62, SEQ ID NO:48, SEQ ID NO:75, SEQ ID NO:12, SEQ ID NO:102, SEQ ID NO:54, SEQ ID NO:80, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:92, SEQ ID NO:10 8, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:79. , (vi) SEQ ID NO: 105, SEQ ID NO: 101, SEQ ID NO: 4, SEQ ID NO: 72, SEQ ID NO: 28, SEQ ID NO: 64, SEQ ID NO: 25, SEQ ID NO: 60, SEQ ID NO: 55, SEQ ID NO: 52, SEQ ID NO: 27, SEQ ID NO: 43, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 14, SEQ ID NO: 85, SEQ ID NO: 13, SEQ ID NO: 61, SEQ ID NO: 42, SEQ ID NO: 39, SEQ ID NO: 10, SEQ ID NO: 49, SEQ ID NO: 24, SEQ ID NO: 40, SEQ ID NO: 63, SEQ ID NO: 78, SEQ ID NO: 2 , SEQ ID NO:94, and SEQ ID NO:5, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:86, SEQ ID NO:22, SEQ ID NO:69, SEQ ID NO:41, SEQ ID NO:3, SEQ ID NO:66, SEQ ID NO:37, SEQ ID NO:56, SEQ ID NO:21, SEQ ID NO:38, SEQ ID NO:90, SEQ ID NO:100, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:83, SEQ ID NO:1, and SEQ ID NO:19; (viii) a sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:87, SEQ ID NO:82, and SEQ ID NO:104. 0%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence of SEQ ID NO:218, SEQ ID NO:249, SEQ ID NO:230, SEQ ID NO:2 79, SEQ ID NO:316, SEQ ID NO:237, SEQ ID NO:322, SEQ ID NO:225, SEQ ID NO:318, SEQ ID NO:233, SEQ ID NO:305, SEQ ID NO:280, SEQ ID NO:283, SEQ ID NO:242, SEQ ID NO:286, SEQ ID NO:297, SEQ ID NO:309, and SEQ ID NO:246.5%, at least 99.9%, or 100% identical to a sequence selected from: (ii) at least 80%, at least 85% to a sequence selected from SEQ ID NO:222, SEQ ID NO:258, SEQ ID NO:219, SEQ ID NO:313, SEQ ID NO:294, SEQ ID NO:303, SEQ ID NO:317, SEQ ID NO:273, SEQ ID NO:266, SEQ ID NO:315, SEQ ID NO:257, SEQ ID NO:288, SEQ ID NO:301, SEQ ID NO:221, SEQ ID NO:240, SEQ ID NO:299, SEQ ID NO:247, SEQ ID NO:263, and SEQ ID NO:274; (iii) sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to SEQ ID NO:231, SEQ ID NO:250, SEQ ID NO:260, SEQ ID NO:226, SEQ ID NO:271, SEQ ID NO:256, SEQ ID NO:272, SEQ ID NO:278, SEQ ID NO:302, SEQ ID NO:320, SEQ ID NO:295, SEQ ID NO: 292, SEQ ID NO:229, SEQ ID NO:264, SEQ ID NO:252, SEQ ID NO:267, SEQ ID NO:304, SEQ ID NO:300, SEQ ID NO:311, and SEQ ID NO:324, 0% identical to a sequence selected from SEQ ID NO:259, SEQ ID NO:239, SEQ ID NO:281, SEQ ID NO:228, SEQ ID NO:217, SEQ ID NO:227, and SEQ ID NO:251; (iv) a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO:307, SEQ ID NO:262, SEQ ID NO:253, SEQ ID NO:276, SEQ ID NO:323, SEQ ID NO:234, SEQ ID NO:261, SEQ ID NO:312, and SEQ ID NO:290; (v) at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group consisting of SEQ ID NO:307, SEQ ID NO:262, SEQ ID NO:253, SEQ ID NO:276, SEQ ID NO:323, SEQ ID NO:234, SEQ ID NO:261, SEQ ID NO:312, and SEQ ID NO:290. , at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:254, SEQ ID NO:289, SEQ ID NO:238, SEQ ID NO:268, SEQ ID NO:248, SEQ ID NO:284, SEQ ID NO:244, SEQ ID NO:310, SEQ ID NO:243, SEQ ID NO:285, SEQ ID NO:220, SEQ ID NO:255, SEQ ID NO:293, SEQ ID NO:298, SEQ ID NO:235, SEQ ID NO:319, SEQ ID NO:245, SEQ ID NO:224, SEQ ID NO:291, SEQ ID NO:277, and SEQ ID NO:232. 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical, as well as (vii) sequences identical to SEQ ID NO:282, SEQ ID NO:308, SEQ ID NO:287, SEQ ID NO:321, SEQ ID NO:236, SEQ ID NO:265, SEQ ID NO:270, At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5 ...and a light chain comprising a sequence selected from a sequence identical to, or ...

[0011] The present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence selected from any one of SEQ ID NOs: 1-108.

[0012] The present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the light chain comprises a sequence selected from any one of SEQ ID NOs: 217-324.

[0013] The present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence selected from any one of SEQ ID NOs:1-108, and the light chain comprises a sequence selected from any one of SEQ ID NOs:217-324.

[0014] The present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises (i) a heavy chain (HC) CDR1 comprising a sequence selected from SEQ ID NOs: 433-485, (ii) a HC CDR2 comprising a sequence selected from SEQ ID NOs: 486-546, and (iii) a HC CDR3 comprising a sequence selected from SEQ ID NOs: 547-653, and wherein the antibody or antigen-binding fragment thereof binds to CLEC2D.

[0015] The present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the light chain comprises (i) a light chain (LC) CDR1 comprising a sequence selected from SEQ ID NOs: 654-726, (ii) a LC CDR2 comprising a sequence selected from SEQ ID NOs: 727-783, and (iii) a LC CDR3 comprising a sequence selected from SEQ ID NOs: 784-885, and wherein the antibody or antigen-binding fragment thereof binds to CLEC2D.

[0016] The present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain comprising an HC CDR1 sequence selected from SEQ ID NOs: 433 to 485, an HC CDR2 sequence selected from SEQ ID NOs: 486 to 546, and an HC CDR3 sequence selected from SEQ ID NOs: 547 to 653, and a light chain comprising an LC CDR1 sequence selected from SEQ ID NOs: 654 to 726, an LC CDR2 sequence selected from SEQ ID NOs: 727 to 783, and an LC CDR3 sequence selected from SEQ ID NOs: 784 to 885, or a combination thereof.

[0017] In some embodiments of the antibodies or antigen-binding fragments thereof of the present disclosure, the antibodies or antigen-binding fragments thereof bind to a human CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 886-909, a human CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 930-1003, a cynomolgus monkey CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 918-920, a mouse CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 911-915, a rat CLEC2D polypeptide comprising the sequence of SEQ ID NO: 910, and / or a canine CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 916-917.

[0018] The disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises heavy chain complementarity determining region (CDRH) 1, CDRH2, and CDRH3 amino acid sequences of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Table 9A, and the light chain comprises light chain complementarity determining region (CDRL) 1, CDRL2, and CDRL3 amino acid sequences of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Table 9A.

[0019] The disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable heavy chain amino acid sequence of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Table 9A, and the light chain comprises a variable light chain amino acid sequence of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Table 9A.

[0020] The present disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain framework region sequence of an anti-CLEC2D antibody germline family selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9B disclosed herein, and the light chain comprises a framework region sequence of a light chain germline family of anti-CLEC2D antibodies selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9B disclosed herein.

[0021] In some embodiments of the antibodies or antigen-binding fragments thereof of the present disclosure, the antibodies or antigen-binding fragments thereof are monoclonal antibodies.

[0022] In some embodiments of the antibodies or antigen-binding fragments thereof of the present disclosure, the antibodies or antigen-binding fragments thereof block binding of CLEC2D to a receptor. In some embodiments, the receptor comprises a CD161 receptor, and the CD161 receptor comprises a sequence selected from SEQ ID NOs:921-929.

[0023] In some embodiments of the antibodies or antigen-binding fragments thereof of the present disclosure, the antibodies or antigen-binding fragments thereof are human, murine, or chimeric. In some embodiments, the antigen-binding fragments are selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, and diabody fragments. In some embodiments, the antibodies or antigen-binding fragments thereof bind to human CLEC2D with an affinity (KD) of less than 100 nM.

[0024] The present disclosure provides pharmaceutical compositions comprising a peptide (eg, an antibody or antigen-binding fragment thereof) or a nucleic acid described in this disclosure.

[0025] The present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present disclosure.

[0026] The present disclosure provides pharmaceutical compositions comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure.

[0027] In some embodiments of the pharmaceutical compositions of the present disclosure, the pharmaceutical composition further comprises at least one of a buffer, a pharma- ceutically acceptable diluent, a carrier, a solubilizer, an emulsifier, and a preservative.

[0028] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence that encodes an amino acid heavy chain sequence selected from SEQ ID NOs:109-216.

[0029] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding an amino acid light chain sequence selected from SEQ ID NOs:325-432.

[0030] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding a heavy chain comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in the CDRH1, CDRH2, and CDRH3 amino acid sequences, respectively, of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9A disclosed herein.

[0031] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding a light chain comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in the CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9A disclosed herein.

[0032] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding a heavy chain amino acid sequence set forth in the variable heavy chain amino acid sequence of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9A disclosed herein.

[0033] The disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding a light chain amino acid sequence set forth in the variable light chain amino acid sequence of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9A disclosed herein.

[0034] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding a heavy chain comprising a framework region amino acid sequence set forth in the heavy chain framework region amino acid sequences of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9B disclosed herein.

[0035] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding a light chain comprising a framework region amino acid sequence set forth in the light chain framework region amino acid sequences of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9B disclosed herein.

[0036] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding the heavy chain amino acid sequence of an antibody or antigen-binding fragment thereof of the present disclosure.

[0037] The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding the light chain amino acid sequence of an antibody or antigen-binding fragment thereof of the present disclosure.

[0038] The present disclosure provides a composition comprising a first nucleic acid encoding a polypeptide selected from SEQ ID NOs: 109-216, and a second nucleic acid encoding a polypeptide selected from SEQ ID NOs: 325-432.

[0039] The present disclosure provides a vector comprising a nucleic acid of the present disclosure.

[0040] The present disclosure provides a cell comprising a nucleic acid, nucleic acid composition, or vector of the disclosure. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is selected from the group consisting of a CHO cell, a 293 cell, an NSO cell, a PER.C6 cell, and a B cell. In some embodiments, the mammalian cell is a 293-6E cell or a DG44 cell. In some embodiments, the cell expresses an antibody or antigen-binding fragment thereof of the disclosure. In some embodiments, the cell is a germline cell.

[0041] The present disclosure provides a cell that produces an antibody or antigen-binding fragment thereof of the present disclosure.

[0042] The present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure.

[0043] The present disclosure provides a composition for use in a subject in need of treatment for a disease comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, or a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure.

[0044] The present disclosure provides a composition for use in the manufacture of a medicament for a subject in need of prevention or treatment of a disease, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, or a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure.

[0045] In some embodiments of the method or composition for use of the present disclosure, the disease is rheumatoid arthritis.In some embodiments, the subject shows bone loss caused by having rheumatoid arthritis.In some embodiments, administering a therapeutically effective amount of the antibody or its antigen-binding fragment slows or reverses bone loss in the subject.

[0046] In some embodiments of the methods or compositions for use of the present disclosure, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, endometrial cancer, uterine cancer, bladder cancer, kidney cancer, esophageal cancer, squamous cell carcinoma, uveal melanoma, glioma, glioblastoma, myeloma, pheochromocytoma, paraganglioma, follicular lymphoma, renal cell carcinoma, cendcal cancer, ovarian cancer, cervical cancer, lung cancer, colorectal cancer, brain cancer, pancreatic cancer, gastric cancer, intestinal cancer, testicular cancer, skin cancer, thyroid cancer, thymoma, head and neck cancer, liver cancer, pharyngeal cancer, adrenocortical carcinoma, cholangiocarcinoma, mesothelioma, sarcoma, leukemia, lymphoma, Hodgkin's disease, multiple myeloma, melanoma, astrocytoma, stomach cancer, and lung adenocarcinoma. In some embodiments, the cells of the cancer express CLEC2D on the cell surface. In some embodiments, administration of a therapeutically effective amount of an antibody or antigen-binding fragment thereof results in an anti-tumor effect in a subject.

[0047] In some embodiments of the method or composition for use of the present disclosure, the antibody or its antigen-binding fragment is administered as a single agent therapy.In some embodiments, the antibody or its antigen-binding fragment is administered in combination with at least one of a T cell-directed immunomodulator, a second immunomodulator, a cancer vaccine, an adoptive cell therapy agent, an oncolytic virus, a second antibody therapy agent, a radiotherapy agent, an antibody drug conjugate, a small interfering RNA, a chemotherapy agent, an immunotherapy agent, an immune checkpoint inhibitor, a mitotic inhibitor, or a combination thereof.In some embodiments, the adoptive cell therapy agent comprises a CAR-T therapy agent.In some embodiments, administration of a therapeutically effective amount of the antibody or its antigen-binding fragment alleviates the signs or symptoms of a disease.

[0048] The present disclosure provides an antibody library comprising at least about 10 8 unique monoclonal antibody clones, at least about 80% of the antibody clones detectably and specifically bind to the CLEC2D antigen.

[0049] In some embodiments of the antibody library of the present disclosure, the CLEC2D antigen comprises an amino acid sequence selected from SEQ ID NOs: 886-920 and 930-1003. In some embodiments of the antibody library of the present disclosure, the CLEC2D antigen comprises an amino acid sequence selected from SEQ ID NOs: 886-909 and 930-1003. In some embodiments, the CLEC2D antigen comprises a CLEC2D antigen expressed on the surface of a tumor cell, a variant of the CLEC2D antigen, or a homolog of the CLEC2D antigen. In some embodiments, the variant of the CLEC2D antigen comprises a fragment of the CLEC2D protein. In some embodiments, the homolog of the CLEC2D antigen comprises human, mouse, dog, rat, or cynomolgus CLEC2D.

[0050] The present disclosure provides a method for effecting immune modulation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment of the disclosure, or a nucleic acid encoding an antibody or antigen-binding fragment thereof of the disclosure.

[0051] The present disclosure provides a method for modulating (e.g., enhancing) innate immunity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment of the disclosure, or a nucleic acid encoding an antibody or antigen-binding fragment thereof of the disclosure.

[0052] The present disclosure provides a method for improving natural killer cell cytotoxicity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, or a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure.

[0053] The present disclosure provides a method for modulating (e.g., enhancing) adaptive immunity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment of the disclosure, or a nucleic acid encoding an antibody or antigen-binding fragment thereof of the disclosure.

[0054] The present disclosure provides a method for screening a high diversity antibody gene library with antibodies against a CLEC2D antibody, comprising: (a) inserting a library of antibody genes into a phage protein gene and transforming a plurality of phages to generate a phage library, the phages in the phage library displaying the library of antibody genes on their surface; (b) panning the phage library with the CLEC2D antigen against individual phages that bind to the CLEC2D antigen to generate an enriched phage library enriched for antibody genes encoding antibodies that bind to the CLEC2D antigen; (c) repeating step (b) at least once or at least twice; (d) introducing the antibody genes from the enriched phage library into a yeast surface display library; (e) isolating individual yeast cells that bind to the CLEC2D antigen from the yeast surface display library; (f) culturing the isolated individual yeast cells that bind to the CLEC2D antigen to generate yeast surface display library clones; and (g) isolating the antibody genes that bind to the CLEC2D antigen by sequencing the yeast surface display library clones.

[0055] In some embodiments of the screening method of the present disclosure, the panning step (b) comprises panning the phage library with magnetic beads coated with CLEC2D. In some embodiments, the introducing step (d) comprises cloning the antibody gene into a yeast expression vector and transforming the yeast cells. In some embodiments, the method further comprises analyzing the surface expression of the antibody gene using a FLAG tag, a c-Myc tag, a polyhistidine tag, or a V5 tag. In some embodiments, the testing step (e) comprises isolating yeast cells expressing an antibody gene that binds to the CLEC2D antigen using flow cytometry. In some embodiments, the method further comprises repeating the flow cytometry isolation at least 1×, at least 2×, at least 3×, at least 4×, or at least 5×. In some embodiments, the method further comprises cloning the antibody gene that binds to CLEC2D into a mammalian expression vector.

[0056] The present disclosure provides a method for producing a composition comprising an anti-CLEC2D antibody or antigen-binding fragment thereof, comprising: (a) transforming a mammalian cell with a vector comprising a sequence encoding a promoter and a sequence encoding an anti-CLEC2D antibody or antibody fragment, wherein the sequence encoding the promoter and the sequence encoding the anti-CLEC2D antibody or antibody fragment are operably linked; (b) culturing the mammalian cell under conditions suitable for expression of the anti-CLEC2D antibody or antibody fragment; (c) centrifuging the cultured mammalian cells to produce a supernatant; (d) filtering the supernatant; and (e) purifying the filtered supernatant using liquid chromatography.

[0057] In some embodiments of the disclosed method, the filtration step (d) comprises a 3 μm to 30 μm filter. In some embodiments, the filtration step (d) further comprises a 0.22 μm filter. In some embodiments, the purification step (e) comprises a Protein A column. In some embodiments, the Protein A column is treated with a high salt wash buffer to remove host cell proteins. In some embodiments, the anti-CLEC2D antibody or antibody fragment thereof is eluted using a 30 mM phosphate buffer at pH 3.0 to 4.0. In some embodiments, the purification step (e) further comprises an anion exchange chromatography (AEX) step. In some embodiments, the AEX step comprises a Q Sepharose column. In some embodiments, the Q Sepharose is pre-equilibrated with a pre-equilibration buffer comprising 10 to 100 mM histidine. In some embodiments, the pre-equilibration buffer further comprises citrate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), acetate, or a combination thereof. In some embodiments, the pre-equilibration buffer comprises a pH of 4.5 to 6.5. In some embodiments, the anti-CLEC2D antibody is eluted in step (e) with an elution buffer comprising 200 to 1000 mM NaCl, KCl, or a combination thereof. In some embodiments, the elution buffer comprises a pH of 4.5 to 6.5.

[0058] In one aspect, the present disclosure relates to the isolation of novel monoclonal antibodies that specifically bind to the CLEC2D antigen, which modulate (e.g., inhibit) the interaction of CD161 and CLEC2D, thereby altering NK cell / immune cell-mediated cytotoxicity and / or cytokine production.

[0059] In another aspect, the present disclosure relates to cancer cells expressing CLEC2D, which are specifically recognized by these novel antibodies that can kill tumor cells by ADCC (antibody-dependent cellular cytotoxicity) and / or CDC (complement-dependent cytotoxicity) and / or ADCP (antibody-dependent cellular phagocytosis).

[0060] In a related aspect, the disclosure relates to a method for producing an anti-CLEC2D antibody, comprising selecting an anti-CLEC2D antibody from a high diversity antibody gene library. In one embodiment, the high diversity antibody gene library is displayed by phage surface display and / or yeast surface display. In one embodiment, the phage and / or yeast displayed high diversity antibody gene library is selected using purified CLEC2D antigen as a target. In one embodiment, the selected anti-CLEC2D antibody gene is expressed in mammalian cells (e.g., Chinese Hamster Ovary (CHO) cells). In one embodiment, a single cell clone expressing the anti-CLEC2D antibody is developed into a cell line to confirm anti-CLEC2D antibody expression. In one embodiment, overexpression of the selected antibody clone is achieved by a specific medium, supplement, and specific bioreactor process, cumulatively described herein as upstream process development. In one embodiment, the anti-CLEC2D antibody expressed from the cell line is purified to homogeneity, for example, via various filtration and chromatography, herein referred to as downstream purification process.

[0061] The present disclosure provides a method for treating a disease in a subject in need thereof, comprising measuring the level of CLEC2D protein in the subject, and administering a therapeutically effective amount of an anti-CLEC2D antibody to the subject.

[0062] In some embodiments of the methods of the present disclosure, the disease is cancer. In some embodiments, the cancer is breast cancer, prostate cancer, endometrial cancer, uterine cancer, bladder cancer, kidney cancer, esophageal cancer, squamous cell carcinoma, or the like. carcinoma), uveal melanoma, glioma, glioblastoma, myeloma, pheochromocytoma, paraganglioma, follicular lymphoma, renal cell carcinoma, cervical cancer, ovarian cancer, cervical cancer, lung cancer, colorectal cancer, brain cancer, pancreatic cancer, gastric cancer, intestinal cancer, testicular cancer, skin cancer, thyroid cancer, thymoma, head and neck cancer, liver cancer, pharyngeal cancer, adrenal cortical carcinoma, bile duct carcinoma, mesothelioma, sarcoma, leukemia, lymphoma, Hodgkin's disease, multiple myeloma, melanoma, astrocytoma, gastric cancer, lung adenocarcinoma, adenocarcinoma, acinar cell adenocarcinoma, adrenal cortical carcinoma, alveolar cell carcinoma, undifferentiated carcinoma, basaloid carcinoma, basal cell carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, renal adenocarcinoma (renaladinol carcinoma), embryonal carcinoma, endometrioid carcinoma (anometroid carcinoma) carcinoma, fibrolamellar hepatocellular carcinoma, follicular carcinoma, giant cell carcinoma, hepatocellular carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, leptomanigio carcinoma, medullary carcinoma, melanoma, meningeal carcinoma, mesometonephric carcinoma, oat cell carcinoma, squamous cell carcinoma, sweat gland carcinoma, transitional cell carcinoma, tubular cell carcinoma, ameloblastic sarcoma, psammomatous tumor, botryoid sarcoma, endometrial stromal sarcoma, Ewing's sarcoma, fascicular sarcoma, giant cell sarcoma, granulocytic sarcoma, immunoblastic sarcoma, parosteal osteogenic sarcoma, coppices sarcoma sarcoma), leukocytic sarcoma (leukemia), lymphocytic sarcoma (lymphosarcoma), medullary sarcoma, myeloid sarcoma (granulocytic sarcoma), austiogenci sarcoma, periosteal sarcoma, reticulum cell sarcoma (histiocytic lymphoma), round cell sarcoma, spindle cell sarcoma, synovial sarcoma, telangiectatic audiogenic sarcoma, Burkitt's lymphoma, NPDL, NML, NH, diffuse lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell lymphoma, T cell lymphoma, diffuse large B cell lymphoma, acute myeloid lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, mantle cell lymphoma, and follicular lymphoma. In some embodiments, the subject's cancer cells have elevated levels of CLEC2D protein when compared to normal non-cancer cells.In some embodiments, high levels of CLEC2D are associated with a poor prognostic effect.

[0063] In some embodiments of the method of the present disclosure, the disease is an autoimmune disorder or an inflammatory disorder.In some embodiments, the autoimmune disorder or the inflammatory disorder is type I diabetes, rheumatoid arthritis, lupus, inflammatory bowel disease, celiac disease, Crohn's disease, ulcerative colitis, psoriasis, or multiple sclerosis.

[0064] In some embodiments of the method of the present disclosure, the disease is an autoimmune disorder or an inflammatory disorder.In some embodiments, the autoimmune disorder is type I diabetes, rheumatoid arthritis, lupus, inflammatory bowel disease, celiac disease, Crohn's disease, ulcerative colitis, psoriasis, or multiple sclerosis.

[0065] In some embodiments of the method of the present disclosure, the disease is an infectious disease. In some embodiments, the disease is HIV infection, human cytomegalovirus infection, hepatitis B infection, hepatitis C infection, Ebola virus infection, dengue fever, yellow fever, listeriosis, tuberculosis, cholera, malaria, leishmaniasis, or trypanosoma infection.

[0066] In another embodiment, novel antibodies produced from CHO cell lines are characterized using multiple in vitro and in vivo assays, including various biophysical parameters, antigen recognition, tumor cell surface binding, tumor cell death, cytokine production, and downstream genetic analysis to define the mechanism of action. These monoclonal antibodies are also tested for long-term stability, various formulations suitable for therapeutic, prognostic, and diagnostic applications in cancer, infectious diseases, autoimmune diseases, and chronic diseases. In another embodiment, in vivo tumor suppression assays are performed to establish the antitumor activity of selected antibodies as monotherapeutics or in combination with other therapeutic products.

[0067] In one aspect, the disclosure further relates to the isolation of novel and unique monoclonal antibodies that specifically bind to the CLEC2D antigen. In some aspects, the novel antibodies affect the interaction of CD161 and CLEC2D to alter immune cell (e.g., NK, B, or T cell)-mediated cytotoxicity and / or cytokine production. In some aspects, various cancer cells expressing CLEC2D are recognized by these novel antibodies and have demonstrated cytotoxic effects by various methods including ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement-dependent cytotoxicity) and / or ADCP (antibody-dependent cellular phagocytosis). In one aspect, the disclosure provides a focus and hypothesis regarding the role of CLEC2D in lymphocyte crosstalk and immune tolerance. In another aspect, the methods provided herein for identifying novel antibody molecules and related compositions in the field of approved therapeutics or therapeutics in preclinical or clinical trials include pharmaceutical characteristics suitable for manufacturability / development.

[0068] In one aspect, the disclosure relates to a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, wherein the antibody is an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Tables 9A and 9B disclosed herein.

[0069] In one aspect, the disclosure relates to a method for effecting immune modulation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment, wherein the antibody is an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Tables 9A and 9B disclosed herein.

[0070] In one aspect, the disclosure relates to a method for modulating (e.g., improving) innate immunity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment, wherein the antibody is an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Tables 9A and 9B disclosed herein.

[0071] In one aspect, the disclosure relates to a method for modulating (e.g., improving) adaptive immunity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment, wherein the antibody is an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Tables 9A and 9B disclosed herein.

[0072] In one aspect, the disclosure relates to a method for increasing natural killer cell cytotoxicity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment, wherein the antibody is an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Tables 9A and 9B disclosed herein. In certain embodiments, for example, the following are provided: (Item 1) 1. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, the heavy chain comprising: a. a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:46, SEQ ID NO:65, SEQ ID NO:59, and SEQ ID NO:99; b. a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:57, SEQ ID NO:91, SEQ ID NO:98, SEQ ID NO:84, SEQ ID NO:58, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:47, SEQ ID NO:17, and SEQ ID NO:8; c. a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:93, SEQ ID NO:53, SEQ ID NO:95, SEQ ID NO:23, SEQ ID NO:103, and SEQ ID NO:7; d. a sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:45, SEQ ID NO:15, SEQ ID NO:51, SEQ ID NO:44, SEQ ID NO:73, SEQ ID NO:36, SEQ ID NO:77, SEQ ID NO:50, and SEQ ID NO:6; e. SEQ ID NO:97, SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:9, SEQ ID NO:89, SEQ ID NO:107, SEQ ID NO:68, SEQ ID NO:29, SEQ ID NO:67, SEQ ID NO:74, SEQ ID NO:32, SEQ ID NO:81, SEQ ID NO:106, SEQ ID NO:31, SEQ ID NO:62, SEQ ID NO:48, SEQ ID NO:75, SEQ ID NO:12, SEQ ID NO:102, SEQ ID NO:54, SEQ ID NO:80, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:92, SEQ ID NO:108, and SEQ ID NO:79 a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical, or 100% identical to a sequence selected from f. SEQ ID NO:105, SEQ ID NO:101, SEQ ID NO:4, SEQ ID NO:72, SEQ ID NO:28, SEQ ID NO:64, SEQ ID NO:25, SEQ ID NO:60, SEQ ID NO:55, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:43, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:14, SEQ ID NO:85, SEQ ID NO:13, SEQ ID NO:61, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:10, SEQ ID NO:49, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:63, SEQ ID NO:78, SEQ ID NO:2, SEQ ID NO:94, and a sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical, or 100% identical to a sequence selected from sequence no. 5; g. A sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical, or 100% identical to a sequence selected from SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:86, SEQ ID NO:22, SEQ ID NO:69, SEQ ID NO:41, SEQ ID NO:3, SEQ ID NO:66, SEQ ID NO:37, SEQ ID NO:56, SEQ ID NO:21, SEQ ID NO:38, SEQ ID NO:90, SEQ ID NO:100, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:83, SEQ ID NO:1, and SEQ ID NO:19, and h. a sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical, or 100% identical to a sequence selected from SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:87, SEQ ID NO:82, and SEQ ID NO:104; comprising a sequence selected from the group consisting of the antibody or antigen-binding fragment thereof binds to C-type lectin domain family 2 member D (CLEC2D); The isolated antibody or antigen-binding fragment thereof. (Item 2) 1. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, the light chain comprising: a. a sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:218, SEQ ID NO:249, SEQ ID NO:230, SEQ ID NO:279, SEQ ID NO:316, SEQ ID NO:237, SEQ ID NO:322, SEQ ID NO:225, SEQ ID NO:318, SEQ ID NO:233, SEQ ID NO:305, SEQ ID NO:280, SEQ ID NO:283, SEQ ID NO:242, SEQ ID NO:286, SEQ ID NO:297, SEQ ID NO:309, and SEQ ID NO:246; b. a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:222, SEQ ID NO:258, SEQ ID NO:219, SEQ ID NO:313, SEQ ID NO:294, SEQ ID NO:303, SEQ ID NO:317, SEQ ID NO:273, SEQ ID NO:266, SEQ ID NO:315, SEQ ID NO:257, SEQ ID NO:288, SEQ ID NO:301, SEQ ID NO:221, SEQ ID NO:240, SEQ ID NO:299, SEQ ID NO:247, SEQ ID NO:263, and SEQ ID NO:274; c. a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:231, SEQ ID NO:250, SEQ ID NO:260, SEQ ID NO:226, SEQ ID NO:271, SEQ ID NO:256, SEQ ID NO:272, SEQ ID NO:278, SEQ ID NO:302, SEQ ID NO:320, SEQ ID NO:295, SEQ ID NO:292, SEQ ID NO:229, SEQ ID NO:264, SEQ ID NO:252, SEQ ID NO:267, SEQ ID NO:304, SEQ ID NO:300, SEQ ID NO:311, and SEQ ID NO:324; d. a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:259, SEQ ID NO:239, SEQ ID NO:281, SEQ ID NO:228, SEQ ID NO:217, SEQ ID NO:227, and SEQ ID NO:251; e. a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:307, SEQ ID NO:262, SEQ ID NO:253, SEQ ID NO:276, SEQ ID NO:323, SEQ ID NO:234, SEQ ID NO:261, SEQ ID NO:312, and SEQ ID NO:290; f. a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:254, SEQ ID NO:289, SEQ ID NO:238, SEQ ID NO:268, SEQ ID NO:248, SEQ ID NO:284, SEQ ID NO:244, SEQ ID NO:310, SEQ ID NO:243, SEQ ID NO:285, SEQ ID NO:220, SEQ ID NO:255, SEQ ID NO:293, SEQ ID NO:298, SEQ ID NO:235, SEQ ID NO:319, SEQ ID NO:245, SEQ ID NO:224, SEQ ID NO:291, SEQ ID NO:277, and SEQ ID NO:232, and g. a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:282, SEQ ID NO:308, SEQ ID NO:287, SEQ ID NO:321, SEQ ID NO:236, SEQ ID NO:265, SEQ ID NO:270, SEQ ID NO:275, SEQ ID NO:306, SEQ ID NO:296, SEQ ID NO:241, SEQ ID NO:314, and SEQ ID NO:223; comprising a sequence selected from the group consisting of the antibody or antigen-binding fragment thereof binds to CLEC2D; The isolated antibody or antigen-binding fragment thereof. (Item 3) a. i. a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:46, SEQ ID NO:65, SEQ ID NO:59, and SEQ ID NO:99; ii. a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:57, SEQ ID NO:91, SEQ ID NO:98, SEQ ID NO:84, SEQ ID NO:58, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:47, SEQ ID NO:17, and SEQ ID NO:8; iii. a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:93, SEQ ID NO:53, SEQ ID NO:95, SEQ ID NO:23, SEQ ID NO:103, and SEQ ID NO:7; iv. a sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:45, SEQ ID NO:15, SEQ ID NO:51, SEQ ID NO:44, SEQ ID NO:73, SEQ ID NO:36, SEQ ID NO:77, SEQ ID NO:50, and SEQ ID NO:6; v. SEQ ID NO:97, SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:9, SEQ ID NO:89, SEQ ID NO:107, SEQ ID NO:68, SEQ ID NO:29, SEQ ID NO:67, SEQ ID NO:74, SEQ ID NO:32, SEQ ID NO:81, SEQ ID NO:106, SEQ ID NO:31, SEQ ID NO:62, SEQ ID NO:48, SEQ ID NO:75, SEQ ID NO:12, SEQ ID NO:102, SEQ ID NO:54, SEQ ID NO:80, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:92, SEQ ID NO:108, and SEQ ID NO: a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from vi. SEQ ID NO:105, SEQ ID NO:101, SEQ ID NO:4, SEQ ID NO:72, SEQ ID NO:28, SEQ ID NO:64, SEQ ID NO:25, SEQ ID NO:60, SEQ ID NO:55, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:43, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:14, SEQ ID NO:85, SEQ ID NO:13, SEQ ID NO:61, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:10, SEQ ID NO:49, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:63, SEQ ID NO:78, SEQ ID NO:2, SEQ ID NO:94, and a sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:5, vii. a sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:86, SEQ ID NO:22, SEQ ID NO:69, SEQ ID NO:41, SEQ ID NO:3, SEQ ID NO:66, SEQ ID NO:37, SEQ ID NO:56, SEQ ID NO:21, SEQ ID NO:38, SEQ ID NO:90, SEQ ID NO:100, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:83, SEQ ID NO:1, and SEQ ID NO:19; and viii. a sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:87, SEQ ID NO:82, and SEQ ID NO:104; and a heavy chain comprising a sequence selected from b. i. a sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:218, SEQ ID NO:249, SEQ ID NO:230, SEQ ID NO:279, SEQ ID NO:316, SEQ ID NO:237, SEQ ID NO:322, SEQ ID NO:225, SEQ ID NO:318, SEQ ID NO:233, SEQ ID NO:305, SEQ ID NO:280, SEQ ID NO:283, SEQ ID NO:242, SEQ ID NO:286, SEQ ID NO:297, SEQ ID NO:309, and SEQ ID NO:246; ii. a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:222, SEQ ID NO:258, SEQ ID NO:219, SEQ ID NO:313, SEQ ID NO:294, SEQ ID NO:303, SEQ ID NO:317, SEQ ID NO:273, SEQ ID NO:266, SEQ ID NO:315, SEQ ID NO:257, SEQ ID NO:288, SEQ ID NO:301, SEQ ID NO:221, SEQ ID NO:240, SEQ ID NO:299, SEQ ID NO:247, SEQ ID NO:263, and SEQ ID NO:274; iii. a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:231, SEQ ID NO:250, SEQ ID NO:260, SEQ ID NO:226, SEQ ID NO:271, SEQ ID NO:256, SEQ ID NO:272, SEQ ID NO:278, SEQ ID NO:302, SEQ ID NO:320, SEQ ID NO:295, SEQ ID NO:292, SEQ ID NO:229, SEQ ID NO:264, SEQ ID NO:252, SEQ ID NO:267, SEQ ID NO:304, SEQ ID NO:300, SEQ ID NO:311, and SEQ ID NO:324; iv. a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:259, SEQ ID NO:239, SEQ ID NO:281, SEQ ID NO:228, SEQ ID NO:217, SEQ ID NO:227, and SEQ ID NO:251; v. a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:307, SEQ ID NO:262, SEQ ID NO:253, SEQ ID NO:276, SEQ ID NO:323, SEQ ID NO:234, SEQ ID NO:261, SEQ ID NO:312, and SEQ ID NO:290; vi. a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:254, SEQ ID NO:289, SEQ ID NO:238, SEQ ID NO:268, SEQ ID NO:248, SEQ ID NO:284, SEQ ID NO:244, SEQ ID NO:310, SEQ ID NO:243, SEQ ID NO:285, SEQ ID NO:220, SEQ ID NO:255, SEQ ID NO:293, SEQ ID NO:298, SEQ ID NO:235, SEQ ID NO:319, SEQ ID NO:245, SEQ ID NO:224, SEQ ID NO:291, SEQ ID NO:277, and SEQ ID NO:232, and vii. a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a sequence selected from SEQ ID NO:282, SEQ ID NO:308, SEQ ID NO:287, SEQ ID NO:321, SEQ ID NO:236, SEQ ID NO:265, SEQ ID NO:270, SEQ ID NO:275, SEQ ID NO:306, SEQ ID NO:296, SEQ ID NO:241, SEQ ID NO:314, and SEQ ID NO:223; a light chain comprising a sequence selected from 1. An isolated antibody or antigen-binding fragment thereof comprising: the antibody or antigen-binding fragment thereof binds to CLEC2D; The isolated antibody or antigen-binding fragment thereof. (Item 4) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence selected from any one of SEQ ID NOs:1 to 108, and the antibody or antigen-binding fragment thereof binds to CLEC2D. (Item 5) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the light chain comprises an sequence selected from any one of SEQ ID NOs: 217 to 324, and the antibody or antigen-binding fragment thereof binds to CLEC2D. (Item 6) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence selected from any one of SEQ ID NOs: 1 to 108 and the light chain comprises a sequence selected from any one of SEQ ID NOs: 217 to 324, and the antibody or antigen-binding fragment thereof binds to CLEC2D. (Item 7) 1. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, the heavy chain comprising: (i) a heavy chain (HC) CDR1 comprising a sequence selected from SEQ ID NOs: 433 to 485; (ii) HC CDR2 comprising a sequence selected from SEQ ID NOs: 486 to 546, and (iii) HC CDR3 comprising a sequence selected from SEQ ID NOs: 547 to 653; Including, the antibody or antigen-binding fragment thereof binds to CLEC2D; The isolated antibody or antigen-binding fragment thereof. (Item 8) 1. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, the light chain comprising: (i) a light chain (LC) CDR1 comprising a sequence selected from SEQ ID NOs: 654 to 726; (ii) LC CDR2 comprising a sequence selected from SEQ ID NOs: 727 to 783, and (iii) LC CDR3 comprising a sequence selected from SEQ ID NOs: 784 to 885; Including, the antibody or antigen-binding fragment thereof binds to CLEC2D; The isolated antibody or antigen-binding fragment thereof. (Item 9) a. a heavy chain comprising an HC CDR1 sequence selected from SEQ ID NOs: 433 to 485, an HC CDR2 sequence selected from SEQ ID NOs: 486 to 546, and an HC CDR3 sequence selected from SEQ ID NOs: 547 to 653; b. A light chain comprising an LC CDR1 sequence selected from SEQ ID NOs: 654 to 726, an LC CDR2 sequence selected from SEQ ID NOs: 727 to 783, and an LC CDR3 sequence selected from SEQ ID NOs: 784 to 885, or c. combinations of these, 1. An isolated antibody or antigen-binding fragment thereof comprising: the antibody or antigen-binding fragment thereof binds to CLEC2D; The isolated antibody or antigen-binding fragment thereof. (Item 10) a. a human CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 886 to 909 and 930 to 1003; b. A cynomolgus CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 918-920; c. A mouse CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 911 to 915; d. a rat CLEC2D polypeptide comprising the sequence of SEQ ID NO: 910, and / or e. A canine CLEC2D polypeptide comprising a sequence selected from SEQ ID NOs: 916-917; 10. The antibody or antigen-binding fragment thereof according to any one of items 1 to 9, which binds to (Item 11) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises heavy chain complementarity determining region (CDRH) 1, CDRH2, and CDRH3 amino acid sequences of any one of anti-CLEC2D antibody Nos. A1 to N2 in Table 9A, and the light chain comprises light chain complementarity determining region (CDRL) 1, CDRL2, and CDRL3 amino acid sequences of any one of anti-CLEC2D antibody Nos. A1 to N2 in Table 9A. (Item 12) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain amino acid sequence of any one of anti-CLEC2D antibody Nos. A1 to N2 in Table 9A, and the light chain comprises the light chain amino acid sequence of any one of anti-CLEC2D antibody Nos. A1 to N2 in Table 9A. (Item 13) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises heavy chain complementarity determining region (CDRH) 1, CDRH2, and CDRH3 amino acid sequences of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9A, and the light chain comprises light chain complementarity determining region (CDRL) 1, CDRL2, and CDRL3 amino acid sequences of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9A. (Item 14) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable heavy chain amino acid sequence of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9A, and the light chain comprises a variable light chain amino acid sequence of an anti-CLEC2D antibody selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9A. (Item 15) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain framework region sequence of an anti-CLEC2D antibody germline family selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9B, and the light chain comprises a framework region sequence of a light chain germline family of anti-CLEC2D antibodies selected from the group consisting of A1, B1, E1, P1, U1, Y1, E2, I2, and L2 of Table 9B. (Item 16) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: A1 in Tables 9A and 9B. (Item 17) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: B1 in Tables 9A and 9B. (Item 18) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: E1 in Tables 9A and 9B. (Item 19) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: P1 in Tables 9A and 9B. (Item 20) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: U1 in Tables 9A and 9B. (Item 21) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: Y1 in Tables 9A and 9B. (Item 22) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: E2 in Tables 9A and 9B. (Item 23) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: I2 in Tables 9A and 9B. (Item 24) 65. The isolated antibody or antigen-binding fragment thereof according to any one of items 62 to 64, wherein the anti-CLEC2D antibody is anti-CLEC2D antibody number: L2 in Tables 9A and 9B. (Item 25) 25. The antibody or antigen-binding fragment thereof according to any one of items 1 to 24, which is a monoclonal antibody. (Item 26) 25. The antibody or antigen-binding fragment thereof of any one of items 1 to 24, which modulates or blocks the binding of CLEC2D to its receptor. (Item 27) 27. The antibody or antigen-binding fragment thereof according to Item 26, wherein the receptor comprises a CD161 receptor, and the CD161 receptor comprises a sequence selected from SEQ ID NOs: 921 to 929. (Item 28) 28. The antibody or antigen-binding fragment thereof of any one of items 1 to 27, which is human, murine, or chimeric. (Item 29) 29. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, wherein the antigen-binding fragment is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, and diabody fragments. (Item 30) 30. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 29, wherein the antibody is afucosylated. (Item 31) 31. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 30, wherein the antibody or antigen-binding fragment thereof is afucosylated. (Item 32) 32. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 31, wherein the antibody or antigen-binding fragment thereof is afucosylated in the antibody region. (Item 33) 33. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 32, wherein the antibody or antigen-binding fragment thereof comprises an IgG1 Fc region. (Item 34) 33. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 32, wherein the antibody or antigen-binding fragment thereof comprises an IgG4 Fc region. (Item 35) 33. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 32, wherein the antibody or antigen-binding fragment thereof comprises an IgG1 N-A Fc region. (Item 36) 33. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 32, wherein the antibody or antigen-binding fragment thereof comprises an IgG2 Fc region. (Item 37) 36. The antibody or antigen-binding fragment thereof of any one of items 1 to 35, which binds to human CLEC2D with an affinity (KD) of less than 100 nM. (Item 38) 38. The antibody or antigen-binding fragment thereof according to any one of items 1 to 37, which recognizes and binds to a structural epitope of the CLEC2D antigen and consists of amino acid sites that overlap and / or do not overlap with CD161 receptor-interacting amino acid residues. (Item 39) 38. The antibody or antigen-binding fragment thereof according to any one of items 1 to 37, comprising a variable heavy chain sequence and a variable light chain sequence that inhibits or suppresses or competes with another antibody that recognizes and binds to a structural epitope of the CLEC2D antigen and that consists of amino acid sites that either overlap and / or do not overlap with CD161 receptor-interacting amino acid residues. (Item 40) 38. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 37, comprising a variable heavy chain sequence and a variable light chain sequence that bind to a structural epitope of the CLEC2D antigen comprising any of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, or any combination thereof. (Item 41) 38. The antibody or antigen-binding fragment thereof of any one of items 1 to 37, comprising a variable heavy chain sequence and a variable light chain sequence that inhibits, abrogates or competes with binding of another antibody to a structural epitope of the CLEC2D antigen comprising any of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, or any combination thereof. (Item 42) 38. The antibody or antigen-binding fragment thereof according to any one of items 1 to 37, comprising a variable heavy chain sequence and a variable light chain sequence that bind to a structural epitope of the CLEC2D antigen, comprising at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95 of SEQ ID NOs: 886 to 920 and 930 to 1003, and thereby block the interaction between the CLEC2D receptor and the CD161 receptor by constituting a nonlinear scaffold for CD161 receptor-interacting amino acid residues. (Item 43) SEQ ID NOs: 886 to 920 and 930 to 1003: amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PH 38. The antibody or antigen-binding fragment thereof according to any one of items 1 to 37, comprising a variable heavy chain sequence and a variable light chain sequence that bind to a structural epitope of the CLEC2D antigen, including at least one of E116, ASN95, and form an allosteric and nonlinear scaffold for non-CD161 receptor-interacting amino acid residues, thereby blocking the interaction between the CLEC2D receptor and the CD161 receptor. (Item 44) 44. The antibody or antigen-binding fragment thereof according to any one of items 1 to 43, comprising a variable heavy chain sequence and a variable light chain sequence that, when binding to a CLEC2D selected from SEQ ID NOs: 886 to 920 and 930 to 1003, binds to at least one of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95, and induces tumor killing or cytotoxicity, either alone or in combination. (Item 45) 45. The antibody or antigen-binding fragment thereof of item 44, which induces cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibody or antigen-binding fragment thereof. (Item 46) 46. ​​A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of items 1 to 45. (Item 47) 47. The pharmaceutical composition according to item 46, further comprising at least one of a buffer, a pharma- ceutically acceptable diluent, a carrier, a solubilizer, an emulsifier, and a preservative. (Item 48) An isolated nucleic acid comprising a polynucleotide sequence encoding an amino acid heavy chain sequence selected from SEQ ID NOs: 109-216. (Item 49) An isolated nucleic acid comprising a polynucleotide sequence encoding an amino acid light chain sequence selected from SEQ ID NOs: 325-432. (Item 50) An isolated nucleic acid comprising a polynucleotide sequence encoding the heavy chain amino acid sequence according to any one of items 1, 3, 4, 6, 7, and 9 to 37. (Item 51) An isolated nucleic acid comprising a polynucleotide sequence encoding the light chain amino acid sequence according to any one of items 2, 3, 5, 6, 8, and 9 to 37. (Item 52) A composition comprising a first nucleic acid encoding a polypeptide selected from SEQ ID NOs: 109 to 216, and a second nucleic acid encoding a polypeptide selected from SEQ ID NOs: 325 to 432. (Item 53) 52. A vector comprising the nucleic acid according to any one of items 48 to 51. (Item 54) A cell comprising the nucleic acid of any one of items 48 to 51, the composition of item 52, or the vector of item 53, wherein the cell is a eukaryotic cell. (Item 55) 55. The cell of item 54, wherein the eukaryotic cell is a mammalian cell. (Item 56) 56. The cell of item 55, wherein the mammalian cell is selected from the group consisting of a CHO cell, a 293 cell, an NSO cell, a PER.C6 cell, and a B cell. (Item 57) 57. The cell of item 56, wherein the mammalian cell is a 293-6E cell or a DG44 cell. (Item 58) A cell producing the antibody or antigen-binding fragment thereof described in any one of items 1 to 45. (Item 59) 46. ​​A method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of items 1 to 45. (Item 60) 60. The method of claim 59, wherein the disease or disorder is associated with specific or aberrant expression of CLEC2D on various cell surfaces in a subject in need of treatment of the disease or disorder. (Item 61) 60. The method of claim 59, wherein the disease or disorder is associated with high expression of CLEC2D on various cell surfaces in a subject in need of treatment of the disease or disorder. (Item 62) 62. The method of any one of items 60 to 61, wherein the cell is an immune cell. (Item 63) 63. The method of claim 62, wherein the immune cells are NK cells. (Item 64) 62. The method of any one of items 60 to 61, wherein the cell is a cancer cell. (Item 65) 62. The method of any one of items 60 to 61, wherein the cell is a cell infected with a microorganism. (Item 66) 66. The method of claim 65, wherein the microorganism is a bacterium, a virus, a fungus, a parasitic microorganism, or a protozoan. (Item 67) 67. The method of claim 66, wherein the microorganism is an intracellular bacterium. (Item 68) The method according to any one of items 59 to 67, wherein the disease is rheumatoid arthritis. 70. The method of claim 68, wherein the subject exhibits bone loss resulting from having rheumatoid arthritis. (Item 70) 70. The method of claim 69, wherein administration of a therapeutically effective amount of the antibody or antigen-binding fragment thereof slows or reverses the bone loss in the subject. (Item 71) 68. The method of any one of items 59 to 67, wherein the disease is cancer. (Item 72) Item 73. The method according to item 71, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, esophageal cancer, squamous cell carcinoma, uveal melanoma, follicular lymphoma, renal cell carcinoma, cervical cancer, ovarian cancer, lung cancer, colorectal cancer, brain cancer, pancreatic cancer, head and neck cancer, liver cancer, leukemia, lymphoma, Hodgkin's disease, multiple myeloma, melanoma, astrocytoma, gastric cancer, and lung adenocarcinoma. 72. The method according to item 71, wherein the cancer is selected from the group consisting of adrenocortical carcinoma, bladder urothelial carcinoma, invasive breast carcinoma, cervical squamous cell carcinoma and adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, lymphoid tumor diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, chromophobe renal carcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain low grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, skin cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid carcinoma, thymoma, uterine endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma. (Item 74) 74. The method of any one of claims 71 to 73, wherein administration of a therapeutically effective amount of the antibody or antigen-binding fragment thereof results in an anti-tumor effect in the subject. (Item 75) 75. The method of any one of items 59 to 74, wherein administration of a therapeutically effective amount of the antibody or antigen-binding fragment thereof ameliorates a sign or symptom of the disease. (Item 76) The method of any one of items 59 to 75, wherein the therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of items 1 to 45 modulates or inhibits the interaction of CLEC2D with its associated receptor CD161. (Item 77) 46. ​​A method for modulating the activation of an immune response against a disease, disorder, or infection caused by a microorganism in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of items 1 to 45, wherein the immune response is an innate or adaptive immune response or a combination thereof. (Item 78) 46. ​​A method for increasing natural killer cell cytotoxicity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of items 1 to 45. (Item 79) An antibody library comprising at least about 10 8 unique monoclonal antibody clones, wherein at least about 80% of said antibody clones detectably and specifically bind to a CLEC2D antigen. (Item 80) 80. The antibody library according to Item 79, wherein the CLEC2D antigen comprises an amino acid sequence selected from SEQ ID NOs: 886 to 920 and 930 to 1003. (Item 81) The antibody library according to item 79 or 80, wherein the CLEC2D antigen comprises a CLEC2D antigen expressed on the surface of a tumor cell, a variant of the CLEC2D antigen, or a homolog of the CLEC2D antigen. (Item 82) 82. The antibody library of claim 81, wherein the variant of the CLEC2D antigen comprises a fragment of a CLEC2D protein. (Item 83) 83. The antibody library of claim 82, wherein the homolog of the CLEC2D antigen comprises human, mouse, dog, rat, or cynomolgus CLEC2D. (Item 84) 1. A method for screening a high diversity antibody gene library for antibodies that bind to a CLEC2D antigen, comprising: a) inserting a library of antibody genes into a phage protein gene to transform a plurality of phages to generate a phage library, wherein the phages in the phage library display the library of antibody genes on the surface of the phage; b) generating an enriched phage library enriched for antibody genes encoding antibodies that bind to the CLEC2D antigen by panning the phage library with the CLEC2D antigen against individual phages that bind to the CLEC2D antigen; c) repeating step (b) at least once or at least twice; d) introducing the antibody genes from the enriched phage library into a yeast surface display library; e) isolating individual yeast cells from said yeast surface display library that bind said CLEC2D antigen; f) culturing the isolated individual yeast cells that bind to the CLEC2D antigen to generate yeast surface display library clones; and g) sequencing the yeast surface display library clones; isolating an antibody gene that binds to the CLEC2D antigen by The method comprising: (Item 85) 85. The method of claim 84, wherein the panning step (b) comprises panning the phage library with CLEC2D-coated magnetic beads. (Item 86) 85. The method of claim 84, wherein the introducing step (d) comprises cloning the antibody gene into a yeast expression vector. (Item 87) 87. The method of claim 86, further comprising analyzing the surface expression of the antibody gene using a FLAG tag, a c-Myc tag, a polyhistidine tag, or a V5 tag. (Item 88) 85. The method according to Item 84, wherein the testing step (e) comprises isolating yeast cells expressing an antibody gene that binds to the CLEC2D antigen using flow cytometry. (Item 89) 90. The method of claim 88, further comprising repeating the flow cytometry isolation at least 1x, at least 2x, at least 3x, at least 4x, or at least 5x. (Item 90) 90. The method of any one of items 84 to 89, further comprising cloning the antibody gene that binds to the CLEC2D antigen into a mammalian expression vector. (Item 91) 1. A method for making a composition comprising an anti-CLEC2D antibody or antigen-binding fragment thereof, comprising: a) transforming a mammalian cell with a vector comprising a sequence encoding a promoter and a sequence encoding the anti-CLEC2D antibody or antigen-binding fragment thereof, wherein the sequence encoding the promoter and the sequence encoding the anti-CLEC2D antibody or antigen-binding fragment thereof are operably linked; b) culturing said mammalian cells under conditions suitable for expression of said anti-CLEC2D antibody or antigen-binding fragment thereof; and c) obtaining the anti-CLEC2D antibody or antigen-binding fragment thereof from the cultured mammalian cells and producing a supernatant; The method comprising: (Item 92) 92. The method of claim 91, wherein step (c) comprises recovering the supernatant of the cultured mammalian cells. (Item 93) 93. The method of claim 91 or 92, further comprising the step (d) filtering the supernatant after step (c). (Item 94) 94. The method of any one of claims 91 to 93, further comprising step (e) purifying the filtered supernatant. (Item 95) 1. A method for treating a disease in a subject in need thereof, comprising: a. measuring the level of CLEC2D protein in said subject; and b. administering to the subject a therapeutically effective amount of a CLEC2D antibody; The method comprising: (Item 96) 96. The method of claim 95, wherein the disease is cancer. (Item 97) 97. The method of claim 96, wherein the subject's cancer cells have high levels of CLEC2D protein when compared to normal non-cancer cells. (Item 98) 96. The method of item 95, wherein high levels of CLEC2D are associated with poor prognostic effect. (Item 99) 96. The method of item 95, wherein the disease is an autoimmune disorder or an inflammatory disorder.

[0073] The features of the present disclosure will become more fully apparent from the following description taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee. The present disclosure is further described using the accompanying drawings, with the understanding that the figures represent only some embodiments according to the present disclosure and are not to be considered as limiting the scope of the present disclosure. [Brief description of the drawings]

[0074] [Figure 1] AC show the present disclosure in a schematic format. A shows a scenario in which CLEC2D and CD161 interact, resulting in immune cell evasion of tumor cells. B shows a scenario in which the interaction between CLEC2D and CD161 is blocked using an anti-CLEC2D antibody, resulting in tumor cell killing following a lytic signal. C shows a scenario in which binding of the CLEC2D antigen with an anti-CLEC2D antibody results in activation of NK cells and increased cytokine expression, followed by enhanced target cell elimination, either by direct killing or by the involvement of other immune cells. [Figure 2A] Figure 1 shows the expression and purification of CLEC2D antigen in mammalian cells. Figure 2 shows the construction of a mammalian expression plasmid for expressing the CLEC2D ectodomain as a soluble antigen. The construct was made by gene synthesis and then verified by restriction digestion and Sanger sequencing. [Figure 2B] Figure 1 shows the expression and purification of CLEC2D antigen in mammalian cells.IMAC chromatography profile showing purification of soluble CLEC2D (Q72-V191) (inset shows elution profile of CLEC2D antigen). [Figure 2C] Figure 1 shows the expression and purification of CLEC2D antigen in mammalian cells. SDS-PAGE profiles of the loaded, washed and finally eluted CLEC2D protein are shown, indicating that the purified CLEC2D protein is homogenous and pure and suitable for further downstream experiments. [Figure 2D] Figure 2 shows expression and purification of the CLEC2D antigen in mammalian cells.Figure 3 shows a Western blot of purified CLEC2D protein probed with a commercially available antibody against the CLEC2D antigen. [Figure 2E] Figure 1 shows the expression and purification of CLEC2D antigen in mammalian cells.Figure 2 shows an ELISA assay showing the binding specificity of a commercial antibody to different concentrations of purified CLEC2D antigen. [Figure 2F] Figure 2 shows expression and purification of CLEC2D antigen in mammalian cells. Figure 3 shows SDS-PAGE analysis of purified CLEC2D antigen incubated with PNGase enzyme for 3 or 6 hours under reducing conditions, revealing deglycosylation of CLEC2D antigen. [Diagram 3] FIG. 1 shows a schematic of an antibody library screening strategy to screen a naive antibody library against a target CLEC2D antigen using phage and yeast surface display systems. [Figure 4A] Figure 1 shows phage panning of an antibody library using CLEC2D antigen coated on magnetic beads. Figure 2 shows estimation of magnetic bead conjugation efficiency by flow cytometry. [Figure 4B] Figure 1 shows phage panning of an antibody library using CLEC2D antigen coated on magnetic beads.Restriction enzyme digestion of individual heavy chain clones after panning of a Fab library. [Figure 4C] Figure 1 shows phage panning of an antibody library using CLEC2D antigen coated on magnetic beads.Restriction enzyme digestion of individual kappa light chain clones after panning of a Fab library. [Figure 4D] Figure 1 shows phage panning of an antibody library using CLEC2D antigen coated on magnetic beads.Figure 2 shows restriction enzyme digestion of individual heavy chain clones after panning of an ScFv library. [Figure 4E]Figure 1 shows phage panning of an antibody library using CLEC2D antigen coated on magnetic beads.Figure 2 shows restriction enzyme digestion of individual kappa light chain clones after panning of an ScFv library. [Figure 5A] 1 shows screening of antibodies against CLEC2D using yeast surface display, and plate images depicting yeast colonies for generation of an ScFv antibody library by electroporation. [Figure 5B] 1 shows screening of antibodies against CLEC2D using yeast surface display. 2 shows plate images depicting the generation of haploid heavy and light chain antibody libraries. [Figure 5C] 1 shows screening of antibodies against CLEC2D using yeast surface display. Plate images showing mating efficiency of haploid yeast strains containing heavy or light chain antibody libraries are shown (mating efficiency was estimated to be about 29%). [Figure 5D] 1 shows the screening of antibodies against CLEC2D using yeast surface display. A representative flow cytometry analysis of binding of antibody molecules expressed on the surface of yeast cells to the CLEC2D antigen is shown (the ScFv library was sorted multiple times to enrich for high affinity yeast clones). [Figure 5E] Screening of antibodies against CLEC2D using yeast surface display. Representative flow cytometry analysis of binding of antibody molecules expressed on the surface of yeast cells to the CLEC2D antigen (Fab library was sorted multiple times to enrich for high affinity yeast clones). [Figure 5F] 1 shows the screening of antibodies against CLEC2D using yeast surface display. Representative data on the enrichment of yeast clones after multiple rounds of sorting, both in terms of expression and antigen recognition, are shown. [Figure 5G] 1 shows screening of antibodies against CLEC2D using yeast surface display. Individual yeast clones were isolated and tested with the CLEC2D antigen to identify yeast cell lines expressing high affinity antibody clones. [Figure 5H] Screening of antibodies against CLEC2D using yeast surface display. Representative flow cytometry data is shown to show the percentage of binding of soluble CLEC2D antigen to monoclonal antibody clones. At least about 80% of the clones detectably and specifically bound to the CLEC2D antigen. [Figure 6A] Figure 1 shows the peer group sequence analysis of clones screened by yeast display platform.Figure 2 shows a bar graph depicting the CDRH3 length distribution of selected molecules. [Figure 6B] Peer group sequence analysis of clones screened by yeast display platform.Bar graph showing relative amino acid frequency distribution of heavy chain CDRH3 (Kabat nomenclature). [Figure 6C] Figure 1 shows peer group sequence analysis of clones screened by yeast display platform.Figure 2 shows a pie chart depicting heavy chain consensus family distribution. [Figure 6D] Figure 1 shows peer group sequence analysis of clones screened by yeast display platform.Figure 2 shows a pie chart depicting light chain consensus family distribution. [Figure 7A] A shows mammalian expression constructs used to generate full-length monoclonal antibodies. A vector designed for cloning selected antibody variable heavy chain genes after screening with phage and yeast display platforms. Constructs were generated by gene synthesis and then confirmed by restriction digestion and Sanger sequencing. B shows mammalian expression constructs used to generate full-length monoclonal antibodies. A vector designed for cloning selected antibody variable light chain (kappa) genes after screening with phage and yeast display platforms. Constructs were generated by gene synthesis and then confirmed by restriction digestion and Sanger sequencing. [Figure 7B]A shows mammalian expression constructs used to generate full-length monoclonal antibodies. A vector designed for cloning selected antibody variable heavy chain genes after screening with phage and yeast display platforms. Constructs were generated by gene synthesis and then confirmed by restriction digestion and Sanger sequencing. B shows mammalian expression constructs used to generate full-length monoclonal antibodies. A vector designed for cloning selected antibody variable light chain (kappa) genes after screening with phage and yeast display platforms. Constructs were generated by gene synthesis and then confirmed by restriction digestion and Sanger sequencing. [Figure 8A] Figure 1 shows a mammalian expression system for expressing full-length CLEC2D on the cell surface. A CLEC2D gene expression construct was generated by gene synthesis and then confirmed by restriction digestion and Sanger sequencing. [Figure 8B] Figure 1 shows a mammalian expression system for expressing full-length CLEC2D on the cell surface.Figure 2 shows flow cytometry using a commercially available anti-CLEC2D antibody (4C7) showing expression of CLEC2D on the surface of transfected CHO cells (C4548). [Figure 8C] Figure 1 shows a mammalian expression system for expressing full-length CLEC2D on the cell surface. Surface expression of CLEC2D monitored with anti-CLEC2D (4C7) antibody on fixed and non-permeabilized cells by confocal microscopy (60x). Binding of anti-CLEC2D antibody was observed on C4548 cells, whereas no binding was observed on non-transfected CHO cells. Nuclei were counterstained with DAPI (blue). Scale bar is 10 μm. [Figure 9A]Anti-CLEC2D monoclonal antibody clones purified from transiently transfected CHO cells are shown. Antibodies were purified using protein A column chromatography. Representative SDS-PAGE profiles of anti-CLEC2D antibodies are shown. Purified antibodies were subjected to SDS-PAGE analysis under both non-reducing and reducing conditions. Anti-CLEC2D antibody clones purified from C3566 are shown in lane 9, top panel in reducing and non-reducing gels. All clones in the bottom panel except for the clone in lane 4 revealed good profiles in reducing and non-reducing gels. Clones showing degradation products were not considered for further studies. Similar criteria were adopted for other clones as described in the Examples section. [Figure 9B] Figure 1 shows anti-CLEC2D monoclonal antibody clones purified from transiently transfected CHO cells. Antibodies were purified using Protein A column chromatography. Figure 2 shows the interaction of purified anti-CLEC2D antibodies with CLEC2D antigen expressed on the surface of CHO cells by flow cytometry. Representative antibody clones exemplified by C4577, C2907, C3566, C5582, C5397 were evaluated for CLEC2D binding on CHO cell lines either untransfected or transfected with full-length CLEC2D constructs. A shift in MFI to the right indicated binding of the corresponding clone to surface-expressed CLEC2D antigen. [Figure 9C-1]Anti-CLEC2D monoclonal antibody clones purified from transiently transfected CHO cells. Antibodies were purified using protein A column chromatography. Representative images of anti-CLEC2D antibody interaction with CLEC2D antigen expressed on PC3 tumor cells are shown. Qualitative assessment of binding was performed (from "+" indicating low binding to "+++" indicating very high binding) as shown in Table 22. As an example, antibody C4252 showed no detectable surface binding, whereas antibody C0610 showed low binding and was therefore rated (+), while other clones showed specific and even significant surface binding. Nuclei were counterstained with DAPI (purple). Scale bar is 10 μm. [Figure 9C-2] Anti-CLEC2D monoclonal antibody clones purified from transiently transfected CHO cells. Antibodies were purified using protein A column chromatography. Representative images of anti-CLEC2D antibody interaction with CLEC2D antigen expressed on PC3 tumor cells are shown. Qualitative assessment of binding was performed (from "+" indicating low binding to "+++" indicating very high binding) as shown in Table 22. As an example, antibody C4252 showed no detectable surface binding, whereas antibody C0610 showed low binding and was therefore rated (+), while other clones showed specific and even significant surface binding. Nuclei were counterstained with DAPI (purple). Scale bar is 10 μm. [Figure 10A] Figure 1 shows the development of a stable CHO cell line expressing an anti-CLEC2D antibody. Binding studies performed with surface-expressed CLEC2D and monitored using flow cytometry with supernatants from CHO minipool samples transfected with anti-CLEC2D antibody expression plasmids. Histograms represent the degree of binding observed in different clones to surface CLEC2D antigen expressed on C4548 cells. Fold change in MFI is plotted against binding of individual minipools. Higher fold change indicates stronger binding of anti-CLEC2D antibody to CLEC2D antigen. [Figure 10B]Figure 1 shows the development of a stable CHO cell line expressing anti-CLEC2D antibody. Single cell clone screening is shown (anti-CLEC2D antibody expressed from single cell clone line was purified and then used for flow cytometry experiments). Higher fold change in fluorescent signal indicates stronger binding of anti-CLEC2D antibody to CLEC2D antigen. [Figure 10C] Development of stable CHO cell lines expressing anti-CLEC2D antibodies. Flow cytometry analysis of monoclonal antibodies produced from stable CHO cell lines is shown (anti-CLEC2D antibodies expressed from single cell clonal lines were purified and then used for flow cytometry experiments). Anti-CLEC2D antibodies expressed by multiple monoclonal cell lines (e.g., C4608, C5093, C5511, C6481, C6726, C7720, C9103, C5848, and C3452, etc.) were tested for binding to the CLEC2D antigen expressed on the surface of CHO cells by flow cytometry. Estimates of the fold increase in mean fluorescence intensity in multiple stable clones were observed to range from 3 to 10 fold. [Figure 10D] 1 shows the development of a stable CHO cell line expressing an anti-CLEC2D antibody. Representative images of the interaction of anti-CLEC2D monoclonal antibodies produced from clonal CHO cell lines with the CLEC2D antigen expressed on a PC3 tumor cell line are shown. As described herein, various anti-CLEC2D antibodies showed specific and significant surface binding to the CLEC2D antigen on the surface of PC3 cells. Nuclei were counterstained with DAPI (purple). Scale bar is 10 μm. [Figure 10E] Figure 1 shows the development of a stable CHO cell line expressing an anti-CLEC2D antibody. Quantitative RT PCR was performed on anti-CLEC2D antibody-stable cell clones C4608 and C5511 to confirm stable transfer of antibody heavy and light chain genes. GAPDH housekeeping gene was used as an internal standard. 60 generations of CHO monoclonal lines expressing anti-CLEC2D antibody were tested. [Figure 11A]1 shows functional characterization of monoclonal anti-CLEC2D antibodies. Anti-CLEC2D antibodies C4608, C5511, C6481, C2438, C3452, C0949 bind to surface-expressed CLEC2D on the prostate cancer cell line PC3. A shift in MFI to the right indicates antibody binding to the surface-expressed CLEC2D antigen on the PC3 cell line. [Figure 11B] Figure 1 shows functional characterization of monoclonal anti-CLEC2D antibodies. Representative flow cytometry analysis of cytotoxicity assays performed on PC3 target cells using PBMCs as effector cells at a ratio of 1:5 and a fixed concentration of 100ug / mL of anti-CLEC2D antibodies. Clones evaluated here for functionality were C5511, C4608, and C6481 with PBMCs from donor 1, while antibodies purified from clones C5392 and C3452 were tested with PBMCs from donor 2. The percentage of PC3 live cells is indicated by APC (eFluor 670) positive cells, and dead cells indicate Sytox green positive cells. Corresponding single cell clones were labeled for each plot. [Figure 11C] Figure 1 shows functional characterization of monoclonal anti-CLEC2D antibodies.Representative flow cytometry analysis of cytotoxicity assays performed on PC3 target cells using PBMCs as effector cells (1:5) with increasing concentrations of anti-CLEC2D antibody (C5511) from 10μg / mL to 200ug / mL, revealing high dose-dependent tumor cell cytotoxicity. [Figure 11D] Figure 1 shows functional characterization of monoclonal anti-CLEC2D antibodies. Representative flow cytometry analysis of cytotoxicity assays performed against PC3 target cells using PBMCs as effector cells and a fixed concentration of anti-CLEC2D antibody C5511. The tumor:effector cell ratio (T:E) was increased from 1:5 to 1:10. Data revealed that increasing the effector cell ratio resulted in higher levels of tumor cell cytotoxicity. [Figure 11E-1]Functional characterization of monoclonal anti-CLEC2D antibodies. End-point cytotoxicity assays reveal significant cytotoxicity of tumor cells at 10 μg / mL. The assay also uses confocal microscopy to identify optimal concentrations of anti-CLEC2D antibodies to kill target cells. The top panel shows all control treatments with no cytotoxicity as expected, and the bottom panel shows high PC3 tumor cell death upon treatment with increasing concentrations of anti-CLEC2D antibody (C6726) in the presence of PBMCs (T:E=1:5). Maximum cell death was observed at a concentration of 50ug / ml of anti-CLEC2D antibody. PC3 tumor cells-green, PBMCs-red, dead cells-blue. [Figure 11E-2] Functional characterization of monoclonal anti-CLEC2D antibodies. End-point cytotoxicity assays reveal significant cytotoxicity of tumor cells at 10 μg / mL. The assay also uses confocal microscopy to identify optimal concentrations of anti-CLEC2D antibodies to kill target cells. The top panel shows all control treatments with no cytotoxicity as expected, and the bottom panel shows high PC3 tumor cell death upon treatment with increasing concentrations of anti-CLEC2D antibody (C6726) in the presence of PBMCs (T:E=1:5). Maximum cell death was observed at a concentration of 50ug / ml of anti-CLEC2D antibody. PC3 tumor cells-green, PBMCs-red, dead cells-blue. [Figure 11F-1] Functional characterization of monoclonal anti-CLEC2D antibodies. End-point cytotoxicity assay using selected anti-CLEC2D antibodies to kill target cells using confocal microscopy. No cytotoxicity was observed in control treatments such as PC3 tumor cells alone, PBMCs alone, and PBMCs with isotype human IgG1 antibody. Cytotoxicity of PC3 tumor cells was observed using anti-CLEC2D antibody (C6726, C5848, C4608, C5511, and C6481) clones. Enlarged images revealed that PC3 tumor cells were surrounded by effector cells inducing tumor cell death. PC3 tumor cells-green, PBMCs-red, dead cells-blue. [Figure 11F-2] Functional characterization of monoclonal anti-CLEC2D antibodies. End-point cytotoxicity assay using selected anti-CLEC2D antibodies to kill target cells using confocal microscopy. No cytotoxicity was observed in control treatments such as PC3 tumor cells alone, PBMCs alone, and PBMCs with isotype human IgG1 antibody. Cytotoxicity of PC3 tumor cells was observed using anti-CLEC2D antibody (C6726, C5848, C4608, C5511, and C6481) clones. Enlarged images revealed that PC3 tumor cells were surrounded by effector cells inducing tumor cell death. PC3 tumor cells-green, PBMCs-red, dead cells-blue. [Figure 12A] Figure 1 shows NK cell mediated cytotoxicity of tumor cells using anti-CLEC2D antibodies. Cytotoxicity of PC3 tumor cells upon treatment with purified NK cells and 100ug / ml of anti-CLEC2D antibodies (C6481 and C5511). Data revealed 86% NK cell mediated cytotoxicity of PC3 tumor cells in 1:1 T:E. Percentage of PC3 dead cells indicates Sytox green positive cells. [Figure 12B] NK cell-mediated cytotoxicity of tumor cells with anti-CLEC2D antibody. No target cell death was observed when cultured with either isotype control (human IgG1 antibody) or NK cells alone at increasing T:E ratios starting from 1:0.5 to 1:10. Scale bar is 10 μm. [Figure 12C] NK cell-mediated cytotoxicity of tumor cells using anti-CLEC2D antibodies. Anti-CLEC2D antibodies alone are unable to induce cytotoxicity of PC3 tumor cells. Scale bar is 10 μm. [Figure 12D] Figure 1 shows NK cell-mediated cytotoxicity of tumor cells using anti-CLEC2D antibodies. Anti-CLEC2D antibody C5511 (50ug / mL) revealed increasingly more PC3 tumor cell death with increasing T:E ratio starting from 1:0.5 to 1:5 and 1:10. Scale bar is 10μm. [Figure 13] Cytotoxicity of isolated T cells and PC3 tumor cells treated with 100ug / ml of anti-CLEC2D antibodies (C5511 and C6481) is shown. The percentage of dead PC3 tumor cells was indicated by Sytox green positive cells. [Figure 14A] Live cell imaging with anti-CLEC2D antibody-dependent cellular cytotoxicity of PC3 tumor cells. Live cell imaging reveals cytotoxicity of PC3 tumor cells over the incubation period with human PBMC cells and 200 μg / ml of anti-CLEC2D antibody. The assay was performed for 20 hours in a humidified chamber maintained at 37° C. and 5% CO2 during image acquisition. In contrast, incubation with a control human IgG1 antibody (200 μg / ml) did not result in cytotoxicity of tumor cells. Live PC3 tumor cells-green, PBMC-red, dead cells-blue. Scale bar is 20 μm. [Figure 14B] Live cell imaging with anti-CLEC2D antibody-dependent cellular cytotoxicity of PC3 tumor cells. Live cell imaging reveals cytotoxicity of PC3 tumor cells over the incubation period with human NK cells and 200 μg / ml of anti-CLEC2D antibody. The assay was performed for 20 hours in a humidified chamber maintained at 37° C. and 5% CO2 during image acquisition. In contrast, incubation with a control human IgG1 antibody (200 μg / ml) did not result in cytotoxicity of tumor cells. Live PC3 tumor cells-green, NK cells-red, dead cells-blue. Scale bar is 20 μm. [Figure 15A] A predicted model of the anti-CLEC2D antibody is shown. A cartoon representation of the epitope recognition (chain A - dark blue, chain B - cyan) & CD161 (chain C - orange red, chain D - purple) complex (PDB ID 5MGT) is shown. [Figure 15B] A predicted model of the anti-CLEC2D antibody is shown, with the red selection representing residues within 6 Å of the NKR-P1 chain. [Figure 15C]1 shows a predicted model of an anti-CLEC2D antibody. 2 shows a ribbon representation of the purified anti-CLEC2D antibody structure. 3 shows the corresponding clones for a particular anti-CLEC2D monoclonal antibody appropriately labeled. 4 shows the variable light chain in a darker shade, while the heavy chain variable region is in white. [Figure 15D] 1 shows the predicted model of anti-CLEC2D antibodies. Selected structures after PIZSA scoring and structure clustering method that belong to C4608 and contribute to one of the interacting clusters towards CLEC2D (darker shade). [Figure 15E] 1 shows a predicted model of an anti-CLEC2D antibody, and a visualization of residues selected for mutation to confirm whether the combination of G00001-G00004-G00007-G00010-G00015 clusters derived from C4608 contains a binding site for the CLEC2D antigen. [Figure 15F] 1 shows the predicted model of anti-CLEC2D antibodies. Selected structures after PIZSA scoring and structure clustering method that belong to C5511 and contribute to one of the interacting clusters for CLEC2D (darker shade). [Figure 15G] 1 shows a predicted model of an anti-CLEC2D antibody, and a visualization of residues selected for mutation to confirm whether the combination of the G00001-G00005-G00011-G00019-G00020 cluster derived from C5511 contains a binding site for the CLEC2D antigen. [Figure 16A] Figure 1 shows the identified epitope sections on the CLEC2D antigen for anti-CLEC2D antibody clones C4608 and C5511. Figure 2 shows a surface representation of the contact points of anti-CLEC2D antibody C4608 with the CLEC2D antigen. In the depiction, darker shading indicates the location of residues that interact with the CLEC2D antigen. [Figure 16B]1 shows the identified epitope sections on the CLEC2D antigen for anti-CLEC2D antibody clones C4608 and C5511. The contact points of anti-CLEC2D antibody C4608 with the CLEC2D antigen overlap with the CD161 binding region on CLEC2D. In the depiction, darker shading indicates the location of residues that interact with the CLEC2D antigen. [Figure 16C] Figure 1 shows the identified epitope sections on the CLEC2D antigen for anti-CLEC2D antibody clones C4608 and C5511. Figure 2 shows a surface representation of the contact points of anti-CLEC2D antibody C5511 with the CLEC2D antigen. In the depiction, darker shading indicates the location of residues that interact with the CLEC2D antigen. [Figure 16D] 1 shows the identified epitope sections on the CLEC2D antigen for anti-CLEC2D antibody clones C4608 and C5511. The contact points of anti-CLEC2D antibody C5511 with the CLEC2D antigen overlap with the CD161 binding region on CLEC2D. In the depiction, darker shading indicates the location of residues that interact with the CLEC2D antigen. [Figure 16E] Figure 1 shows the identified epitope compartments on the CLEC2D antigen for anti-CLEC2D antibody clones C4608 and C5511. Figure 2 shows anti-CLEC2D antibody mediated inhibition of the interaction of CLEC2D and CD161 (monitoring confirmation of CLEC2D antigen bead conjugate efficiency). [Figure 16F] 1 shows the epitope sections identified on the CLEC2D antigen for the anti-CLEC2D antibody clones C4608 and C5511. It shows that binding of CD161-FC to the CLEC2D antigen on magnetic beads was observed in a concentration-dependent manner. [Figure 16G] Figure 1 shows the identified epitope compartments on the CLEC2D antigen for anti-CLEC2D antibody clones C4608 and C5511. Flow cytometric monitoring of CD161 binding in the presence or absence of anti-CLEC2D antibodies compared to controls as an indication of inhibition of CD161 and CLEC2D binding, indicated by solid black arrows. [Figure 17A]Figure 1 shows NK cell activation by anti-CLEC2D antibody. Figure 2 shows that anti-CLEC2D antibody C5511 induces CD69 expression, indicating that NK cell activation results in cytotoxicity. The corresponding experimental conditions are described for each plot. IL2 treatment was performed as a positive control for CD69 overexpression. [Figure 17B] Figure 2 shows NK cell activation by anti-CLEC2D antibody, showing that anti-CLEC2D antibody-mediated CD69 expression is higher on NK cells compared to PC3 cell-primed CD69 expression levels. [Figure 18A] 1 shows the effect of anti-CLEC2D antibody C5511 on cytokine expression by effector cells, and shows that increased IFNγ expression levels were monitored using anti-CLEC2D antibody C5511 at concentrations of 10 μg / mL and 100 μg / mL. [Figure 18B] Figure 1 shows the effect of anti-CLEC2D antibody C5511 on cytokine expression by effector cells. Anti-CLEC2D antibody C5511 was used at a concentration of 100ug / mL in the presence or absence of PC3 cells (E:T=10:1). IFNγ expression of the CD3+ gated population was monitored. [Figure 18C] Figure 1 shows the effect of anti-CLEC2D antibody C5511 on cytokine expression by effector cells. Anti-CLEC2D antibody C5511 was used at a concentration of 100ug / mL in the presence or absence of PC3 cells (E:T=10:1). IFNγ expression of the CD3-gated population was monitored. [Figure 18D] 1 shows the effect of anti-CLEC2D antibody C5511 on cytokine expression by effector cells. Anti-CLEC2D antibody C5511 was used at a concentration of 100 μg / mL in the presence or absence of isolated NK cells. IFNγ overexpression was observed with anti-CLEC2D antibody C5511. [Figure 19A] Figure 1 shows mammalian expression constructs used to generate full-length monoclonal antibodies. Constructs were generated by gene synthesis and then verified by restriction digestion and Sanger sequencing. Figure 2 shows vectors designed for cloning select antibody variable heavy chain genes into an IgG4 backbone. [Figure 19B] Figure 1 shows mammalian expression constructs used to generate full-length monoclonal antibodies. Constructs were generated by gene synthesis and then verified by restriction digestion and Sanger sequencing. Figure 2 shows vectors designed for cloning select antibody variable heavy chain genes into an IgG1 N-A backbone. [Figure 19C] Figure 1 shows mammalian expression constructs used to generate full-length monoclonal antibodies. Constructs were generated by gene synthesis and then verified by restriction digestion and Sanger sequencing. Flow cytometry analysis of binding of anti-CLEC2D antibodies with IgG4 isotype backbone (C3256 and C3276) to CLEC2D antigen expressed on the surface of CHO cells. Binding estimated from peak shift to the right was compared to non-transfected CHO cells. [Figure 19D] Figure 1 shows mammalian expression constructs used to generate full-length monoclonal antibodies. Constructs were generated by gene synthesis and then confirmed by restriction digestion and Sanger sequencing. Cytotoxicity of anti-CLEC2D antibodies using various antibody isotypes. IgG1 (C3452 and C4608) and IgG4 (C3256 and C3276) anti-CLEC2D antibodies showed significant cytotoxicity when cultured with freshly isolated PBMCs and PC3 tumor cells. [Figure 19E] Figure 1 shows mammalian expression constructs used to generate full-length monoclonal antibodies. Constructs were generated by gene synthesis and then verified by restriction digestion and Sanger sequencing. Figure 2 shows that anti-CLEC2D antibodies generated as afucosylated monoclonal antibodies C0613, C1301, C6268, C1699, C2437, C9832, C8900, and C7749 demonstrated binding to the CLEC2D antigen expressed on the surface of CHO cells using flow cytometry. [Figure 19F]Figure 1 shows mammalian expression constructs used to generate full-length monoclonal antibodies. Constructs were generated by gene synthesis and then confirmed by restriction digestion and Sanger sequencing. Figure 1 shows NK cell-mediated cytotoxicity of PC3 tumor cells by afucosylated anti-CLEC2D antibodies (C7749, C8800, C9832) used at 5x lower concentrations compared to C5511. Data revealed that afucosylated anti-CLEC2D antibodies achieved nearly equivalent cell killing at 5x lower concentrations, indicating that afucosylated anti-CLEC2D antibodies are more cytotoxic. [Figure 19G] Figure 1 shows the mammalian expression constructs used to generate full-length monoclonal antibodies. Constructs were generated by gene synthesis and then verified by restriction digestion and Sanger sequencing. Ramos and PC3 tumor cell lines were used to measure CDC-mediated cytotoxicity of anti-CLEC2D antibody C5511. Rituximab was used as a positive control. [Figure 20A] Figure 1 shows anti-tumor effects in a cancer xenograft mouse model. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. Plots of tumor volume versus time are shown showing the significant anti-tumor effects observed with anti-CLEC2D antibodies alone or in combination with anti-PDL1 antibodies. [Figure 20B] Antitumor efficacy in a cancer xenograft mouse model. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. Images showing immune cell infiltration in the tumor microenvironment by staining for CD3+ T cells are shown. [Figure 20C] Figure 1 shows anti-tumor efficacy in a cancer xenograft mouse model. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. Images of xenograft-bearing mice are shown showing Alexa 647-labeled anti-CLEC2D antibodies injected intratumorally over a 96 hour period. [Figure 20D]Antitumor efficacy in a cancer xenograft mouse model. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. The effect of test compounds on tumor volume in humanized (huNOG-EXL) mice bearing subcutaneous PC-3 tumor xenografts is shown (up to day 36). Each treatment group consisted of 5 animals and was named the C5511 mAb group, the solvent control IgG1 group, and the C6481 mAb group. Values ​​are represented as the mean of 2-5 animals in each group. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test using Graph Pad Prism (version 8.3.0). **p<0.01, statistically significant (day 36) when comparing the C5511 mAb group with the solvent control IgG1 group. [Figure 20E] Antitumor effects in a cancer xenograft mouse model are shown. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. The effect of test compounds on tumor volume in humanized (huNOG-EXL) mice bearing subcutaneous PC-3 tumor xenografts is shown (up to day 24). Each treatment group consisted of 5 animals and was named the C5511 mAb group, the vehicle control IgG1 group, and the C6481 mAb group. Values ​​are represented as the mean of 2-5 animals in each group. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test using Graph Pad Prism (version 8.3.0). ***p<0.001 and *p<0.05, statistically significant (day 24) when comparing the C5511 mAb group and the C6481 mAb group with the vehicle control IgG1 group, respectively. [Figure 20F]Antitumor efficacy in a cancer xenograft mouse model. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. Effect of test compounds on delta tumor volume in humanized (huNOG-EXL) mice bearing subcutaneous PC-3 tumor xenografts (up to day 36). Each treatment group consisted of 5 animals and was named C5511 mAb group, solvent control IgG1 group, and C6481 mAb group. Values ​​are represented as the mean of 2-5 animals in each group. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test using Graph Pad Prism (version 8.3.0). **p<0.01, statistically significant (day 36) when comparing C5511 mAb group with solvent control IgG1 group. [Figure 20G] Antitumor efficacy in a cancer xenograft mouse model. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. Effect of test compounds on delta tumor volume in humanized (huNOG-EXL) mice bearing subcutaneous PC-3 tumor xenografts (up to day 24). Each treatment group consisted of 5 animals and was named C5511 mAb group, vehicle control IgG1 group, and C6481 mAb group. Values ​​are represented as the mean of 2-5 animals in each group. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test using Graph Pad Prism (version 8.3.0). ***p<0.001 and *p<0.05, statistically significant (day 24) when comparing C5511 mAb group and C6481 mAb group with vehicle control IgG1 group, respectively. [Figure 20H]Antitumor efficacy in a cancer xenograft mouse model is shown. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. The effect of test compounds on relative tumor volume in humanized (huNOG-EXL) mice bearing subcutaneous PC-3 tumor xenografts is shown (up to day 36). Each treatment group consisted of 5 animals and was named the C5511 mAb group, the solvent control IgG1 group, and the C6481 mAb group. Values ​​are represented as the mean of 2-5 animals in each group. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test using Graph Pad Prism (version 8.3.0). *p<0.05, statistically significant (day 36) when comparing the C5511 mAb group with the solvent control IgG1 group. [Figure 20I] Antitumor efficacy in a cancer xenograft mouse model. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. The effect of test compounds on relative tumor volume in humanized (huNOG-EXL) mice bearing subcutaneous PC-3 tumor xenografts is shown (up to day 24). Each treatment group consisted of 5 animals and was named the C5511 mAb group, the vehicle control IgG1 group, and the C6481 mAb group. Values ​​are represented as the mean of 2-5 animals in each group. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test using Graph Pad Prism (version 8.3.0). ***p<0.001 and *p<0.05, statistically significant (day 24) when comparing the C5511 mAb group and the C6481 mAb group with the vehicle control IgG1 group, respectively. [Figure 20J]Antitumor efficacy in a cancer xenograft mouse model is shown. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. The effect of test compounds on delta relative tumor volume in humanized (huNOG-EXL) mice bearing subcutaneous PC-3 tumor xenografts is shown (up to day 36). Each treatment group consisted of 5 animals and was named the C5511 mAb group, the solvent control IgG1 group, and the C6481 mAb group. Values ​​are represented as the mean of 2-5 animals in each group. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test using Graph Pad Prism (version 8.3.0). *p<0.05, statistically significant (day 36) when comparing the C5511 mAb group with the solvent control IgG1 group. [Figure 20K] Antitumor efficacy in a cancer xenograft mouse model. huNOG-EXL mice were used for PC3 xenografts, and tumor-bearing animals were randomized and used to inject anti-CLEC2D antibody products. Effect of test compounds on delta relative tumor volume in humanized (huNOG-EXL) mice bearing subcutaneous PC-3 tumor xenografts (up to day 24). Each treatment group consisted of 5 animals and was named C5511 mAb group, vehicle control IgG1 group, and C6481 mAb group. Values ​​are represented as the mean of 2-5 animals in each group. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-hoc test using Graph Pad Prism (version 8.3.0). ***p<0.001 and *p<0.05, statistically significant (day 24) when comparing C5511 mAb group and C6481 mAb group with vehicle control IgG1 group, respectively. [Figure 21A] Figure 2 shows the characterization of purified anti-CLEC2D antibody products.Figure 2 shows SDS-PAGE analysis of purified C5511 antibody under non-reducing and reducing conditions. [Figure 21B] Characterization of purified anti-CLEC2D antibody products.TIC chromatograms (three replicates) of intact mass spectrometry analysis of anti-CLEC2D antibodies are shown. [Figure 21C] Characterization of purified anti-CLEC2D antibody products. WCX chromatogram analysis of anti-CLEC2D antibodies. [Figure 21D] 1 shows the characterization of purified anti-CLEC2D antibody products. 2 shows size exclusion chromatograms of anti-CLEC2D antibodies. [Figure 21E] Characterization of purified anti-CLEC2D antibody products is shown. ELISA assay expression of anti-CLEC2D antibodies against CLEC2D purified biotinylated antigen is shown. Data was fitted to a one-site binding model to calculate the Kd of anti-CLEC2D antibodies. CLEC2D antigen affinity-based binding studies of representative anti-CLEC2D antibodies are shown. [Figure 21F] 1 shows characterization of purified anti-CLEC2D antibody products. 1 shows CLEC2D antigen affinity-based binding studies of representative anti-CLEC2D antibodies. [Figure 21G] Figure 1 shows the characterization of purified anti-CLEC2D antibody products. Purified CLEC2D antigen ectodomain was used as a source of antigen in the BIACORE assay. Monitored responses are plotted against time. [Fig. 21H] Figure 2 shows characterization of purified anti-CLEC2D antibody products. Figure 3 shows affinity-based binding studies of representative anti-CLEC2D antibody molecules with FcRn at pH 5.9. [Figure 21I] Figure 2 shows characterization of purified anti-CLEC2D antibody products. Figure 3 shows affinity-based binding studies of representative anti-CLEC2D antibody molecules with FcRn at pH 7.4. [Figure 22A] Anti-CLEC2D antibodies for valid diagnostic and prognostic applications. Selection of an anti-CLEC2D antibody (C0949) based on binding characteristics. Four anti-CLEC2D antibodies (C2779, C2438, C0949, and C2543) were evaluated for binding to CLEC2D on PC3 target cells. C0949 showed good binding and a shift to the center of the peak. [Figure 22B] Figure 1 shows anti-CLEC2D antibodies for valid diagnostic and prognostic applications. Figure 2 shows that anti-CLEC2D antibody C0949 recognizes the CLEC2D antigen on multiple prostate cancer cell lines. [Figure 22C] Figure 2 shows anti-CLEC2D antibodies for valid diagnostic and prognostic applications. Figure 2 shows that anti-CLEC2D antibody C0949 recognizes the CLEC2D antigen on multiple tumor cell lines. Specific binding and fold change in mean fluorescence were calculated by the ratio of mean FITC fluorescence between test and control. [Figure 23A] Figure 2 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on prostate cancer tumor cells.Figure 3 shows the expression levels of CLEC2D antigen in prostate cancer disease stages after TCGA data analysis. [Figure 23B] 1 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on prostate cancer tumor cells. 2 shows the expression levels of the CLEC2D antigen on prostate cancer cell lines PC3, DU145, 22RV1, and LnCap. [Figure 23C-1] 1 shows that anti-CLEC2D antibody recognizes CLEC2D antigen on prostate cancer tumor cells. CLEC2D antigen expression levels on prostate cancer cell lines PC3, LnCap, 22RV1, and DU145 induced using LPS, poly I:C, IFN-γ, PBMC supernatant, PBMC cells, NK cells, and T cells. The upper panel shows anti-CLEC2D antibody, and the lower panel shows merged images. [Figure 23C-2] 1 shows that anti-CLEC2D antibody recognizes CLEC2D antigen on prostate cancer tumor cells. CLEC2D antigen expression levels on prostate cancer cell lines PC3, LnCap, 22RV1, and DU145 induced using LPS, poly I:C, IFN-γ, PBMC supernatant, PBMC cells, NK cells, and T cells. The upper panel shows anti-CLEC2D antibody, and the lower panel shows merged images. [Figure 23C-3] 1 shows that anti-CLEC2D antibody recognizes CLEC2D antigen on prostate cancer tumor cells. CLEC2D antigen expression levels on prostate cancer cell lines PC3, LnCap, 22RV1, and DU145 induced using LPS, poly I:C, IFN-γ, PBMC supernatant, PBMC cells, NK cells, and T cells. The upper panel shows anti-CLEC2D antibody, and the lower panel shows merged images. [Figure 23C-4] 1 shows that anti-CLEC2D antibody recognizes CLEC2D antigen on prostate cancer tumor cells. CLEC2D antigen expression levels on prostate cancer cell lines PC3, LnCap, 22RV1, and DU145 induced using LPS, poly I:C, IFN-γ, PBMC supernatant, PBMC cells, NK cells, and T cells. The upper panel shows anti-CLEC2D antibody, and the lower panel shows merged images. [Figure 23D] Figure 2 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on prostate cancer tumor cells.Figure 3 shows a human tissue microarray slide stained with anti-CLEC2D antibody C2685, showing staining of tumor cells within malignant prostate cancer tissue. [Figure 24A] Figure 2 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on various other tumor cells.Figure 3 shows TCGA data analysis of CLEC2D antigen expression in various cancers. [Figure 24B] 1 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on various other tumor cells. 2 shows the expression levels of the CLEC2D antigen on various tumor cell lines, including HepG2 (liver cancer), LN229 (glioblastoma), SKOV3 (ovarian cancer), BT474 (breast cancer), NCI-H929 (myeloma), and Ramos (lymphoma). [Figure 24C-1] 1 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on various other tumor cells. 2 shows the expression levels of the CLEC2D antigen on BT474 (breast cancer), SKOV3 (ovarian cancer), LN229 (glioblastoma), Ramos (lymphoma), NCI-H929 (myeloma), and HepG2 (liver cancer) upon induction with LPS, poly I:C, and IFNγ. [Figure 24C-2] 1 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on various other tumor cells. 2 shows the expression levels of the CLEC2D antigen on BT474 (breast cancer), SKOV3 (ovarian cancer), LN229 (glioblastoma), Ramos (lymphoma), NCI-H929 (myeloma), and HepG2 (liver cancer) upon induction with LPS, poly I:C, and IFNγ. [Figure 24C-3]1 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on various other tumor cells. 2 shows the expression levels of the CLEC2D antigen on BT474 (breast cancer), SKOV3 (ovarian cancer), LN229 (glioblastoma), Ramos (lymphoma), NCI-H929 (myeloma), and HepG2 (liver cancer) upon induction with LPS, poly I:C, and IFNγ. [Figure 24D] Figure 2 shows that anti-CLEC2D antibodies recognize the CLEC2D antigen on various other tumor cells. Figure 2 shows cytotoxicity mediated by anti-CLEC2D antibody C5511 at 100 μg / ml on SKOV3 (ovarian cancer) and anti-CLEC2D antibodies C5511 and C6481 at 100 μg / ml on HepG2 (liver cancer) cell line. The percentage of dead cells was indicated by Sytox green positive cells. [Figure 25A] 1 shows a lymphocyte proliferation assay using anti-CLEC2D antibodies with flow cytometry analysis. 2 shows the antibody wet coating protocol. [Figure 25B] 1 shows a lymphocyte proliferation assay with anti-CLEC2D antibody using flow cytometry analysis. 2 shows the air-dried antibody coating protocol. [Figure 25C] 1 shows a lymphocyte proliferation assay using anti-CLEC2D antibody with flow cytometry analysis. [Figure 25D] Lymphocyte proliferation assay with anti-CLEC2D antibodies using flow cytometry analysis. Measurement of IFNγ cytokine secretion from effector cells upon long-term culture of PBMCs with anti-CLEC2D antibodies (C5511, C4608, C6481). Treatment with OKT3 antibody was used as a positive control. PBMCs were treated with anti-CD3 antibody OKT3 (1 μg / ml), anti-CLEC2D antibodies C4608, C5511, and C6481 (1 μg / ml, 10 μg / ml, 50 μg / ml, and 100 μg / ml) and then cultured for 4 days. Fluorescent proliferation dye status was monitored using a flow cytometer. Untreated PBMCs were used as control. [Figure 25E]Lymphocyte proliferation assay with anti-CLEC2D antibodies using flow cytometry analysis. Measurement of IL2 cytokine secretion from effector cells upon long-term culture of PBMCs with anti-CLEC2D antibodies (C5511, C4608, C6481). Treatment with OKT3 antibody was used as a positive control. PBMCs were treated with anti-CD3 antibody OKT3 (1 μg / ml), anti-CLEC2D antibodies C4608, C5511, and C6481 (1 μg / ml, 10 μg / ml, 50 μg / ml, and 100 μg / ml) and then cultured for 4 days. Fluorescent proliferation dye status was monitored using a flow cytometer. Untreated PBMCs were used as control. [Figure 26] FIG. 1 shows histograms overlaying the binding of anti-CLEC2D antibodies (C3566 and C5511) to CLEC2D antigen homologs from rat, mouse, and cynomolgus monkey expressed on the surface of CHO cells using flow cytometry analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Modulation of immune cell checkpoint receptors by antibody-based / directed therapeutic approaches has attracted some interest over the past few years. Most efforts have been directed towards modulating checkpoints in T cells. However, more and more attention is being paid to modulating checkpoints in B cells, NK cells, and myeloid cells as well. The innate immune system includes natural killer (NK) cells, which have the ability to recognize and induce cytotoxicity in a wide range of target cells, such as tumor cells or virus-infected cells. NK cells do not require any prior antigen sensitization. In addition to direct cytotoxicity, NK cells are also involved in the induction and progression of adaptive immune responses through the production and secretion of cytokines. In general, these responses are regulated by an appropriate balance of signals induced by the interaction of a wide variety of surface activating and inhibitory receptors with ligands on the surface of target cells. Modulation of the number of NK cells and their associated functions via various agents, such as monoclonal antibodies, cytokines, etc., can result in improved antitumor activity. These agents, either alone or in combination, can serve as potential therapeutic agents. Therefore, by inhibiting both activating and / or inhibitory surface receptors, the anti-cancer activity of NK cells can be unleashed.

[0076] Blocking these interactions could represent a novel therapeutic option for treating some cancers, however, there remains an unmet need to coordinate discovery, understanding, and design, and to further tailor therapeutic interventions to individual receptors as targets for various cancers.

[0077] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular, where appropriate for the context and / or application. Various singular / plural interchanges may be expressly set forth herein for clarity. In general, the technical terms used in connection with the biotechnology, immunology, molecular cell biology, and recombinant DNA techniques described herein are those well known and commonly used in the art. Specific references and other documents cited herein are expressly incorporated herein by reference. In case of conflict, the present specification (including definitions) shall control. The materials, methods, diagrams, and examples are illustrative only and are not intended to be limiting.

[0078] Furthermore, the methods, preparation, and use of the disclosed naive antibody libraries employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA technology, polymerase chain reaction (PCR), and related fields, the principles and conditions of which are explained in the literature and are well known to those of skill in the art.

[0079] Before disclosing and describing the antibody naive library and the method for generating a nucleic acid encoding the antibody naive library, as well as other embodiments of the present disclosure, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0080] In one embodiment, the terms "library" and "libraries" are used interchangeably within this disclosure and refer to the constructs of this disclosure. In one embodiment, it refers to a collection or pool of nucleic acid sequences. In one embodiment, it refers to a collection or pool of amino acid sequences. In some embodiments, it refers to a collection or pool of organisms that contain the collection or pool of amino acid or nucleic acid sequences. In some embodiments, the organism is a bacteriophage (phage) or yeast (e.g., Saccharomyces cerevisiae).

[0081] In one embodiment, the terms "pooling", "pooled", "pool" and "pools" in the context of the present disclosure refer to mixing samples / nucleic acid sequences / nucleic acid fragments / gene clones / amplification products / antibodies obtained from multiple donors, i.e., two or more donors, by employing the methods of the present disclosure.

[0082] In one embodiment, the term "PBMC" refers to any peripheral blood cell with a round nucleus, consisting of lymphocytes (T cells, B cells, NK cells) and monocytes, erythrocytes, platelets, and granulocytes (neutrophils, basophils, and eosinophils).

[0083] antigen As used herein, the term "antigen" or "immunogen" refers to any foreign substance that induces an immune response in the body. In one embodiment, the antigen is a cellular protein. In one embodiment, the antigen is a cell surface protein.

[0084] Antigens may be isolated or derived from any species. Representative species include Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupis familiaris, and Cynomolgus macaca. fascicularis. In some embodiments, the antigen is a fragment of a wild-type protein isolated or derived from Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupis familiaris, or Cynomolgus macaca fascicularis. In some embodiments, the antigen is a mutant variant of a protein derived from Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupis familiaris, or Cynomolgus macaca fascicularis. In some embodiments, the antigen can be mutated to improve the solubility and / or stability of the antigen. For example, the CLEC2D antigen may contain a mutation at H176C to introduce an additional disulfide bridge to the Cys163 amino acid to improve the stability and homogeneity of the expressed protein.

[0085] In some embodiments, the antigen comprises an epitope tag at either the N-terminus or C-terminus of the polypeptide. Exemplary tags include, but are not limited to, polyhistidine tags and FLAG tags. Any epitope tag known in the art is considered to be within the scope of this disclosure.

[0086] C-type lectin domain family 2 member D (CLEC2D), also called CLAX, lectin-like transcript-1 (LLT1), and OCIL, is a member of the natural killer cell receptor C-type lectin family. CLEC2D binds to killer cell lectin-like receptor B1 (KLRB1). KLRB1 is also known as CD161, CLEC5B, NKR, NKR-P1, NKR-P1A, NKRP1A, and hNKR-P1A. All orthologs and isoforms of CLEC2D and CD161 are considered within the scope of this disclosure.

[0087] In some embodiments, a C-type lectin domain family 2 member D (CLEC2D) protein known in the art or any of its aliases or homologs, whether of human or other species origin, represents the target antigen of antibodies generated by the methods described herein.

[0088] In some embodiments, the antigen is a CLEC2D antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a CLEC2D sequence isolated or derived from Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupis familiaris, and Cynomolgus macaca fascicularis.

[0089] In some embodiments, the CD161 protein or any of its aliases or homologs known in the art, whether of human or other species origin, represents the target antigen for antibodies generated by the methods described herein.

[0090] In some embodiments, the CD161 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a CD161 sequence isolated or derived from Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupis familiaris, and Cynomolgus macaca fascicularis.

[0091] Exemplary antigens are shown in Table 1 below. [Table 1-1] [Table 1-2]

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

[0092] Antibody In one embodiment, the term "antibody" refers to an immunoglobulin that may be derived in whole or in part from natural sources or may be synthetically produced. The terms "antibody" and "immunoglobulin" are used interchangeably throughout the specification, unless otherwise specified.

[0093] In one embodiment, the term "antibody" includes both polyclonal and monoclonal antibody preparations, including, below, chimeric antibody molecules, F(ab')2 and F(ab) fragments, Fv molecules, single chain Fv molecules (ScFv), dimeric and trimeric antibody fragments, bispecific antibodies, minibodies, humanized monoclonal antibody molecules, human antibodies, fusion proteins comprising the Fc region of an antibody and any functional fragments resulting from these molecules, where the derivative molecules retain the immunological function of the parent antibody molecule. The antibodies of the present disclosure may be human, humanized, chimeric, or further engineered antibodies, so long as the properties of the present disclosure are retained.

[0094] "Native antibodies and immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592 (1985)).

[0095] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, called "hypervariable regions", are arranged between more conserved adjacent stretches known as "framework regions" or "FR". Thus, the term "FR" refers to the naturally occurring amino acid sequences between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three-dimensional space relative to each other to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs".

[0096] The term "variable" refers to the fact that the sequences of certain parts of the variable domains differ widely among antibodies and that these parts are utilized for the binding and specificity of each individual antibody to its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy variable domains. The more highly conserved parts of the variable domains are called frameworks (FRs). Naturally occurring heavy and light chain variable domains each contain four FR regions that adopt a predominantly β-sheet structure, connected by three CDRs that form loops that bind to the β-sheet structure and, in some cases, by three CDRs that form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the target binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen but exert various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0097] An "antibody fragment" comprises a portion of a full-length antibody, preferably a variable domain thereof, or at least an antigen-binding site thereof. scFv antibodies are described, for example, in Huston, JS, Methods in Enzymol. 203 (1991) 46-88. In one embodiment, an "antibody fragment" is a portion of a complete antibody that retains the ability to exhibit antigen-binding activity. In addition, an antibody fragment comprises a single-chain polypeptide that has the properties of a VH domain (i.e., capable of assembling with a VL domain) or a VL domain (capable of assembling with a VH domain) that binds to the corresponding antigen, a functional antigen-binding site, thereby resulting in the properties of the antibodies of the present disclosure.

[0098] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment, the designation of which means it is readily crystallizable. Pepsin treatment produces an F(ab')2 fragment that still has two antigen-binding sites capable of cross-linking to antigen.

[0099] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region is composed of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. In this structure, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv, containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity compared to the complete binding site.

[0100] Single-chain Fv ("scFv") polypeptide molecules are covalently linked VH:VL heterodimers that can be expressed from gene fusions containing VH- and VL-encoding genes linked by a peptide-encoding linker. Numerous methods have been described to identify chemical structures for converting the naturally aggregated but chemically separate light and heavy polypeptide chains derived from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,946,778.

[0101] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0102] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (l), based on the amino acid sequences of their constant domains.

[0103] As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent interactions that occur between immunoglobulin molecules and the antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, with a smaller Kd representing a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Therefore, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio Koff / Kon allows all parameters that are not related to affinity to be cancelled out and corresponds to the dissociation constant Kd. In some embodiments, the antibodies of the disclosure bind to CLEC2D with a Kd of ≦10 μM, preferably ≦1 μM, more preferably ≦100 nM, e.g., ≦90 nM, ≦80 nM, ≦70 nM, ≦60 nM, ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, ≦10 nM, ≦5 nM, or ≦1 nM, as measured using an assay such as a radioligand binding assay or a similar assay known to one of skill in the art. -5 M~10 -12 For example, the binding affinity of the antibodies of the present disclosure is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10-9 M~10 -11 M、10 -10 M~10 -11 M、10 -5 M~10 -10 M、10 -6 M~10 -10 M、10 -7 M~10 -10 M、10 -8 M~10 -10 M、10 -9 M~10 -10 M、10 -5 M~10 -9 M、10 -6 M~10 -9 M、10 -7 M~10 -9 M、10 -8 M~10 -9 M、10 -5 M~10 -8 M、10 -6 M~10 -8 M、10 -7 M~10 -8 M、10 -5 M~10 -7 M、10 -6 M~10 -7 M、または10 -5 M~10 -6 Mである。

[0104] The present disclosure also features antibodies that have a certain percentage of identity or similarity to the amino acid or nucleotide sequence of the CLEC2D antibodies described herein. For example, the antibody may have at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more percent identity when compared to a specific region or the full length of any one of the CLEC2D antibodies described herein. Preferably, the antibody may have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more percent identity when compared to a specific region or the full length of any one of the CLEC2D antibodies described herein. More preferably, the antibody may have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more percent identity when compared to a specific region or the full length of any one of the CLEC2D antibodies described herein. Even more preferably, the antibody may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more percent identity when compared to a specific region or the full length of any one of the CLEC2D antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the present disclosure can be determined by sequence comparison and / or alignment using methods well known in the art. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence identity or similarity to the nucleic acids and proteins of the present disclosure.

[0105] With respect to amino acid sequences, those skilled in the art will readily understand that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that change, add, delete, or replace a single amino acid or a small percentage of amino acids in the coding sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the changes result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0106] Depending on the amino acid sequence of the constant domain of the immunoglobulin heavy chain, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, ∂, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0107] In one embodiment, humanized antibodies may be used in the compositions and methods provided herein. In some embodiments, the term "humanized antibody" or "humanized form of antibody" refers to an antibody whose framework or "complementarity determining regions" (CDRs) have been modified to include CDRs of an immunoglobulin of different specificity compared to the specificity of the parent immunoglobulin. In other embodiments, the CDRs of VH and VL are grafted into the framework regions of a human antibody to prepare a "humanized antibody." See, for example, Riechmann, L., et al, Nature 332 (1988) 323-327; and Neuberger, MS, et al, Nature 314 (1985) 268-270. The heavy chain variable framework region and the light chain variable framework region can be derived from the same or different human antibody sequences. The human antibody sequences may be sequences of natural human antibodies. Human heavy chain variable framework regions and human light chain variable framework regions are listed, for example, in Lefranc, M.-P., Current Protocols in Immunology (2000)-Appendix IP A.1P.1-A.1P.37 and are available via IMGT, the international ImMunoGeneTics information system® (http: / / imgt.cines.fr) or http: / / vbase.mrc-cpe.cam.ac.uk. Optionally, the framework regions may be modified by further mutations. Particularly preferred CDRs correspond to the CDRs that represent sequences recognizing the above-mentioned antigens for the chimeric antibody. The term "humanized antibody" as used herein also includes such antibodies whose constant regions have been modified, for example by "class switching", i.e. by alteration or mutation of the Fc part (e.g. IgG1 to IgG4 and / or IgG1 / IgG4 mutations), to produce the properties of the present disclosure, in particular with regard to C1q binding and / or FcR binding. The term "human antibody", as used herein, is meant to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.Human antibodies are well known in the state of the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be generated using transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. Introduction of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge (e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature. 362 (1993) 255-258; Brueggemann, MD, et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be made using phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al, J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A., et al. and Boerner, P., et al. are also available for the preparation of human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, AL, p. 77 (1985); and Boerner, P., et al, J. Immunol. 147 (1991) 86-95). As already mentioned in the humanized antibodies of this disclosure, the term "human antibody" as used herein also includes such antibodies in which the constant region has been modified to produce the properties of the present disclosure.

[0108] In one embodiment, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The antibody is not limited by the species or source of the antibody, or by the method of making the antibody. In another embodiment, the term encompasses complete immunoglobulins as well as fragments, such as Fab, F(ab')2, Fv, and other fragments, as well as chimeric and humanized homogeneous antibody populations that exhibit the immunological binding properties of the parent monoclonal antibody molecule. In another embodiment, the term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single amino acid composition. In another embodiment, the terms "Fab" or "ScFv" are used to specifically refer to antibody fragments.

[0109] In some embodiments, chimeric antibodies may be used in the compositions and methods provided herein. In one embodiment, the term "chimeric antibody" refers to a monoclonal antibody, generally prepared using recombinant DNA techniques, that comprises a variable region, i.e., a binding region, from one species (e.g., mouse or rat) and at least a portion of a constant region from a different source or species (e.g., human). Chimeric antibodies comprising a mouse variable region and a human constant region are particularly preferred. Such chimeric antibodies are the expression product of immunoglobulin genes that comprise a DNA segment encoding an immunoglobulin variable region from one species and a DNA segment encoding an immunoglobulin constant region from a different species. Other forms of "chimeric antibodies" encompassed by the present disclosure are chimeric antibodies whose class or subclass has been altered or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies". Methods for making chimeric antibodies include conventional recombinant DNA and gene transfection techniques currently well known in the art. See, e.g., Morrison, SL, et al, Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238, and US 5,204,244.

[0110] In one embodiment, "antibody display library" refers to a platform or platforms that express antibodies on the surface of cells or acellular suitable for screening methods against a target antigen. Phage display library and yeast display library are used herein with precise specifications unless otherwise stated.

[0111] In one embodiment, the term "naive library" refers to a collection of nucleic acid sequences encoding natural VH repertoires derived from a non-immunized source.

[0112] In one embodiment, the term "VH" refers to a single heavy chain variable domain of an antibody of a type that may exist in a mammal that naturally lacks a light chain or a portion of a light chain, whereby a native VH may be appropriately understood.

[0113] In one embodiment, the term "VL" refers to a single light chain variable domain of an antibody, which exists in two types based on the constant domain sequence: Vk (kappa constant region) and Vl (lambda constant region), as appropriate understood.

[0114] In one embodiment, the term "CDR" refers to the complementarity determining regions of the antibody structure.

[0115] In one embodiment, the term "repertoire" refers to a collection and refers to genetic diversity.

[0116] In one embodiment, the term "framework region" is used herein to mean the regions of nucleic acid sequence of an antibody molecule that encode the structural elements of the molecule.

[0117] In another embodiment, the term "vector" refers to a DNA associated cloning or expression system for delivering antibody genes into specific designated restriction sites, where phagemid vectors (applicable for phage display systems) or yeast vectors (applicable for yeast display systems) or mammalian expression vectors (applicable for mammalian expression systems) are understood accordingly.

[0118] The present disclosure provides antibodies and antibody fragments that bind to the CLEC2D antigens of the disclosure.

[0119] The present disclosure provides VH and VL domains of antibodies or antibody fragments that bind to a CLEC2D antigen or an epitope of CLEC2D according to this disclosure.

[0120] The present disclosure provides sequences of the CDR1, CDR2, and CDR3 of the VH domain, and the CDR1, CDR2, and CDR3 of the VL domain of an antibody that binds to a CLEC2D antigen or an epitope of CLEC2D described in this disclosure.

[0121] Any combination of the VH and VL sequences of the present disclosure is considered to be within the scope of the present disclosure. Any combination of the CDR1, CDR2, and CDR3 sequences of a VH domain or the CDR1, CDR2, and CDR3 sequences of a VL domain is considered to be within the scope of the present disclosure.

[0122] One of skill in the art will appreciate that if a monoclonal antibody has the same specificity as a monoclonal antibody of the present disclosure, this can be determined without undue experimentation by determining whether the former inhibits the latter from binding to CLEC2D. If the monoclonal antibody being tested competes with a monoclonal antibody of the present disclosure, as indicated by reduced binding by the monoclonal antibody of the present disclosure, then the two monoclonal antibodies are likely to bind to the same epitope or closely related epitopes.

[0123] Another method to confirm whether a monoclonal antibody has the specificity of the monoclonal antibody of the present disclosure is to preincubate the monoclonal antibody of the present disclosure with the CLEC2D protein, and if it reacts normally, add the monoclonal antibody to be tested to confirm whether the ability of the monoclonal antibody to be tested to bind to CLEC2D is inhibited. If the monoclonal antibody to be tested is inhibited, the monoclonal antibody most likely has the same or a functionally equivalent epitope specificity as the monoclonal antibody of the present disclosure. Screening of the monoclonal antibodies of the present disclosure can also be performed by utilizing CLEC2D to confirm whether the test monoclonal antibody is capable of neutralizing CLEC2D.

[0124] Various techniques well known in the art may be used to generate polyclonal or monoclonal antibodies directed to a protein of the present disclosure or its derivatives, fragments, analogs, homologs, or orthologs (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0125] Antibodies may be purified using well-known techniques such as affinity chromatography using protein A or protein G, which mainly yields the IgG fraction of immune serum. Subsequently or alternatively, immunoaffinity chromatography may be used to purify immune specific antibodies by immobilizing the specific antigen or epitope that is the target of the immunoglobulin being sought on a column. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, Vol. 14, No. 8 (April 17, 2000), pp. 25-28, published by The Scientist, Inc., Philadelphia PA).

[0126] Monoclonal antibodies can be prepared using hybridoma methods such as those described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, typically a mouse, hamster, or other suitable host animal is immunized with an immunizing agent to induce lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0127] The immunizing agent typically includes a protein antigen, a fragment thereof, or a fusion protein thereof. Typically, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Usually, rat or mouse myeloma cells are employed. The hybridoma cells may be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas usually contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances inhibit the growth of HGPRT-deficient cells.

[0128] Preferred immortalized cell lines are those that fuse efficiently, sustain stable high-level expression of antibody by selected antibody-producing cells, and are amenable to culture media such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63).

[0129] The culture medium in which the hybridoma cells are cultured may then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is measured by immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are well known in the art. The binding affinity of the monoclonal antibody can be measured, for example, using the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity for the target antigen.

[0130] After desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods. (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1999.) Press, (1986) pp. 59-103. Suitable media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

[0131] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid using conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0132] Monoclonal antibodies can also be produced using recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the present disclosure can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). In some embodiments, hybridoma cells of the present disclosure serve as the source of such DNA. In some embodiments, antibody gene sequences, isolated and cloned using methods of the present disclosure (e.g., phage library display and yeast library display), serve as the source of such DNA. After isolation, the DNA may be placed into an expression vector and then the expression vector may be transfected into host cells that do not otherwise produce immunoglobulin proteins, such as ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. The DNA may also be modified, for example, by substituting the coding sequence with human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368,812-13 (1994)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide may be substituted for the constant domains of an antibody of the present disclosure to create a chimeric bivalent antibody, or may be substituted for the variable domains of one antigen-binding site of an antibody of the present disclosure.

[0133] All cell lines suitable for expression and purification of antibodies or antibody fragments are considered within the scope of this disclosure. In some embodiments, the cell line is a mammalian cell line. The cell line may be isolated or derived from any source, including human, mouse, and hamster. Suitable cell lines include Chinese Hamster Ovary (CHO) cells, HEK 293 cells, HEK293T cells, BHK21 cells, NSO cells, PER.C6 cells, B cells, HEK Examples include, but are not limited to, 293-6E cells, Sp2 / 0-Ag14 cells, and DG44 cells. In some embodiments, the cell line is a hybridoma cell line.

[0134] Antibodies may also be expressed using vectors containing a DNA segment encoding the single chain antibodies described herein.

[0135] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA viral vectors or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0136] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney Murine Leukemia Virus. DNA viral vectors are preferred. These vectors include pox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci USA 87:1149 (1990)), adenovirus vectors (LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 259:1149 (1993)), and the like. 3:219(1993);Yang,et al., J. Virol. 69:2004 (1995)), and adeno-associated virus vectors (Kaplitt, MG. et al., Nat. Genet. 8:148 (1994)).

[0137] Poxvirus vectors introduce genes into the cytoplasm of cells. Avian poxvirus vectors only result in short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. Adenovirus vectors provide a shorter period of expression (about 2 months) compared to adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector selected depends on the target cell and the condition to be treated. The introduction may be by standard techniques, such as infection, transfection, transduction, or transformation. Examples of gene introduction methods include, for example, naked DNA, CaPO4 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0138] Exemplary VH amino acid sequences of CLEC2D antibodies of the disclosure are shown below in Table 2. VH amino acid sequences having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity to the sequences listed in Table 2 are considered to be within the scope of the disclosure. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0139] The VH amino acid sequences of the disclosure may be encoded by the polynucleotides shown in Table 3 below. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

[0140] Exemplary VL amino acid sequences of CLEC2D antibodies of the disclosure are shown below in Table 4. VL amino acid sequences having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity to the sequences listed in Table 4 are considered to be within the scope of the disclosure. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10]

[0141] The VL amino acid sequences of the disclosure may be encoded by the polynucleotides shown in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] [Table 5-13] [Table 5-14] [Table 5-15] [Table 5-16] [Table 5-17] [Table 5-18] [Table 5-19] [Table 5-20] [Table 5-21]

[0142] Exemplary CDR amino acid sequences for CLEC2D antibodies of the disclosure are shown in Table 6 below. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10] [Table 6-11]

Table 6-12

Table 6-13

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

[0143] In some embodiments, the nucleotide sequence encoding the antibody, antibody fragment, VH domain, VL domain or CDR of the present disclosure is a wild type sequence.In some embodiments, the nucleotide sequence is codon-optimized for expression in mammalian cells.In some embodiments, the nucleotide sequence is codon-optimized for expression in human cells.

[0144] In some embodiments, the present invention relates to an antibody capable of binding to CLEC2D and blocking the interaction between CLEC2D and CD161 (Figure 1). In some embodiments, the anti-CLEC2D antibody disclosed herein is a monoclonal antibody. In some embodiments, the anti-CLEC2D antibody disclosed herein is a polyclonal antibody.

[0145] In some embodiments, the present invention relates to antibodies capable of binding to CLEC2D and capable of eliminating CLEC2D expressing cells by antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) by blocking the interaction between CLEC2D and CD161. In some embodiments, the present invention relates to antibodies capable of binding to CLEC2D and capable of promoting cytokine production and NK cell-mediated cytotoxicity by blocking the interaction between CLEC2D and CD161.

[0146] In some embodiments, the anti-CLEC2D antibodies disclosed herein are humanized antibodies. In some embodiments, the anti-CLEC2D antibodies disclosed herein are human IgG1, IgG1 N296A is an anti-CLEC2D antibody of the IgG2, IgG3, or IgG4 isotype. In some embodiments, the anti-CLEC2D antibody is of the murine IgG1, IgG2a, IgG2b, or IgG3 isotype.

[0147] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable heavy chain (VH) comprising an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108.

[0148] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable light chain (VL) comprising an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 217-324.

[0149] In some embodiments, the anti-CLEC2D antibodies disclosed herein have an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108. and a variable light chain (VL) comprising an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 217-324.

[0150] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable heavy chain (VH) comprising an amino acid sequence encoded by a nucleic acid selected from the group consisting of SEQ ID NOs:109-216.

[0151] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable light (VL) chain comprising an amino acid sequence encoded by a nucleic acid selected from the group consisting of SEQ ID NOs: 325-432.

[0152] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable heavy chain (VH) comprising an amino acid sequence encoded by a nucleic acid selected from the group consisting of SEQ ID NOs: 109-216, and a variable light chain (VL) comprising an amino acid sequence encoded by a nucleic acid selected from the group consisting of SEQ ID NOs: 325-432.

[0153] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108.

[0154] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable light chain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 217-324.

[0155] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-108, and a variable light chain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 217-324.

[0156] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:44, and a VL comprising the amino acid sequence set forth in SEQ ID NO:260.

[0157] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:45, and a VL comprising the amino acid sequence set forth in SEQ ID NO:261.

[0158] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:42, and a VL comprising the amino acid sequence set forth in SEQ ID NO:258.

[0159] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:1, and a VL comprising the amino acid sequence set forth in SEQ ID NO:217.

[0160] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:73, and a VL comprising the amino acid sequence set forth in SEQ ID NO:289.

[0161] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:21, and a VL comprising the amino acid sequence set forth in SEQ ID NO:237.

[0162] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:35, and a VL comprising the amino acid sequence set forth in SEQ ID NO:251.

[0163] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:58, and a VL comprising the amino acid sequence set forth in SEQ ID NO:274.

[0164] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL comprising the amino acid sequence set forth in SEQ ID NO:223.

[0165] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable heavy (VH) complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 433-485.

[0166] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable heavy (VH) complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 486-546.

[0167] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable heavy (VH) complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 547-653.

[0168] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable light (VL) complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 654-726.

[0169] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable light (VL) complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 727-783.

[0170] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a variable light (VL) chain complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 784-885.

[0171] In some embodiments, the anti-CLEC2D antibody disclosed herein comprises a variable heavy chain (VH) complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 433 to 485, a VH complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 486 to 546, and a VH complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 547 to 653.

[0172] In some embodiments, the anti-CLEC2D antibody disclosed herein comprises a variable light chain (VL) complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 654 to 726, a VL complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 727 to 783, and a VL complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 784 to 885.

[0173] In some embodiments, the anti-CLEC2D antibody disclosed herein comprises a variable heavy chain (VH) complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 433 to 485, a VH complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 486 to 546, and a VH complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 547 to 653, as well as a variable light chain (VL) complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 654 to 726, a VL complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 727 to 783, and a VL complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 784 to 885.

[0174] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 439, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 492, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 589, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 687, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 729, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 827.

[0175] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 439, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 492, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 590, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 688, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 755, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 828.

[0176] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:473, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:495, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:587, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:655, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:732, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:825.

[0177] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 433, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 486, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 547, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 654, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 727, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 784.

[0178] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 439, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 492, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 618, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 678, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 730, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 852.

[0179] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:446, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:501, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:567, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:655, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:735, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:804.

[0180] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 435, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 488, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 581, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 680, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 782, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 818.

[0181] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:466, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:521, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:603, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:662, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:732, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:814.

[0182] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 439, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 492, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 553, as well as a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 660, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 733, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 790.

[0183] The present disclosure provides an antibody library comprising at least about 108 unique monoclonal antibody clones, at least about 80% of the antibody clones detectably and specifically bind to the CLEC2D antigen. Various anti-CLEC2D antibodies having specific combinations of heavy chains, light chains, heavy chain CDR1-3 (i.e., CDRH1, CDRH2, and CDRH3), and light chain CDR1-3 (i.e., CDRL1, CDRL2, and CDRL3) are listed in Table 9A. [Table 9A-1] [Table 9A-2] [Table 9A-3] [Table 9A-4] [Table 9B] [Table 9C]

[0184] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:A1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:A1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No.:A1 disclosed in Table 9B comprises a variable heavy chain having framework region sequences of germline gene families: IGHV4, IGHD3, and IGHJ2. In some embodiments, the anti-CLEC2D antibody No.:A1 disclosed in Table 9B comprises a variable light chain having framework region sequences of germline gene families: IGKV3 and IGKJ4.

[0185] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:B1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:B1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No.:B1 disclosed in Table 9B comprises a variable heavy chain having framework region sequences from germline gene families: IGHV4, IGHD3, and IGHJ5. In some embodiments, the anti-CLEC2D antibody No.:B1 disclosed in Table 9B comprises a variable light chain having framework region sequences from germline families: IGKV1 and IGKJ1.

[0186] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:C1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:C1 disclosed in Table 9A.

[0187] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:D1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:D1 disclosed in Table 9A.

[0188] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:E1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:E1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No.:E1 disclosed in Table 9B comprises a variable heavy chain having framework region sequences of germline gene families: IGHV3, IGHD5, and IGHJ4. In some embodiments, the anti-CLEC2D antibody No.:E1 disclosed in Table 9B comprises a variable light chain having framework region sequences of germline gene families: IGKV3 and IGKJ5.

[0189] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:F1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:F1 disclosed in Table 9A.

[0190] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:G1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:G1 disclosed in Table 9A.

[0191] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:H1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:H1 disclosed in Table 9A.

[0192] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No. I1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No. I1 disclosed in Table 9A.

[0193] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:J1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:J1 disclosed in Table 9A.

[0194] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:K1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:K1 disclosed in Table 9A.

[0195] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:L1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:L1 disclosed in Table 9A.

[0196] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:M1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:M1 disclosed in Table 9A.

[0197] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:N1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:N1 disclosed in Table 9A.

[0198] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:O1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:O1 disclosed in Table 9A.

[0199] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No. P1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No. P1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No. P1 disclosed in Table 9B comprises a variable heavy chain having framework region sequences from germline gene families: IGHV1, IGHD6, and IGHJ4. In some embodiments, the anti-CLEC2D antibody No. P1 disclosed in Table 9B comprises a variable light chain having framework region sequences from germline gene families: IGKV3 and IGKJ5.

[0200] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.: Q1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.: Q1 disclosed in Table 9A.

[0201] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:R1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:R1 disclosed in Table 9A.

[0202] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 of anti-CLEC2D antibody No.: S1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.: S1 disclosed in Table 9A.

[0203] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:T1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:T1 disclosed in Table 9A.

[0204] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:U1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:U1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No.:U1 disclosed in Table 9B comprises a variable heavy chain having framework region sequences of germline gene families: IGHV4, IGHD1, and IGHJ4. In some embodiments, the anti-CLEC2D antibody No.:U1 disclosed in Table 9B comprises a variable light chain having framework region sequences of germline gene families: IGKV4 and IGKJ4.

[0205] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.: V1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.: V1 disclosed in Table 9A.

[0206] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:W1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:W1 disclosed in Table 9A.

[0207] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:X1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:X1 disclosed in Table 9A.

[0208] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:Y1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:Y1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No.:Y1 disclosed in Table 9B comprises a variable heavy chain having framework region sequences of germline gene families: IGHV5, IGHD5, and IGHJ4. In some embodiments, the anti-CLEC2D antibody No.:Y1 disclosed in Table 9B comprises a variable light chain having framework region sequences of germline gene families: IGKV3 and IGKJ4.

[0209] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.: Z1 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.: Z1 disclosed in Table 9A.

[0210] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:A2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:A2 disclosed in Table 9A.

[0211] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:B2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:B2 disclosed in Table 9A.

[0212] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:C2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:C2 disclosed in Table 9A.

[0213] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:D2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:D2 disclosed in Table 9A.

[0214] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:E2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:E2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No.:E2 disclosed in Table 9B comprises a variable heavy chain having framework region sequences from germline gene families: IGHV1, IGHD5, and IGHJ4. In some embodiments, the anti-CLEC2D antibody No.:E2 disclosed in Table 9B comprises a variable light chain having framework region sequences from germline families: IGKV1 and IGKJ1.

[0215] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:F2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:F2 disclosed in Table 9A.

[0216] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:G2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:G2 disclosed in Table 9A.

[0217] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:H2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:H2 disclosed in Table 9A.

[0218] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:I2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:I2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No.:I2 disclosed in Table 9B comprises a variable heavy chain having framework region sequences from germline gene families: IGHV6, IGHD1, and IGHJ4. In some embodiments, the anti-CLEC2D antibody No.:I2 disclosed in Table 9B comprises a variable light chain having framework region sequences from germline gene families: IGKV3 and IGKJ1.

[0219] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No. J2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No. J2 disclosed in Table 9A.

[0220] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:K2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:K2 disclosed in Table 9A.

[0221] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:L2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:L2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibody No.:L2 disclosed in Table 9B comprises a variable heavy chain having framework region sequences from germline gene families: IGHV4, IGHD3, and IGHJ4. In some embodiments, the anti-CLEC2D antibody No.:L2 disclosed in Table 9B comprises a variable light chain having framework region sequences from germline families: IGKV1 and IGKJ3.

[0222] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:M2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:M2 disclosed in Table 9A.

[0223] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 as set forth in the amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 of anti-CLEC2D antibody No.:N2 disclosed in Table 9A. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the amino acid sequences of heavy and light chains of anti-CLEC2D antibody No.:N2 disclosed in Table 9A.

[0224] In some embodiments, any one or all of the anti-CLEC2D antibodies disclosed herein (including, for example, any one or all of the antibody numbers: A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A and 9B) comprise a human IgG1 Fc region or backbone. In some embodiments, any one or all of the anti-CLEC2D antibodies disclosed herein (including, for example, any one or all of the antibody numbers: A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A and 9B) comprise a human IgG4 Fc region or backbone. In some embodiments, any one or all of the anti-CLEC2D antibodies disclosed herein (including, for example, any one or all of the antibody numbers disclosed in Tables 9A and 9B: A1, B1, E1, P1, U1, Y1, E2, I2, and L2) comprise a human IgG1 N-A Fc region or backbone. In some embodiments, any one or all of the anti-CLEC2D antibodies disclosed herein (including, for example, any one or all of the antibody numbers disclosed in Tables 9A and 9B: A1, B1, E1, P1, U1, Y1, E2, I2, and L2) comprise a human IgG2 Fc region or backbone. In some embodiments, any one or all of the anti-CLEC2D antibodies disclosed herein (including, for example, any one or all of the antibody numbers disclosed in Tables 9: A1, B1, E1, P1, U1, Y1, E2, I2, and L2) are afucosylated. In some embodiments, any one or all of the anti-CLEC2D antibodies disclosed herein (e.g., any one or all of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Table 9) comprise an afucosylated antibody region.

[0225] In some embodiments, any one or all of the anti-CLEC2D antibodies selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A and 9B comprise a human IgG1 Fc region or backbone. In some embodiments, any one or all of the anti-CLEC2D antibodies selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Table 9C comprise a human IgG4 Fc region or backbone. In some embodiments, any one or all of the anti-CLEC2D antibodies selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A and 9B comprise a human IgG1 N-A Fc region or backbone. In some embodiments, any one or all of the anti-CLEC2D antibodies selected from the group consisting of antibody Nos. A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Table 9C comprise a human IgG2 Fc region or main chain.

[0226] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No:A1 disclosed in Table 9C.

[0227] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No: B1 disclosed in Table 9C.

[0228] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No:E1 disclosed in Table 9C.

[0229] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No: P1 disclosed in Table 9C.

[0230] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No:U1 disclosed in Table 9C.

[0231] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No: Y1 disclosed in Table 9C.

[0232] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No:E2 disclosed in Table 9C.

[0233] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No:I2 disclosed in Table 9C.

[0234] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a combination of heavy and light chains as set forth in the heavy and light chain amino acid sequences of IgG1 format anti-CLEC2D antibody No:L2 disclosed in Table 9C.

[0235] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No:A1 disclosed in Table 9C.

[0236] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No: B1 disclosed in Table 9C.

[0237] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No:E1 disclosed in Table 9C.

[0238] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No: P1 disclosed in Table 9C.

[0239] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No: U1 disclosed in Table 9C.

[0240] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No: Y1 disclosed in Table 9C.

[0241] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No: E2 disclosed in Table 9C.

[0242] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No: I2 disclosed in Table 9C.

[0243] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No: L2 disclosed in Table 9C.

[0244] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise an IgG N2A sequence as set forth in the amino acid sequence of the heavy chain of IgG4 format anti-CLEC2D antibody No:L2 disclosed in Table 9C.

[0245] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No:A1 disclosed in Table 9C.

[0246] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No: B1 disclosed in Table 9C.

[0247] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No:E1 disclosed in Table 9C.

[0248] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No: P1 disclosed in Table 9C.

[0249] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No: U1 disclosed in Table 9C.

[0250] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No: Y1 disclosed in Table 9C.

[0251] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No: E2 disclosed in Table 9C.

[0252] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No: I2 disclosed in Table 9C.

[0253] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No: L2 disclosed in Table 9C.

[0254] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise an IgG N2A sequence as set forth in the amino acid sequence of the heavy chain of IgG2 format anti-CLEC2D antibody No:L2 disclosed in Table 9C.

[0255] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No:A1 disclosed in Table 9C.

[0256] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No: B1 disclosed in Table 9C.

[0257] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No:E1 disclosed in Table 9C.

[0258] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No: P1 disclosed in Table 9C.

[0259] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No:U1 disclosed in Table 9C.

[0260] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No: Y1 disclosed in Table 9C.

[0261] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No: E2 disclosed in Table 9C.

[0262] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No: I2 disclosed in Table 9C.

[0263] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise a heavy chain set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No:L2 disclosed in Table 9C.

[0264] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise an IgG N2A sequence as set forth in the amino acid sequence of the heavy chain of IgG N2A format anti-CLEC2D antibody No:L2 disclosed in Table 9C.

[0265] In some embodiments, any one or all of the anti-CLEC2D antibodies selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C are afucosylated. In some embodiments, any one or all of the anti-CLEC2D antibodies selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C comprise an afucosylated antibody region.

[0266] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody Nos. A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C comprises a variable heavy chain sequence and a variable light chain sequence that recognizes and binds to a human CLEC2D protein having an amino acid sequence set forth in at least one of SEQ ID NOs: 886 to 920 and SEQ ID NOs: 930 to 1003.

[0267] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody Nos. A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C comprises a variable heavy chain sequence and a variable light chain sequence that recognize and bind to a human CLEC2D protein having an amino acid sequence set forth in at least one of SEQ ID NOs: 886 to 909. In some embodiments, the anti-CLEC2D antibody or antibody fragment thereof disclosed herein comprises a variable heavy chain sequence and a variable light chain sequence that recognize and bind to a human CLEC2D protein having an amino acid sequence set forth in at least one of SEQ ID NOs: 930 to 1003.

[0268] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C comprises a variable heavy chain sequence and a variable light chain sequence that recognizes and binds a structural epitope of the CLEC2D antigen and consists of amino acid sites that either overlap and / or do not overlap with CD161 receptor interacting amino acid residues.

[0269] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C comprises variable heavy and variable light chain sequences that inhibit, suppress or compete with another antibody that recognizes and binds to a structural epitope of the CLEC2D antigen and consists of amino acid sites that either overlap and / or do not overlap with CD161 receptor interacting amino acid residues.

[0270] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C comprises a variable heavy chain sequence and a variable light chain sequence that binds to a structural epitope of the CLEC2D antigen comprising any of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, or a combination thereof.

[0271] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C comprises variable heavy and variable light chain sequences that inhibit, abrogate, or compete with binding of another antibody to a structural epitope of the CLEC2D antigen comprising any of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, or any combination thereof.

[0272] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is selected from the group consisting of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP108, ASP109, ASP200, ASP201, ASP202, ASP203, ASP204, ASP205, ASP206, ASP207, ASP208, ASP209, ASP210, ASP211, ASP212, ASP213, ASP214, ASP215, ASP216, ASP217, ASP218, ASP220, ASP225, ASP226, ASP227, ASP228, ASP230, ASP231, ASP232, ASP233, ASP234, ASP235, ASP236, ASP237, ASP238, ASP239, ASP240, ASP241, ASP242, ASP243, ASP244, ASP245, ASP246, ASP247, ASP248, ASP249, ASP250, ASP251, ASP252, ASP253, ASP254, ASP255, ASP256, ASP257, ASP258, ASP259, ASP260, ASP261, ASP262, ASP26 and variable heavy chain and variable light chain sequences that bind to structural epitopes of the CLEC2D antigen, including at least one of the following: 92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a nonlinear scaffold for the CD161 receptor-interacting amino acid residues.

[0273] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is selected from the group consisting of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP108, ASP109, ASP200, ASP201, ASP202, ASP203, ASP204, ASP205, ASP206, ASP207, ASP208, ASP209, ASP210, ASP211, ASP212, ASP213, ASP214, ASP215, ASP216, ASP217, ASP218, ASP219, ASP220, ASP221, ASP222, ASP223, ASP224, ASP225, ASP226, ASP230, ASP231, ASP232, ASP233, ASP234, ASP235, ASP236, ASP237, ASP238, ASP239, ASP240, ASP241, ASP242, ASP243, ASP244, ASP245, ASP246, ASP247, ASP248, ASP249, ASP250, ASP251, ASP252, ASP253, ASP254, ASP255, ASP256, ASP257, ASP258, ASP259, ASP26 and variable heavy chain and variable light chain sequences that bind to structural epitopes of the CLEC2D antigen, including at least one of the following: 92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a nonlinear scaffold for the CD161 receptor-interacting amino acid residues.

[0274] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is selected from the group consisting of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, TH The variable heavy chain sequence and variable light chain sequence bind to a structural epitope of the CLEC2D antigen, including at least one of R93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a non-linear scaffold for CD161 receptor-interacting amino acid residues.

[0275] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is selected from the group consisting of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, TH The variable heavy chain sequence and variable light chain sequence bind to a structural epitope of the CLEC2D antigen, including at least one of R93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and non-linear scaffold for non-CD161 receptor-interacting amino acid residues.

[0276] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is selected from the group consisting of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, TH The variable heavy chain sequence and variable light chain sequence bind to a structural epitope of the CLEC2D antigen, including at least one of R93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and non-linear scaffold for non-CD161 receptor-interacting amino acid residues.

[0277] In some embodiments, the anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is selected from the group consisting of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, L The variable heavy chain sequence and variable light chain sequence bind to a structural epitope of the CLEC2D antigen, including at least one of YS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and non-linear scaffold for non-CD161 receptor-interacting amino acid residues.

[0278] In some embodiments, an anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is an anti-CLEC2D antibody that binds to a CLEC2D selected from SEQ ID NOs: 886 to 920 and 930 to 1003 by isoforms of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU1 and variable heavy and light chain sequences that bind to at least one of the following: GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0279] In some embodiments, an anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is an anti-CLEC2D antibody that binds to a CLEC2D selected from SEQ ID NOs: 886 to 909 and 930 to 1003 by isoforms of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU1 and variable heavy and light chain sequences that bind to at least one of the following: GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0280] In some embodiments, an anti-CLEC2D antibody selected from the group consisting of antibody numbers A1, B1, E1, P1, U1, Y1, E2, I2, and L2 disclosed in Tables 9A, 9B, and 9C is an anti-CLEC2D antibody that binds to a CLEC2D selected from SEQ ID NOs: 886 to 890 by isoforms selected from the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GL The antibodies include variable heavy and light chain sequences that bind to at least one of N154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0281] In some embodiments, the anti-CLEC2D antibodies disclosed herein are deglycosylated. In some embodiments, the deglycosylated anti-CLEC2D antibodies disclosed herein exhibit increased cytotoxicity against host cells compared to the glycosylated form of the same anti-CLEC2D antibody. In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise N-linked glycosylation.

[0282] In some embodiments, the anti-CLEC2D antibodies disclosed herein are afucosylated. In some embodiments, the afucosylated anti-CLEC2D antibodies disclosed herein exhibit increased cytotoxicity against host cells compared to a fucosylated form of the same anti-CLEC2D antibody.

[0283] In some embodiments, the anti-CLEC2D antibodies disclosed herein are sialylated. In some embodiments, the sialylated anti-CLEC2D antibodies disclosed herein exhibit increased cytotoxicity against host cells compared to a non-sialylated form of the same anti-CLEC2D antibody.

[0284] In some embodiments, the anti-CLEC2D antibodies disclosed herein are hypergalactosylated. In some embodiments, the hypergalactosylated anti-CLEC2D antibodies disclosed herein exhibit increased cytotoxicity against host cells compared to non-galactosylated or hypogalactosylated forms of the same anti-CLEC2D antibody.

[0285] In some embodiments, the anti-CLEC2D antibodies disclosed herein are hypermannosylated. In some embodiments, the hypermannosylated anti-CLEC2D antibodies disclosed herein exhibit increased cytotoxicity against host cells compared to non-galactosylated or hypomannosylated forms of the same anti-CLEC2D antibody.

[0286] In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the heavy chain CDR1, 2, and 3 of any of the anti-CLEC2D antibodies disclosed herein. In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the light chain CDR1, 2, and 3 of any of the anti-CLEC2D antibodies disclosed herein.

[0287] In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable heavy chain of any of the anti-CLEC2D antibodies disclosed herein. In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the light chain of any of the anti-CLEC2D antibodies disclosed herein.

[0288] In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable heavy chain CDR1, 2, and 3 of any of the anti-CLEC2D antibodies disclosed in Table 9A. In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable light chain CDR1, 2, and 3 of any of the anti-CLEC2D antibodies disclosed in Table 9A.

[0289] In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable heavy chain of any of the anti-CLEC2D antibodies disclosed in Table 9A. In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable light chain of any of the anti-CLEC2D antibodies disclosed in Table 9A.

[0290] In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable heavy chain CDR1, 2, and 3 of any of the anti-CLEC2D antibodies disclosed in Table 9A. In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable heavy chain of any of the anti-CLEC2D antibodies disclosed in Table 9A.

[0291] In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable light chain CDR1, 2, and 3 of any of the anti-CLEC2D antibodies disclosed in Table 9A. In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the variable light chain of any of the anti-CLEC2D antibodies disclosed in Table 9A.

[0292] In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the heavy chain of an anti-CLEC2D antibody disclosed in Table 9A having the germline family framework region sequences disclosed herein. In some embodiments, the invention disclosed herein relates to a nucleic acid sequence encoding the amino acid sequence of the light chain of an anti-CLEC2D antibody disclosed in Table 9A having the germline family framework region sequences disclosed herein.

[0293] In some embodiments, the anti-CLEC2D antibodies or antibody fragments thereof disclosed herein may comprise framework region sequences that are derived from or are from a human, murine, rodent, rabbit, equine, bovine, avian, caprine, porcine, fish, canine, or feline framework germline family. In some embodiments, the anti-CLEC2D antibodies or antibody fragments thereof disclosed herein may comprise framework region sequences that are derived from or are from a human framework germline family.

[0294] In some embodiments, the invention disclosed herein relates to vectors carrying nucleic acids encoding the amino acid sequences of the anti-CLEC2D antibodies disclosed herein. In some embodiments, the invention disclosed herein relates to vectors carrying any one or all of the nucleic acid sequences encoding the amino acid sequences of the anti-CLEC2D antibodies disclosed in Table 9A.

[0295] In some embodiments, the invention disclosed herein relates to a host cell transfected with a vector carrying a nucleic acid encoding the amino acid sequence of an anti-CLEC2D antibody disclosed herein. In some embodiments, the invention disclosed herein relates to a host cell transfected with a vector carrying a nucleic acid sequence encoding the amino acid sequence of an anti-CLEC2D antibody disclosed in Table 9A.

[0296] In some embodiments, the anti-CLEC2D antibodies or antibody fragments thereof disclosed herein may be conjugated to a drug, chemical, or small molecule. In some embodiments, the drug is a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or drug. In some embodiments, the therapeutic agent is a radioisotope. In some embodiments, the drug is a diagnostic agent. In some embodiments, diagnostic agents include, but are not limited to, fluorescent, chemiluminescent, or radioisotopic dyes or agents.

[0297] Epitope Recognition Generally, the term "epitope" refers to an area or region on an antigen to which an antibody specifically binds, i.e., an "epitope" is an area or region that is in physical contact with an antibody. A protein epitope may include amino acid residues in the antigen that are directly involved in binding to the antibody (also referred to as the main antigenic component of the epitope), and other amino acid residues that are not directly involved in binding. In some embodiments, the term "epitope" as used herein includes both types of binding sites of any particular region in CLEC2D that specifically binds to an anti-CLEC2D antibody of the present disclosure or another CLEC2D-specific substance, unless otherwise specified (e.g., in some circumstances, the present disclosure relates to an antibody that directly binds to a particular amino acid residue). More detailed epitope mapping of individual anti-CLEC2D antibodies can be determined by alanine scanning methods.

[0298] In some embodiments, the anti-CLEC2D antibody or antibody fragment thereof disclosed herein comprises a variable heavy chain sequence and a variable light chain sequence that recognizes and binds to a human CLEC2D protein having an amino acid sequence set forth in at least one of SEQ ID NOs: 886 to 920 and 930 to 1003.

[0299] In some embodiments, the anti-CLEC2D antibody or antibody fragment thereof disclosed herein comprises a variable heavy chain sequence and a variable light chain sequence that recognize and bind to a human CLEC2D protein having an amino acid sequence set forth in at least one of SEQ ID NOs: 886 to 909. In some embodiments, the anti-CLEC2D antibody or antibody fragment thereof disclosed herein comprises a variable heavy chain sequence and a variable light chain sequence that recognize and bind to a human CLEC2D protein having an amino acid sequence set forth in at least one of SEQ ID NOs: 930 to 1003.

[0300] In some embodiments, the anti-CLEC2D antibodies or antibody fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that recognize and bind a structural epitope of the CLEC2D antigen and consist of amino acid sites that either overlap and / or do not overlap with CD161 receptor-interacting amino acid residues.

[0301] In some embodiments, the anti-CLEC2D antibodies or antibody fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that inhibit, suppress or compete with another antibody that recognizes and binds to a structural epitope of the CLEC2D antigen, and consist of amino acid sites that either overlap and / or do not overlap with CD161 receptor-interacting amino acid residues.

[0302] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that bind to a structural epitope of the CLEC2D antigen comprising any of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, or any combination thereof.

[0303] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that inhibit, abrogate or compete with binding of another antibody to a structural epitope of the CLEC2D antigen comprising any of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, or any combination thereof.

[0304] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in SEQ ID NOs: 886-920 and 930-1003, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a nonlinear scaffold for the CD161 receptor interacting amino acid residues.

[0305] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in SEQ ID NOs: 886-909 and 930-1003, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a nonlinear scaffold for the CD161 receptor interacting amino acid residues.

[0306] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95 of SEQ ID NOs: 886-890, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a nonlinear scaffold for the CD161 receptor-interacting amino acid residues.

[0307] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in SEQ ID NOs: 886-920 and 930-1003, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and non-linear scaffold for non-CD161 receptor-interacting amino acid residues.

[0308] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in SEQ ID NOs: 886-909 and 930-1003, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and non-linear scaffold for non-CD161 receptor-interacting amino acid residues.

[0309] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95 of SEQ ID NOs: 886-890, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and nonlinear scaffold for non-CD161 receptor-interacting amino acid residues.

[0310] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that, upon binding to a CLEC2D selected from SEQ ID NOs: 886-920 and 930-1003, bind to at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0311] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that, when binding to a CLEC2D selected from SEQ ID NOs: 886-909 and 930-1003, bind to at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0312] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that, when binding to a CLEC2D selected from SEQ ID NOs:886-890, bind to at least one of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0313] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise an antibody that binds to amino acid residues THR178, ASN95, ARG137, GLU179, TYR177, SER98, GLU162, GLN139, ARG101, ALA160, TRP96, CYS176, GLU138, ARG175, CYS176, GLU138, ARG175, CYS177, CYS178, CYS179 ... GLY140, SER136, ASP104, ASP92, THR97, LYS94, GLU150, THR149, GLY148, GLN141, PRO142, LYS144, THR152, TRP151, A SN147, ARG153, TRP143, ILE157, CYS163, SER129, THR93, LYS181, ASP91, ARG180, SER187, LYS194, TYR165, ALA174, L EU110, ASN167, ASP168, ILE146, SER172, GLY161, SER173, LEU135, ASP130, GLN100, PHE155, GLY159, PRO156, LEU158 , GLN117, SER115, GLU114, GLN154, ASN120, PHE116, PHE102, GLN106, SER105, ASP107, LYS186, ASP109, GLN112, VAL1 91, TRP145, LYS169, GLY127, PRO128, GLN83, LYS85, GLU77, GLY170, LEU119, LEU123, TRP182, SER90, ALA108, TYR88, HIS190, ILE189, ALA73, ARG84, SER78, TRP79, PRO76, PHE82, ALA171, ASP188, and CYS75.

[0314] In some embodiments, the anti-CLEC2D antibodies disclosed herein comprise variable heavy and variable light chain sequences that recognize and bind to amino acid residues ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in human CLEC2D of the amino acid sequence set forth in SEQ ID NO: 886, 889, 894, 899, 903, 905, 906, or 907.

[0315] In some embodiments, the anti-CLEC2D antibody or antibody fragment thereof disclosed herein comprises the variable heavy chain and variable light chain sequences of SEQ ID NO: 42 and SEQ ID NO: 258 that recognize and bind to a human CLEC2D protein having an amino acid sequence set forth in at least one of SEQ ID NOs: 886 to 909. In some embodiments, the anti-CLEC2D antibody or antibody fragment thereof disclosed herein comprises the variable heavy chain and variable light chain sequences of SEQ ID NO: 42 and SEQ ID NO: 258 that recognize and bind to a human CLEC2D protein having an amino acid sequence set forth in at least one of SEQ ID NOs: 921 to 909.

[0316] In some embodiments, the anti-CLEC2D antibodies or antibody fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that recognize and bind a structural epitope of the CLEC2D antigen and consist of amino acid sites that either overlap and / or do not overlap with CD161 receptor-interacting amino acid residues.

[0317] In some embodiments, the anti-CLEC2D antibodies or antibody fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that inhibit, suppress or compete with another antibody that recognizes and binds to a structural epitope of the CLEC2D antigen, and consist of amino acid sites that either overlap and / or do not overlap with CD161 receptor-interacting amino acid residues.

[0318] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that bind to a structural epitope of the CLEC2D antigen comprising any of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, or any combination thereof.

[0319] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that inhibit, abrogate or compete with binding of another antibody to a structural epitope of the CLEC2D antigen comprising any of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, or any combination thereof.

[0320] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in SEQ ID NOs: 886-920 and 930-1003, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a nonlinear scaffold for the CD161 receptor interacting amino acid residues.

[0321] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in SEQ ID NOs: 886-909 and 930-1003, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a nonlinear scaffold for the CD161 receptor interacting amino acid residues.

[0322] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95 of SEQ ID NOs: 886-890, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming a nonlinear scaffold for the CD161 receptor-interacting amino acid residues.

[0323] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in SEQ ID NOs: 886-920 and 930-1003, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and non-linear scaffold for non-CD161 receptor-interacting amino acid residues.

[0324] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95 in SEQ ID NOs: 886-909 and 930-1003, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and non-linear scaffold for non-CD161 receptor-interacting amino acid residues.

[0325] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that bind to a structural epitope of the CLEC2D antigen, including at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, and ASN95 of SEQ ID NOs: 886-890, and block the interaction between the CLEC2D receptor and the CD161 receptor by forming an allosteric and nonlinear scaffold for non-CD161 receptor-interacting amino acid residues.

[0326] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that, upon binding to a CLEC2D selected from SEQ ID NOs: 886-920 and 930-1003, bind to at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0327] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that, when binding to a CLEC2D selected from SEQ ID NOs: 886-909 and 930-1003, bind to at least one of amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0328] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise variable heavy and variable light chain sequences that, when binding to a CLEC2D selected from SEQ ID NOs:886-890, bind to at least one of the amino acid sites ARG175, TYR177, GLU179, ARG153, ARG84, HIS190, ARG101, GLU150, GLN154, THR152, GLN141, SER105, ASP107, ASP92, THR93, LYS94, LYS144, GLU138, CYS176, GLN139, ARG180, SER187, LYS181, PHE116, ASN95, and induce tumor killing or cytotoxicity, either alone or in combination. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein induce cytotoxicity in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total number of cells treated with the antibodies or antigen-binding fragments thereof.

[0329] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:42 and SEQ ID NO:258, comprise variable heavy and variable light chain sequences that recognize and bind to amino acids ARG175-XAA176-TYR177-XAA178-GLU179, ARG153, ARG84, HIS190.

[0330] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:44 and SEQ ID NO:260, comprise variable heavy and variable light chain sequences that recognize and bind to amino acids ARG101, GLU150-XAA151-THR152-ARG153-GLN154, ARG175-XAA176-TYR177-XAA178-GLU179.

[0331] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:45 and SEQ ID NO:261, comprise variable heavy and variable light chain sequences that recognize and bind to amino acids GLN141, ARG101-XAA102-XAA-103-XAA104-SER105-XAA106-ASP107, HIS190.

[0332] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:1 and SEQ ID NO:217, comprise variable heavy and variable light chain sequences that recognize and bind to amino acids GLN141, ARG153, ASP92-THR93-LYS94, HIS190.

[0333] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:58 and SEQ ID NO:274, comprise variable heavy and variable light chain sequences that recognize and bind to amino acids GLU138-XAA139-XAA140-GLN141-XAA142-XAA143-LYS144, CYS176.

[0334] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:35 and SEQ ID NO:251, comprise amino acids GLU138-GLN139-XAA140-GLN141, ARG175-XAA176-TYR177-XAA178-XAA179-ARG180, variable heavy chain sequences and variable light chain sequences that recognize and bind to SER187.

[0335] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:21 and SEQ ID NO:237, comprise variable heavy and variable light chain sequences that recognize and bind to amino acids ASP92, TYR177-XAA179-XAA180-LYS181, THR152-ARG153-GLN154.

[0336] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:7 and SEQ ID NO:223, comprise variable heavy and variable light chain sequences that recognize and bind to amino acids THR93-XAA94-ASN95, ARG101, GLN139, PHE116, ARG153.

[0337] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, comprise variable heavy and variable light chain sequences that recognize and bind to amino acids THR93-LYS94, ARG101, GLN141, TYR177-XAA178-GLU179.

[0338] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, are identical to SEQ ID NO: 2561~2567 Amino acids listed in One of The variable heavy and light chain sequences recognize and bind to

[0339] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, comprise variable heavy and variable light chain sequences that recognize and bind to the amino acid set forth in SEQ ID NO:2561.

[0340] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, comprise variable heavy and variable light chain sequences that recognize and bind to the amino acid set forth in SEQ ID NO:2562.

[0341] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, comprise variable heavy and variable light chain sequences that recognize and bind to the amino acid set forth in SEQ ID NO:2563.

[0342] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, comprise variable heavy and variable light chain sequences that recognize and bind to the amino acid set forth in SEQ ID NO:2564.

[0343] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, comprise variable heavy and variable light chain sequences that recognize and bind to the amino acid set forth in SEQ ID NO:2565.

[0344] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, comprise variable heavy and variable light chain sequences that recognize and bind to the amino acid set forth in SEQ ID NO:2566.

[0345] In some embodiments, the anti-CLEC2D antibodies disclosed herein, SEQ ID NO:73 and SEQ ID NO:289, comprise variable heavy and variable light chain sequences that recognize and bind to the amino acid set forth in SEQ ID NO:2567.

[0346] Library screening Without being bound to any particular technique, antibodies that bind to the antigens of the present disclosure can be identified and characterized using the methods described below.

[0347] Provided herein is a naive antibody library as a source of therapeutics for treating diseases including cancer, rheumatoid arthritis, neurological disorders, infectious diseases, and metabolic disorders, or any combination thereof. Antibodies identified using the methods of the present disclosure may be used as diagnostic tools, prognostic tools, for research applications, target discovery, validation in functional genomics, or any application using antibodies or antibody derivatives.

[0348] In one embodiment, the term "panning" refers to an affinity selection technique that selects binding agents for a particular target / antigen.

[0349] In some embodiments, the method for screening a naive antibody gene expression library involves sequentially investigating the expression profile of a pool of gene clones by utilizing two independent scanning tools: 1) phage display technology and 2) yeast display technology (Figure 3). The use of yeast systems for antibody gene expression is advantageous due to the translation, processing of eukaryotic protein, and proper folding of the antibody product on the cell surface. Furthermore, yeast expression allows for proper interaction with antigenic targets with high specificity.

[0350] In some embodiments, the methods disclosed herein maintain diversity in the library, allowing for the identification of unique molecules against a range of antigenic targets.

[0351] In some embodiments of the methods of the present disclosure, the methods also include strategies to transfer and explore diversity between the two platforms as various recombinant antibody formats including, but not limited to, chimeric antibody molecules, Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, ScFab, dimeric and trimeric antibody fragments, minibodies, humanized monoclonal antibody molecules, human antibodies, bispecific antibodies, fusion proteins comprising the Fc region of an antibody and any functional fragments resulting from these molecules (wherein the derivative molecules retain the immunological function of the parent antibody molecule), and all other antibody formats.

[0352] In some embodiments, the candidate antibody molecules obtained by this method are further optimized by rational design guided by structure-function studies of antibody-antigen interactions. The prerequisites for successful manufacturability of monoclonal antibody drugs depend on various biological and / or correlation properties, such as solubility, aggregation, antigenicity, stability, etc. By way of illustration, rational, evidence-based, and more rapid structure-based drug design has greatly contributed to the fields of cancer chemotherapy, drug-resistant infectious diseases, and neurological diseases, among others. The results obtained by these methods are employed in the present disclosure to improve the manufacturability of antibody library construction and selected molecules.

[0353] In some embodiments, the term "isolated" refers to a novel and unique molecule that comprises two protein chains or fragments thereof that are not part of a biological membrane. In particular, the isolated molecules of the present disclosure are soluble and are linked by covalent or non-covalent bonds, either directly or indirectly through a linker molecule. These molecules can include monoclonal or polyclonal antibodies, which can be easily obtained according to methods well known to those skilled in the art.

[0354] In some embodiments, the antigen or antibody disclosed herein may include an affinity tag for isolation or detection purposes. Affinity tags are well known in the art and are used to bind to targets and detect or isolate the targets using molecules that bind to the affinity tag. In general, any peptide or protein that can obtain an antibody or other specific binding substance can be used as an affinity tag. Exemplary affinity tags suitable for use include, but are not limited to, monocyte adaptor protein (MONA)-binding peptides, T7-binding peptides, V5 tags, streptavidin-binding peptides, polyhistidine tracts, protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione S-transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tags (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)), or other antigenic epitopes or binding domains. See generally, Ford et al., Protein Expression and Purification 2:95 (1991). In one embodiment, the methods disclosed herein use a His6 tag. In another embodiment, the methods disclosed herein use a FLAG tag. In another embodiment, the methods and compositions disclosed herein use a V5 tag. DNA molecules encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

[0355] In summary, the methods of the present disclosure focus on identifying, validating, characterizing, and developing novel monoclonal antibodies against the CLEC2D antigenic target for use in therapeutic, diagnostic, and prognostic products applicable to a variety of diseases, including cancer.

[0356] In some embodiments, the antibody library, which may be a naive antibody library, allows for the isolation of unique antibody molecules with desired functional properties against a particular therapeutic target, i.e., antigen, such as the CLEC2D protein disclosed herein or any fragment thereof.

[0357] Combining a diverse library and an appropriate and compatible display platform allows for rapid selection and generation of therapeutic antibodies with higher affinity and superior functionality for specific antigen molecules. Standard screening of directed therapeutic antibody molecules involves selecting molecules from a diverse and large antibody library against the target antigen using a smaller antibody fragment display platform, and then constructing full-length antibody molecules expressed in mammalian cell lines. After expression, a purification process and several functional assays to confirm the purification are performed. The development of selected lead antibodies is further enhanced by optimizing parameters such as epitope identification, formulation, stability testing, and in vivo effect.

[0358] In an exemplary embodiment of the present disclosure, a method for screening, isolating and developing monoclonal antibodies from a human naive antibody library against the CLEC2D antigen comprises the following: designing and generating various CLEC2D antigen constructs, i.e., soluble ectodomains of wild-type and mutant full-length CLEC2D proteins, using appropriately optimized / customized vectors for expression in mammalian systems, followed by purification to homogeneity using affinity chromatography methods.

[0359] In some embodiments, screening of the library of molecules is performed by about 1-3 rounds of phage panning with the CLEC2D antigen. During each round, specific binders are selected from the library by removing non-binders. The pool of selected molecules screened using the phage display platform is introduced to a yeast surface display platform with or without randomization of the selection diversity. This avoids any PCR-based method steps, thereby maintaining the pool of selected molecules against the CLEC2D antigen. The yeast display platform involves expressing various antibody moieties in different formats. The display fragments are screened against a specific antigenic target, and then a specific population showing higher affinity to the target antigen is isolated. The antigen specificity of these selected pools is further tested. Finally, individual clones are isolated and the clonal population is used for sequencing of the individual antibody clones.

[0360] The method for phage panning against antigen is well known in the art. For example, magnetic beads may be used. Antigen coated on magnetic Dynabeads may be prepared, and phage antibody library may be panned against the antigen-coated beads to separate phage particles expressing desired antibody clones.

[0361] The purified DNA may then be digested and ligated into a suitable yeast expression vector to produce the antibody in the desired format, e.g., Fab or ScFv. Standard methods may be used to transform yeast cells and confirm antibody expression. Multiple tags for immunohistochemistry, e.g., FLAG tag, c-Myc tag, and (His) tag, for the heavy and light chains, respectively, may be added. 6The surface expression of the antibody may be analyzed using tags, as well as V5 tags, etc. Flow cytometry may be used to isolate yeast cells that express antibody sequences that exhibit specific antigen binding. Flow cytometry sorting of yeast cell population may be repeated at least 1x, at least 2x, at least 3x, at least 4x, or at least 5x to enrich for antibody clones with higher affinity to labeled antigen.

[0362] The individual yeast clones are sequenced using standard methods in the art and the antibody sequences are then further cloned into a suitable mammalian gene expression vector.

[0363] The present disclosure provides a method for screening a high diversity antibody gene library for antibodies that bind to a CLEC2D antigen. In some embodiments, the method includes inserting a library of antibody genes into a phage protein gene using a vector to transform the phage to generate a phage library that includes a high diversity antibody gene library. The phage in the phage library display the library of antibody genes on the surface of the phage. The phage library is then panned with the CLEC2D antigen against individual phages that bind to the CLEC2D antigen to generate an enriched phage library that is enriched for antibody genes that encode antibodies that bind to the CLEC2D antigen. This panning can be performed, for example, by conjugating the antigen to magnetic beads and can be used to isolate phages that bind to the antigen on the beads. The panning step can be repeated at least once, at least twice, or more times to enrich for phages that express antibodies or antibody fragments that bind to the antigen.

[0364] The antibody or antibody fragment genes from the enriched phage library are then introduced into the yeast surface display library. In some embodiments, this is done by cloning the antibody or antibody fragment genes into a suitable yeast transformation vector and then transforming the yeast cells using methods standard in the art. The yeast cells expressing the antibody or antibody fragment that binds to the CLEC2D antigen are then isolated. In some embodiments, this isolation is done using flow cytometry to sort the yeast cells. In some embodiments, the method further comprises repeating the flow cytometry isolation at least 1×, at least 2×, at least 3×, at least 4×, or at least 5×, or more times to enrich for the yeast cells expressing the antibody or antibody fragment that binds to the antigen. In some embodiments, the method further comprises analyzing the surface expression of the antibody genes using a FLAG tag, a c-Myc tag, a polyhistidine tag, or a V5 tag. In some embodiments, the method further comprises cloning the antibody genes that bind to CLEC2D into a mammalian expression vector.

[0365] Optimization and Refinement In some embodiments, the methods disclosed herein include the design, creation, and optimization of vector constructs for the smooth introduction of selected antibody gene sequences into mammalian cell lines for expression, such as Chinese Hamster Ovary (CHO) cell lines, the generation of stable cell lines, and the subsequent purification of full-length monoclonal antibodies, allowing for the rapid and efficient establishment of stable cell lines expressing monoclonal antibodies with excellent uniformity in terms of conformation and post-translational modifications found in downstream processes.

[0366] All cell lines suitable for expression and purification of antibodies or antibody fragments are considered within the scope of this disclosure. In some embodiments, the cell line is a mammalian cell line. The cell line may be isolated or derived from any source, including human, mouse, and hamster. Suitable cell lines include Chinese Hamster Ovary (CHO) cells, HEK 293 cells, HEK293T cells, BHK21 cells, NSO cells, PER.C6 cells, B cells, HEK These include, but are not limited to, 293-6E cells, Sp2 / 0-Ag14 cells, and DG44 cells.

[0367] In some embodiments, the CDR length and amino acid composition of the antibody clones are analyzed to understand the novelty of these clones. Additional careful analysis is performed to remove clones with unfavorable motifs for purification strategy, stability, and physicochemical properties that directly affect the charge variants present or within the antibody.

[0368] In some embodiments, mass production of the lead antibody clone is achieved using defined media, supplements, and specific bioreactor processes well known in the art and described herein.

[0369] The exemplary purification method of the present disclosure includes multiple steps of chromatographic techniques that utilize the physicochemical properties of the amino acid composition in the antibody molecule. In addition, higher purity can be achieved by effectively removing the host cell proteins / impurities, polymers (or aggregates) of the antibody, improving the antibody recovery rate. The purification of the antibody molecule is completed with a suitable formulation that further improves the stability.

[0370] Therapeutic compositions must be sterile and stable under the conditions of manufacture and storage.

[0371] Antibody purification processes are well known to those of skill in the art. Without being bound to any particular process, an exemplary antibody purification process involves centrifuging a primary cell culture expressing the antibody or antibody fragment to be purified and then further clarifying it using a filter, such as a 3 μm to 30 μm filter. The collected filtrate may then be further filtered, for example, through a 0.22 μm filter. The sample may then be loaded onto a column for further purification using liquid chromatography. An exemplary column includes, but is not limited to, an XK 16 / 20 Protein A column. Liquid chromatography may include treatment with a high salt wash buffer to remove loosely bound host cell proteins and other impurities....

Claims

[Claim 1] The invention described in the specification.