Anti-cd161 antibodies and uses thereof

Abstract

The present invention belongs to the field of biomedicine and relates to anti-CD161 antibodies. The present invention also relates to nucleic acid molecules, vectors and host cells encoding the antibodies, to derivatives of said antibodies, and to their use for the treatment of diseases.

Description

Anti-CD161 antibodies and uses thereof

[0001] The present invention claims priority to a Chinese patent application filed on March 15, 2024 (application number: CN202410305500.X, invention title: anti-CD161 antibodies and their uses). The entire contents of the priority patent application are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedicine and relates to anti-CD161 antibodies and their applications. The present invention also relates to nucleic acid molecules, vectors, and host cells encoding the antibodies and antigen-binding fragments thereof, derivatives of the antibodies and antigen-binding fragments thereof, and their use in treating diseases. Technical Background

[0003] Tumor heterogeneity is a major challenge facing cancer treatment approaches, including immunotherapy. During drug treatment, a small number of tumor cells, influenced by the tumor microenvironment, can develop into drug-resistant clones through acquired mutations and epigenetic remodeling, thereby evading the immune response. T lymphocyte-mediated cellular immunity is the primary mechanism of anti-tumor immunity. Currently, widely used inhibitors of the PD-1 / L1 immune checkpoint target targeting T cells have a clinical response rate of only 20% to 30%. Immune checkpoint drugs targeting natural killer (NK) cells are mostly in clinical trials and have not yet been approved for marketing. Leveraging the synergistic effects of various immune cells (T cells, NK cells, and DCs) in tumor immunotherapy to overcome tumor cell heterogeneity will have significant clinical value. Research has shown that T cells and NK cells can use different recognition strategies to identify cancer cells, complementing each other in tumor immunotherapy. NK cells can kill cancer cells that have escaped CD8+ T cell recognition. Therefore, the dual attack of T cells and NK cells significantly enhances the efficacy of immunotherapy. On the other hand, NK cells play an important role in recruiting dendritic cells (DCs) to tumors, thereby enhancing the induction of CD8+ T cell responses, while IL-2 secreted by T cells activates NK cells.

[0004] Although NK cells and T cells belong to the innate and adaptive branches of the immune system, the biology of T cells in tumors is highly intertwined with that of NK cells. CD161 (also known as CLEC5B and NKR-P1A) and Clec2D (LLT1) are a common pair of immunosuppressive receptor ligands expressed on both T cells and NK cells. Clec2D is highly expressed on activated myeloid cells (DCs, macrophages) and a variety of tumor cells, including B-cell-derived non-Hodgkin's lymphomas, gliomas, and squamous cell carcinomas of the head and neck. In NK cells, CD161 acts as an inhibitory receptor on the cell surface. It can inhibit NK cell IFN-γ release through both cis-action (NK cells express both CD161 and Clec2D) and trans-action (tumor-infiltrating immune cells or tumor cells express Clec2D), thereby reducing NK cell cytotoxicity. In T cells, activation of CD161 signaling can further activate AKT1 / PKB and RPS6KA1 / RSK1 kinases and their downstream signaling, promoting anti-CD3-induced T cell proliferation. Summary of the Invention

[0005] After in-depth research, the inventors of the present invention have obtained a series of anti-CD161 antibodies. In some embodiments, the antibodies can bind to human and cynomolgus monkey CD161 proteins with high affinity at the cellular level and protein level, and can effectively block the binding of CD161 to its receptor Clec2D at the cellular level. In some embodiments, the antibodies of the present invention can effectively promote the activation of T cells mediated by CD3 antibodies, promote the killing ability of PBMCs against tumor cells, dose-dependently enhance NK cell degranulation and promote the killing effect of NK cells on tumor cells. In addition, in some embodiments, the antibodies of the present invention also have species cross-binding activity and activation activity for human and monkey CD161, which is beneficial for carrying out preclinical evaluation studies in animal models.

[0006] Antibody

[0007] In one aspect, the present invention provides an anti-CD161 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), and the anti-CD161 antibody comprises:

[0008] (1) three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in any one of SEQ ID NOs: 69-72; and three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO: 79;

[0009] (2) three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in SEQ ID NO: 68; and three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO: 79 or 80;

[0010] (3) three complementarity determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO:49; and three complementarity determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO:61;

[0011] (4) the three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in any one of SEQ ID NOs: 69-72; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO: 80;

[0012] (5) three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in any one of SEQ ID NOs:73-75; and three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO:61;

[0013] (6) the three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in any one of SEQ ID NOs: 76-78; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO: 79;

[0014] (7) three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in SEQ ID NO:49; and three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO:66;

[0015] (8) three complementarity determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO:49; and three complementarity determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO:67;

[0016] (9) three complementarity determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 50; and three complementarity determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 62;

[0017] (10) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 51; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 63;

[0018] (11) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 52; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 61;

[0019] (12) three complementary determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 53; and three complementary determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 61;

[0020] (13) three complementary determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 54; and three complementary determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 64;

[0021] (14) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 55; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 65;

[0022] (15) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 54; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 61;

[0023] (16) three complementary determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 56; and three complementary determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 61;

[0024] (17) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 57; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 61;

[0025] (18) three complementary determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 58; and three complementary determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 61;

[0026] (19) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 59; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 61; or

[0027] (20) The three complementarity determining regions HCDR1-HCDR3 contained in the VH shown in SEQ ID NO:60; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL shown in SEQ ID NO:61.

[0028] In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT, Abm, Contact or Chothia numbering systems. In certain embodiments, the CDRs are defined according to the Kabat numbering system.

[0029] In certain embodiments, the anti-CD161 antibody, wherein:

[0030] (1) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 5, HCDR2 comprises the sequence of SEQ ID NO: 13, and HCDR3 comprises the sequence of SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of SEQ ID NO: 32, LCDR2 comprises the sequence of SEQ ID NO: 37, and LCDR3 comprises the sequence of SEQ ID NO: 42;

[0031] (2) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 6, and HCDR3 comprises the sequence shown in SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42;

[0032] (3) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 5, HCDR2 comprises the sequence of any one of SEQ ID NOs: 14-16, and HCDR3 comprises the sequence of SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of SEQ ID NO: 32, LCDR2 comprises the sequence of SEQ ID NO: 37, and LCDR3 comprises the sequence of SEQ ID NO: 42;

[0033] (4) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 5, HCDR2 comprises the sequence of any one of SEQ ID NOs: 17-19, and HCDR3 comprises the sequence of SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of SEQ ID NO: 32, LCDR2 comprises the sequence of SEQ ID NO: 37, and LCDR3 comprises the sequence of SEQ ID NO: 42;

[0034] (5) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 6, and HCDR3 comprises the sequence shown in SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 41, and LCDR3 comprises the sequence shown in SEQ ID NO: 47;

[0035] (6) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 6, and HCDR3 comprises the sequence shown in SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 36, LCDR2 comprises the sequence shown in SEQ ID NO: 41, and LCDR3 comprises the sequence shown in SEQ ID NO: 48

[0036] (7) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 7, and HCDR3 comprises the sequence shown in SEQ ID NO: 21; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 33, LCDR2 comprises the sequence shown in SEQ ID NO: 38, and LCDR3 comprises the sequence shown in SEQ ID NO: 43;

[0037] (8) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 8, and HCDR3 comprises the sequence shown in SEQ ID NO: 22; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 33, LCDR2 comprises the sequence shown in SEQ ID NO: 38, and LCDR3 comprises the sequence shown in SEQ ID NO: 44;

[0038] (9) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 23; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42;

[0039] (10) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 24; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42;

[0040] (11) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 10, and HCDR3 comprises the sequence shown in SEQ ID NO: 25; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 34, LCDR2 comprises the sequence shown in SEQ ID NO: 39, and LCDR3 comprises the sequence shown in SEQ ID NO: 45;

[0041] (12) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 8, and HCDR3 comprises the sequence shown in SEQ ID NO: 26; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 35, LCDR2 comprises the sequence shown in SEQ ID NO: 40, and LCDR3 comprises the sequence shown in SEQ ID NO: 46;

[0042] (13) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 10, and HCDR3 comprises the sequence shown in SEQ ID NO: 25; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42;

[0043] (14) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 11, and HCDR3 comprises the sequence shown in SEQ ID NO: 27; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42;

[0044] (15) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 28; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42;

[0045] (16) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 29; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42;

[0046] (17) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 30, and the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; or

[0047] (18) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 12, and HCDR3 comprises the sequence shown in SEQ ID NO: 31; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42.

[0048] In certain embodiments, the antibody, wherein:

[0049] (1) The VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 13, and HCDR3 of SEQ ID NO: 20, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0050] (2) the VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6, and HCDR3 of SEQ ID NO: 20, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0051] (3) the VH of the anti-CD161 antibody comprises the HCDR1 of SEQ ID NO: 5, the HCDR2 of any one of SEQ ID NOs: 14-16, and the HCDR3 of SEQ ID NO: 20, and the VL of the anti-CD161 antibody comprises the LCDR1 of SEQ ID NO: 32, the LCDR2 of SEQ ID NO: 37, and the LCDR3 of SEQ ID NO: 42;

[0052] (4) the VH of the anti-CD161 antibody comprises the HCDR1 of SEQ ID NO: 5, the HCDR2 of any one of SEQ ID NOs: 17-19, and the HCDR3 of SEQ ID NO: 20, and the VL of the anti-CD161 antibody comprises the LCDR1 of SEQ ID NO: 32, the LCDR2 of SEQ ID NO: 37, and the LCDR3 of SEQ ID NO: 42;

[0053] (5) the VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6, and HCDR3 of SEQ ID NO: 20, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 41, and LCDR3 of SEQ ID NO: 47;

[0054] (6) The VH of the anti-CD161 antibody comprises HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6, and HCDR3 shown in SEQ ID NO: 20, and the VL of the anti-CD161 antibody comprises LCDR1 shown in SEQ ID NO: 36, LCDR2 shown in SEQ ID NO: 41, and LCDR3 shown in SEQ ID NO: 48

[0055] (7) the VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 21, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 33, LCDR2 of SEQ ID NO: 38, and LCDR3 of SEQ ID NO: 43;

[0056] (8) The VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 22, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 33, LCDR2 of SEQ ID NO: 38, and LCDR3 of SEQ ID NO: 44;

[0057] (9) the VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 9, and HCDR3 of SEQ ID NO: 23, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0058] (10) The VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 9, and HCDR3 of SEQ ID NO: 24, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0059] (11) The VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 25, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 45;

[0060] (12) the VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 26, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 40, and LCDR3 of SEQ ID NO: 46;

[0061] (13) The VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 25, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0062] (14) the VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 11, and HCDR3 of SEQ ID NO: 27, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0063] (15) The VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 9, and HCDR3 of SEQ ID NO: 28, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0064] (16) The VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 9, and HCDR3 of SEQ ID NO: 29, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0065] (17) the VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 9, and HCDR3 of SEQ ID NO: 30, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42; or

[0066] (18) The VH of the anti-CD161 antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 12, and HCDR3 of SEQ ID NO: 31, and the VL of the anti-CD161 antibody comprises LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 37, and LCDR3 of SEQ ID NO: 42;

[0067] In certain embodiments, the CDRs are defined by the Kabat numbering system.

[0068] In certain embodiments, the present invention provides an anti-CD161 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0069] (1) The VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 68-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises a sequence shown in SEQ ID NOs: 79, 80 or a variant thereof;

[0070] (2) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0071] (3) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 73-75 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0072] (4) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 76-78 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof;

[0073] (5) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 66 or a variant thereof;

[0074] (6) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 67 or a variant thereof;

[0075] (7) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 50 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 62 or a variant thereof;

[0076] (8) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 51 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 63 or a variant thereof;

[0077] (9) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 52 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0078] (10) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 53 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0079] (11) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 54 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 64 or a variant thereof;

[0080] (12) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 55 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 65 or a variant thereof;

[0081] (13) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 54 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0082] (14) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 56 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0083] (15) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 57 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0084] (16) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 58 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0085] (17) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 59 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; or

[0086] (18) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 60 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0087] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived; wherein "having at least 80% sequence identity compared to the sequence from which it is derived" means having at least 80% sequence identity compared to its parent sequence, for example, "a variant of the sequence shown in SEQ ID NO: 49" is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity to SEQ ID NO: 49, the same below. In certain embodiments, the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions, or additions in the framework regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0088] In certain embodiments, the present disclosure provides an anti-CD161 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0089] (1) The VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 69-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof;

[0090] (2) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 68 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof;

[0091] (3) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 68 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 80 or a variant thereof;

[0092] (4) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 69-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 80 or a variant thereof;

[0093] (5) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 73-75 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0094] (6) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 76-78 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof;

[0095] (7) The VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 68-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises a sequence shown in SEQ ID NOs: 79, 80 or a variant thereof;

[0096] (8) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0097] (9) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 66 or a variant thereof;

[0098] (10) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 67 or a variant thereof;

[0099] (11) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 50 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 62 or a variant thereof;

[0100] (12) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 51 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 63 or a variant thereof;

[0101] (13) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 52 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0102] (14) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 53 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0103] (15) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 54 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 64 or a variant thereof;

[0104] (16) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 55 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 65 or a variant thereof;

[0105] (17) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 54 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0106] (18) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 56 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0107] (19) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 57 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0108] (20) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 58 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0109] (21) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 59 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; or

[0110] (22) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 60 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0111] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived; wherein "having at least 80% sequence identity compared to the sequence from which it is derived" means having at least 80% sequence identity compared to its parent sequence, for example, "a variant of the sequence shown in SEQ ID NO: 49" is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity to SEQ ID NO: 49, the same below. In certain embodiments, the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions, or additions in the framework regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0112] In certain embodiments, the antibodies comprise framework region sequences derived from murine immunoglobulins.

[0113] In certain embodiments, the anti-CD161 antibody of any of the preceding items, wherein:

[0114] (1) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0115] (2) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 66 or a variant thereof;

[0116] (3) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 67 or a variant thereof;

[0117] (4) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 50 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 62 or a variant thereof;

[0118] (5) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 51 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 63 or a variant thereof;

[0119] (6) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 52 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0120] (7) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 53 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0121] (8) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 54 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 64 or a variant thereof;

[0122] (9) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 55 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 65 or a variant thereof;

[0123] (10) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 54 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0124] (11) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 56 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0125] (12) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 57 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0126] (13) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 58 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0127] (14) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 59 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0128] (15) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 60 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; or

[0129] (16) The VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 73-75 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof;

[0130] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived; in certain embodiments, the variant is a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant is a sequence having at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions or additions in the framework regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0131] In certain embodiments, the anti-CD161 antibody of any of the foregoing comprises:

[0132] (1) VH set forth in SEQ ID NO:49 or a variant thereof, and VL set forth in SEQ ID NO:61 or a variant thereof;

[0133] (2) the VH set forth in SEQ ID NO:49 or a variant thereof, and the VL set forth in SEQ ID NO:66 or a variant thereof;

[0134] (3) the VH set forth in SEQ ID NO:49 or a variant thereof, and the VL set forth in SEQ ID NO:67 or a variant thereof;

[0135] (4) VH represented by SEQ ID NO: 50 or a variant thereof, and VL represented by SEQ ID NO: 62 or a variant thereof;

[0136] (5) VH represented by SEQ ID NO: 51 or a variant thereof, and VL represented by SEQ ID NO: 63 or a variant thereof;

[0137] (6) VH represented by SEQ ID NO: 52 or a variant thereof, and VL represented by SEQ ID NO: 61 or a variant thereof;

[0138] (7) VH represented by SEQ ID NO: 53 or a variant thereof, and VL represented by SEQ ID NO: 61 or a variant thereof;

[0139] (8) VH represented by SEQ ID NO: 54 or a variant thereof, and VL represented by SEQ ID NO: 64 or a variant thereof;

[0140] (9) VH represented by SEQ ID NO: 55 or a variant thereof, and VL represented by SEQ ID NO: 65 or a variant thereof;

[0141] (10) VH represented by SEQ ID NO: 54 or a variant thereof, and VL represented by SEQ ID NO: 61 or a variant thereof;

[0142] (11) VH represented by SEQ ID NO: 56 or a variant thereof, and VL represented by SEQ ID NO: 61 or a variant thereof;

[0143] (12) VH represented by SEQ ID NO: 57 or a variant thereof, and VL represented by SEQ ID NO: 61 or a variant thereof;

[0144] (13) VH represented by SEQ ID NO: 58 or a variant thereof, and VL represented by SEQ ID NO: 61 or a variant thereof;

[0145] (14) VH represented by SEQ ID NO: 59 or a variant thereof, and VL represented by SEQ ID NO: 61 or a variant thereof;

[0146] (15) the VH set forth in SEQ ID NO: 60 or a variant thereof, and the VL set forth in SEQ ID NO: 61 or a variant thereof; or

[0147] (16) VH represented by any one of SEQ ID NOs: 73-75 or variants thereof, and VL represented by SEQ ID NO: 61 or variants thereof;

[0148] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived. In certain embodiments, the variant is a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant is a sequence having at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions or additions in the framework regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0149] In certain embodiments, the anti-CD161 antibody of any of the foregoing comprises:

[0150] (1) VH represented by SEQ ID NO:49, and VL represented by SEQ ID NO:61;

[0151] (2) VH represented by SEQ ID NO:49, and VL represented by SEQ ID NO:66;

[0152] (3) VH represented by SEQ ID NO:49, and VL represented by SEQ ID NO:67;

[0153] (4) VH shown in SEQ ID NO: 50, and VL shown in SEQ ID NO: 62;

[0154] (5) VH represented by SEQ ID NO: 51, and VL represented by SEQ ID NO: 63;

[0155] (6) VH shown in SEQ ID NO: 52, and VL shown in SEQ ID NO: 61;

[0156] (7) VH shown in SEQ ID NO: 53, and VL shown in SEQ ID NO: 61;

[0157] (8) VH shown in SEQ ID NO: 54, and VL shown in SEQ ID NO: 64;

[0158] (9) VH shown in SEQ ID NO: 55, and VL shown in SEQ ID NO: 65;

[0159] (10) VH represented by SEQ ID NO: 54, and VL represented by SEQ ID NO: 61;

[0160] (11) VH represented by SEQ ID NO: 56, and VL represented by SEQ ID NO: 61;

[0161] (12) VH represented by SEQ ID NO: 57, and VL represented by SEQ ID NO: 61;

[0162] (13) VH represented by SEQ ID NO: 58, and VL represented by SEQ ID NO: 61;

[0163] (14) VH represented by SEQ ID NO: 59, and VL represented by SEQ ID NO: 61;

[0164] (15) the VH represented by SEQ ID NO: 60, and the VL represented by SEQ ID NO: 61; or

[0165] (16) VH shown in any one of SEQ ID NOs: 73-75, and VL shown in SEQ ID NO: 61.

[0166] In certain embodiments, the antibodies are humanized and comprise framework region sequences derived from human immunoglobulins.

[0167] In certain embodiments, the antibody comprises a framework region contained in the amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody comprises: a heavy chain framework region of a human heavy chain germline sequence, and / or a light chain framework region of a human light chain germline sequence. In certain embodiments, the heavy chain framework region of the human heavy chain germline sequence and / or the light chain framework region of the human light chain germline sequence may optionally include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9 or at most 10) back mutations from human residues to mouse residues. In certain embodiments, the heavy chain framework region of the human heavy chain germline sequence and / or the light chain framework region of the human light chain germline sequence include no more than 10 amino acid substitutions.

[0168] In certain embodiments, the anti-CD161 antibody of any of the foregoing items comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0169] (1) The VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 69-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof;

[0170] (2) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 68 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof;

[0171] (3) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 68 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 80 or a variant thereof;

[0172] (4) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 69-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 80 or a variant thereof;

[0173] (5) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 73-75 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; or

[0174] (6) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 76-78 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof;

[0175] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived. In certain embodiments, the variant is a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant is a sequence having at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions or additions in the framework regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0176] In certain embodiments, the anti-CD161 antibody of any of the foregoing comprises:

[0177] (1) VH set forth in SEQ ID NO: 68 or a variant thereof, and VL set forth in SEQ ID NO: 79 or a variant thereof;

[0178] (2) the VH set forth in SEQ ID NO: 68 or a variant thereof, and the VL set forth in SEQ ID NO: 80 or a variant thereof;

[0179] (3) the VH set forth in any one of SEQ ID NOs: 69-72 or a variant thereof, and the VL set forth in SEQ ID NO: 79 or a variant thereof; or

[0180] (4) the VH set forth in any one of SEQ ID NOs: 69-72 or variants thereof, and the VL set forth in SEQ ID NO: 80 or variants thereof;

[0181] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived. In certain embodiments, the variant is a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant is a sequence having at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions or additions in the framework regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0182] In certain embodiments, the anti-CD161 antibody of any of the foregoing comprises:

[0183] (1) VH represented by SEQ ID NO:68, and VL represented by SEQ ID NO:79;

[0184] (2) VH shown in SEQ ID NO:68, and VL shown in SEQ ID NO:80;

[0185] (3) the VH set forth in any one of SEQ ID NOs: 69-72, and the VL set forth in SEQ ID NO: 79; or

[0186] (4) The VH shown in any one of SEQ ID NOs: 69-72, and the VL shown in SEQ ID NO: 80.

[0187] In certain embodiments, the heavy chain variable region of the antibody comprises a mutation in a PTM site to eliminate the post-translational modification of the antibody, thereby maintaining the immunogenicity and structural stability of the antibody.

[0188] In certain embodiments, the anti-CD161 antibody of any of the foregoing comprises:

[0189] (1) the VH set forth in any one of SEQ ID NOs: 73-75 or variants thereof, and the VL set forth in SEQ ID NO: 61 or variants thereof; or

[0190] (2) the VH set forth in any one of SEQ ID NOs: 76-78 or variants thereof, and the VL set forth in SEQ ID NO: 79 or variants thereof;

[0191] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived. In certain embodiments, the variant is a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant is a sequence having at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions or additions in the framework regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0192] In certain embodiments, the anti-CD161 antibody of any of the foregoing comprises:

[0193] (1) the VH set forth in any one of SEQ ID NOs: 73-75, and the VL set forth in SEQ ID NO: 61; or

[0194] (2) VH shown in any one of SEQ ID NOs: 76-78, and VL shown in SEQ ID NO: 79.

[0195] In certain embodiments, the antibody further comprises a constant region derived from a mammalian (eg, human or murine) immunoglobulin.

[0196] In certain embodiments, the heavy chain of the antibody comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3 or IgG4), and / or the light chain of the antibody comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

[0197] In certain embodiments, the heavy chain constant region of the antibody comprises the mutation N297A to reduce ADCC effector function.

[0198] In certain exemplary embodiments, the antibody comprises the heavy chain constant region set forth in SEQ ID NO:81, and / or the light chain constant region set forth in SEQ ID NO:82.

[0199] In certain embodiments, the heavy chain of the anti-CD161 antibody comprises any one of SEQ ID NOs: 85-89 or variants thereof, and the light chain of the anti-CD161 antibody comprises a sequence shown in SEQ ID NOs: 96, 97 or variants thereof.

[0200] In certain embodiments, the anti-CD161 antibody of any of the foregoing comprises a heavy chain and a light chain, wherein:

[0201] (1) The heavy chain comprises any one of SEQ ID NOs: 86-89 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 96 or a variant thereof;

[0202] (2) the heavy chain comprises the sequence shown in SEQ ID NO: 85 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 96 or a variant thereof;

[0203] (3) the heavy chain comprises the sequence shown in SEQ ID NO: 85 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 97 or a variant thereof;

[0204] (4) the heavy chain comprises the sequence shown in SEQ ID NO: 83 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 84 or a variant thereof;

[0205] (5) the heavy chain comprises any one of SEQ ID NOs: 86-89 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 97 or a variant thereof;

[0206] (6) the heavy chain comprises any one of SEQ ID NOs: 90-92 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 84 or a variant thereof; or

[0207] (7) The heavy chain comprises any one of SEQ ID NOs: 93-95 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 96 or a variant thereof;

[0208] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived. In certain embodiments, the variant is a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant is a sequence having at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions or additions in the framework or constant regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0209] In certain embodiments, the anti-CD161 antibody of any of the foregoing comprises:

[0210] (1) the heavy chain represented by any one of SEQ ID NOs: 86-89 or a variant thereof, and the light chain represented by SEQ ID NO: 96 or a variant thereof;

[0211] (2) the heavy chain set forth in SEQ ID NO:85 or a variant thereof, and the light chain set forth in SEQ ID NO:96 or a variant thereof;

[0212] (3) the heavy chain set forth in SEQ ID NO:85 or a variant thereof, and the light chain set forth in SEQ ID NO:97 or a variant thereof;

[0213] (4) the heavy chain represented by SEQ ID NO: 83 or a variant thereof, and the light chain represented by SEQ ID NO: 84 or a variant thereof;

[0214] (5) the heavy chain represented by any one of SEQ ID NOs: 86-89 or a variant thereof, and the light chain represented by SEQ ID NO: 97 or a variant thereof;

[0215] (6) the heavy chain set forth in any one of SEQ ID NOs: 90-92 or a variant thereof, and the light chain set forth in SEQ ID NO: 84 or a variant thereof; or

[0216] (7) the heavy chain represented by any one of SEQ ID NOs: 93-95 or a variant thereof, and the light chain represented by SEQ ID NO: 96 or a variant thereof;

[0217] Wherein, the variant is a sequence having at least 80% (e.g., 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%, or 100%) sequence identity compared to the sequence from which it is derived. In certain embodiments, the variant is a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant is a sequence having at most 10 amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the variant has one or more amino acid substitutions, deletions or additions in the framework or constant regions compared to the sequence from which it is derived. In certain embodiments, the CDRs of the variant are identical to the CDRs of the sequence from which it is derived.

[0218] In certain exemplary embodiments, the anti-CD161 antibody of any of the foregoing comprises:

[0219] (1) the heavy chain represented by SEQ ID NO: 83, and the light chain represented by SEQ ID NO: 84;

[0220] (2) the heavy chain represented by SEQ ID NO:85, and the light chain represented by SEQ ID NO:96;

[0221] (3) the heavy chain represented by SEQ ID NO:85, and the light chain represented by SEQ ID NO:97;

[0222] (4) the heavy chain of any one of SEQ ID NOs: 86-89, and the light chain of SEQ ID NO: 96;

[0223] (5) the heavy chain shown in any one of SEQ ID NOs: 86-89, and the light chain shown in SEQ ID NO: 97;

[0224] (6) the heavy chain of any one of SEQ ID NOs: 90-92, and the light chain of SEQ ID NO: 84; or

[0225] (7) The heavy chain shown in any one of SEQ ID NOs: 93-95, and the light chain shown in SEQ ID NO: 96.

[0226] In certain exemplary embodiments, an anti-CD161 antibody is provided that binds to the same epitope as any of the above anti-CD161 antibodies or competes with any of the above anti-CD161 antibodies for binding to human CD161 antigen.

[0227] In certain exemplary embodiments, the antibody of any of the preceding items, wherein the antibody is a whole antibody or an antigen-binding fragment.

[0228] In certain exemplary embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, and diabody.

[0229] In certain exemplary embodiments, the anti-CD161 antibody is a murine antibody, a humanized antibody, a chimeric antibody, or a multispecific antibody.

[0230] In certain exemplary embodiments, the anti-CD161 antibody has at least one of the following functions:

[0231] (1) specifically binds to human CD161 antigen and / or specifically binds to cynomolgus macaque CD161 antigen;

[0232] (2) blocking the binding of CD161 to its receptor Clec2D;

[0233] (3) Promote CD3 antibody-mediated T cell activation;

[0234] (4) Promote the killing ability of PBMCs against tumor cells;

[0235] (5) Promote NK cell killing of tumor cells and / or enhance NK cell degranulation.

[0236] In certain exemplary embodiments, any of the above antibodies, wherein the anti-CD161 antibody specifically binds to human CD161 antigen, and / or specifically binds to cynomolgus monkey CD161 antigen. In certain exemplary embodiments, the anti-CD161 antibody can bind to human CD161 protein with a KD value less than 10.00E-9M (e.g., less than 10.00E-9M, 5.00E-9M, less than 1.00E-9M, less than 5.00E-10M, less than 1.00E-10M, less than 5.00E-11M, less than 1.00E-11M, less than 1.00E-12M or less, or any value therebetween), and / or the anti-CD161 antibody can bind to human CD161 protein with a KD value less than 10.00E-8M (e.g., less than 10.00E-9M, 5.00E-9M, less than 1.00E-9M, less than 5.00E-10M, less than 1.00E-10M, less than 5.00E-11M, less than 1.00E-11M, less than 1.00E-12M or less, or any value therebetween). For example, a KD value of less than 10.00E-8M, less than 5.00E-8M, less than 1.00E-8M, less than 10.00E-9M, less than 7.00E-9M, less than 1.00E-9M, less than 1.00E-10M, less than 1.00E-11M, less than 1.00E-12M or less, or any value therebetween) binds to the cynomolgus monkey CD161 protein, and the KD value is detected by surface plasmon resonance technology; in some embodiments, the KD value is detected by the method described in Example 4 of the present invention.

[0237] In certain exemplary embodiments, the antibody of any of the preceding items, wherein the anti-CD161 antibody promotes NK cell killing of tumor cells and / or enhances NK cell degranulation. In certain exemplary embodiments, the anti-CD161 antibody can activate NK cells with an EC50 value of less than 1 nM (e.g., less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, less than 0.04 nM, less than 0.03 nM, less than 0.01 nM, less than 0.001 nM or less, or any value therebetween), and / or the anti-CD161 antibody can kill target cells with an EC50 value of less than 1 nM (e.g., less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, less than 0.03 nM, less than 0.02 nM, less than 0.01 nM, less than 0.001 nM or less, or any value therebetween), and the EC50 value is detected by a FACS method; in some embodiments, the target cells are Daudi-HuClec2D; in some embodiments, the EC50 value is detected by the method described in Example 11 of the present invention.

[0238] In certain exemplary embodiments, an antibody is provided that binds to the same epitope as any of the above anti-CD161 antibodies or competes with any of the above anti-CD161 antibodies for binding to the human CD161 antigen.

[0239] In another aspect, the present invention provides a multispecific molecule comprising an anti-CD161 antibody of the present invention.

[0240] In certain embodiments, the multispecific molecule specifically binds CD161 and additionally specifically binds one or more other targets.

[0241] In certain embodiments, the multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody).

[0242] Antibody preparation

[0243] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant techniques. For example, a DNA molecule encoding the antibody of the present invention can be obtained by chemical synthesis or PCR amplification, the resulting DNA molecule can be inserted into an expression vector, and then transfected into a host cell. The transfected host cell can then be cultured under specific conditions to express the antibody of the present invention. The antigen-binding fragment of the present invention can be obtained by hydrolyzing the intact antibody molecule.

[0244] On the other hand, the invention provides a kind of isolated nucleic acid molecules, its encoding antibody of the present invention, or its heavy chain variable region and / or light chain variable region, or its heavy chain and / or light chain.In certain embodiments, described nucleic acid molecules encoding front any one described anti-CD161 antibody.

[0245] In certain embodiments, the nucleic acid molecule comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different nucleic acid molecules, the nucleic acid molecule of the present invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0246] In certain embodiments, the nucleic acid molecule comprises a first nucleotide sequence encoding the heavy chain variable region of an antibody of the present invention, and a second nucleotide sequence encoding the light chain variable region thereof, wherein:

[0247] (1) the first nucleotide sequence comprises: the sequence of SEQ ID NO: 98, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence of SEQ ID NO: 99, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof;

[0248] (2) The first nucleotide sequence comprises: a sequence as shown in any one of SEQ ID NOs: 100-104, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; The second nucleotide sequence comprises: SEQ ID NOs: 100-104, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; The sequence shown in NO:105, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof;

[0249] (3) The first nucleotide sequence comprises: a sequence as set forth in any one of SEQ ID NOs: 100-104, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; and the second nucleotide sequence comprises: a sequence as set forth in any one of SEQ ID NOs: 100-104, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; The sequence shown in NO:106, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof;

[0250] (4) The first nucleotide sequence comprises: a sequence as shown in any one of SEQ ID NOs: 114-116, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; The second nucleotide sequence comprises: SEQ ID NOs: 114-116, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; NO:99, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; or

[0251] (5) The first nucleotide sequence comprises: a sequence as shown in any one of SEQ ID NOs: 101-103, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; The second nucleotide sequence comprises: SEQ ID NOs: 101-103, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity thereto, or a degenerate sequence thereof; The invention relates to a sequence of the invention that is a member of a class I or a class I of the invention, or a sequence having at least 80% (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity compared to the sequence shown in NO:104 ...

[0252] In another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising a nucleic acid molecule as described above. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, or the like.

[0253] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0254] In another aspect, the present invention provides a host cell comprising a nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0255] In another aspect, a method for preparing the antibody of the present invention is provided, comprising the steps of:

[0256] The host cell described in any one of the above items is cultured under conditions that allow protein expression, and the antibody is recovered from the cultured host cell culture.

[0257] Conjugate

[0258] In another aspect, the present invention also provides a conjugate comprising any of the above antibodies and a coupling moiety.

[0259] In certain embodiments, the antibodies of the present invention are conjugated to the coupling moiety, optionally via a linker.

[0260] In certain embodiments, the coupling moiety is selected from a protein tag. Such protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select an appropriate protein tag (e.g., a purification tag, a detection tag, or a tracer tag) according to the desired purpose. In certain exemplary embodiments, the C-terminus of the antibody of the present invention is connected to a purification tag.

[0261] In certain embodiments, the coupling moiety is selected from a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material) or biotin. The detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridinium ester compounds), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable labels described in any of the above items can be attached to the antibodies of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0262] In certain embodiments, the conjugated moiety is selected from a therapeutic agent, such as an anti-tumor drug or an immunosuppressant.

[0263] In certain embodiments, the conjugation moiety is selected from additional biologically active polypeptides.

[0264] Pharmaceutical composition

[0265] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody, nucleic acid molecule, vector, host cell, multispecific molecule, or conjugate as described above, and one or more pharmaceutically acceptable excipients.

[0266] In certain embodiments, the pharmaceutical composition may further comprise additional anti-tumor drugs and / or immunosuppressants.

[0267] In certain embodiments, the antibody, nucleic acid molecule, vector, host cell, multispecific molecule, or conjugate of the present invention and the additional anti-tumor drug and / or immunosuppressant can be provided as separate components or as mixed components in the pharmaceutical composition. Thus, the antibody, nucleic acid molecule, vector, host cell, multispecific molecule, or conjugate of the present invention and the additional anti-tumor drug and / or immunosuppressant can be administered simultaneously, separately, or sequentially.

[0268] In certain embodiments, the one or more pharmaceutically acceptable excipients may comprise a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), a stabilizer, a surfactant, a buffer solution, and any combination thereof.

[0269] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" of an antibody, nucleic acid molecule, vector, host cell, multispecific molecule, or conjugate of the present invention. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered therapies.

[0270] Therapeutic applications

[0271] On the other hand, the present invention provides a method for preventing and / or treating a disease associated with CD161 in a subject, comprising administering to a subject in need thereof an antibody, nucleic acid molecule, vector, host cell, multispecific molecule, conjugate or pharmaceutical composition of the present invention. The present invention also relates to the use of the antibody, nucleic acid molecule, vector, host cell, multispecific molecule, conjugate or pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease associated with CD161 in a subject. The present invention also relates to an antibody, nucleic acid molecule, vector, host cell, multispecific molecule, conjugate or pharmaceutical composition as described above for use as a medicament; the medicament is used to prevent and / or treat a disease associated with CD161 in a subject.

[0272] In some embodiments, the disease is a disease associated with high expression of CD161.

[0273] In certain embodiments, the disease is a solid tumor, a hematological tumor, or an autoimmune disease.

[0274] In certain embodiments, the disease is selected from non-Hodgkin's lymphoma of B-cell origin, glioma, squamous cell carcinoma of the head and neck skin, melanoma, lung cancer, colorectal cancer, and liver cancer.

[0275] In certain embodiments, the antibody, nucleic acid molecule, vector, host cell, multispecific molecule, conjugate or pharmaceutical composition is used alone or in combination with another anti-tumor drug and / or immunosuppressant.

[0276] In certain embodiments, the subject is a mammal, such as a human.

[0277] The antibodies, nucleic acid molecules, vectors, host cells, multispecific molecules, conjugates or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use.

[0278] A preferred dosage form is injection. Such injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: in an appropriate solvent, the antibody of the present invention of the necessary dose is mixed, and optionally, other desired ingredients (including but not limited to, pH regulator, surfactant, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof) are mixed at the same time, followed by filtration sterilization. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), stabilizer, surfactant, buffer solution and any combination thereof.

[0279] The antibodies, nucleic acid molecules, vectors, host cells, multispecific molecules, conjugates or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral (e.g., intravenous or bolus, subcutaneous, intraperitoneal, intramuscular). It will be understood by those skilled in the art that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the antibodies, nucleic acid molecules, vectors, host cells, multispecific molecules, conjugates or pharmaceutical compositions of the present invention are administered by intravenous or bolus injection.

[0280] Detection Application

[0281] On the other hand, the present invention provides a method for detecting the presence or content of CD161 in a sample, which comprises using an antibody or conjugate of the present invention. In certain embodiments, the antibody and conjugate are anti-CD161 antibodies and conjugates as described in any one of the preceding items.

[0282] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0283] In certain embodiments, the conjugate used in the methods comprises an antibody of the invention and a detectable label.

[0284] In certain embodiments, the antibodies used in the methods are detectably labeled.

[0285] In certain embodiments, the antibodies used in the methods are not detectably labeled. Thus, the methods may further comprise detecting the antibodies of the invention using other reagents (such as a second antibody) that are detectably labeled.

[0286] In certain embodiments, the method comprises the steps of:

[0287] (1) contacting the sample with the antibody or conjugate of the present invention;

[0288] (2) detecting the formation of a complex between the antibody or conjugate and the antigen or detecting the amount of the complex.

[0289] The formation of the complex indicates the presence of an antigen or a cell expressing an antigen; wherein the antigen is selected from CD161.

[0290] The methods may be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0291] In certain embodiments, the method is used for the prognostic assessment of a subject suffering from a disease associated with CD161. In such embodiments, the method may further comprise a step of comparing the amount of the CD161 in a sample from the subject with a reference value. The reference value may be the level of CD161 in a sample from a subject (e.g., a healthy control) that is known not to have a disease associated with CD161 (also referred to as a "negative reference value").

[0292] In certain embodiments, the disease associated with CD161 is a solid tumor, a hematologic tumor, or an autoimmune disease. In certain embodiments, the disease associated with CD161 is selected from non-Hodgkin's lymphoma of B cell origin, glioma, head and neck skin squamous cell carcinoma, melanoma, lung cancer, colorectal cancer, and liver cancer.

[0293] In certain embodiments, the sample can be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy tissue, or fine needle aspirate tissue), histological preparations, and the like.

[0294] In certain embodiments, the CD161 is human CD161.

[0295] On the other hand, there is provided the use of an antibody or conjugate of the present invention in the preparation of a detection reagent for detecting the presence or level of CD161 in a sample or for the prognostic assessment of a subject suffering from a disease associated with CD161. In certain embodiments, the antibody and conjugate are any of the anti-CD161 antibodies and conjugates described above.

[0296] In certain embodiments, the conjugate used to prepare the detection reagent comprises an antibody of the invention and a detectable label.

[0297] In certain embodiments, the antibody used to prepare the detection reagent is detectably labeled.

[0298] In certain embodiments, the antibody used to prepare the detection reagent does not carry a detectable label. In such embodiments, the detection reagent may further comprise other reagents (such as a second antibody) capable of detecting the antibody of the present invention.

[0299] Definition of terms

[0300] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0301] When the terms "for example," "such as," "including," "including" or variations thereof are used herein, these terms will be interpreted as meaning "including but not limited to."

[0302] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0303] The term "and / or" is intended to include both "and" and "or". For example, the phrase "A, B and / or C" is intended to include each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0304] As used herein, the term “CD161” refers to a C-type lectin-like receptor encoded by killer cell lectin-like receptor subfamily B member 1 (KLRB1), which is commonly expressed in human natural killer NK cells and various T lymphocyte subsets. In NK cells, CD161 acts as an inhibitory receptor on the cell surface. It can inhibit NK cells from releasing IFN-γ by binding to Clec2D through cis-action (NK by expressing CD161 and Clec2D simultaneously) and trans-action (tumor-infiltrating immune cells or tumor cells express Clec2D), thereby reducing the cytotoxicity of NK cells. In T cells, the activation of CD161 signals can further activate AKT1 / PKB and RPS6KA1 / RSK1 kinases and their downstream signals, promoting anti-CD3-induced T cell proliferation. The sequence of CD161 is well known to those skilled in the art (see, for example, UniProt No.: Q12918).

[0305] As used herein, the term "antibody" refers to a protein molecule that can specifically bind to an antigen. An antibody can bind to the antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, the term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies; full-length antibodies and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. A "full-length antibody" or "intact antibody" is typically composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The typical IgG heavy chain constant region includes three domains (CH1, CH2, and CH3). The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions include highly variable domains (called hypervariable regions or complementarity determining regions (CDRs)), interspersed with more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxyl terminus). The heavy chain / light chain variable region pair (VH and VL) forms the antigen-binding site. The distribution of amino acids in various regions or domains can follow the definitions of Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0306] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs (CDR1, CDR2, and CDR3). The three CDRs of the heavy chain are designated as HCDR1, HCDR2, and HCDR3, or as VH-CDR1, VH-CDR2, and VH-CDR3, respectively. The three CDRs of the light chain are designated as LCDR1, LCDR2, and LCDR3, or as VL-CDR1, VL-CDR2, and VL-CDR3, respectively. The boundaries of a CDR can be defined according to various numbering systems known in the art, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the Abm numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), the Contact numbering system (MacCallum, RM, Martin, ACR, & Thornton, JM (1996). Antibody-antigen interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 262(5), 732-745.) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody variable region, a person skilled in the art can easily determine the CDR according to the definition of the numbering system. In addition, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0307] As used herein, the term "framework region" or "FR" refers to the domains of an antibody variable region other than the CDR residues.

[0308] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0309] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a portion of an intact antibody that is different from the intact antibody and that specifically binds to the antigen to which the intact antibody binds. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, diabodies, and other polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0310] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0311] As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

[0312] As used herein, the term "Fc" means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain bound via disulfide bonds. The Fc fragment of an antibody has a variety of different functions, but does not participate in the binding of antigens. The "effector functions" mediated by the Fc domain include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptors); and B cell activation, etc. The Fc domain can include both a native Fc region and a variant Fc region. The native Fc region comprises an amino acid sequence that is consistent with the amino acid sequence of the Fc region found in nature, for example, the native sequence human Fc region includes a native sequence human IgG1 Fc region; a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. The variant Fc region comprises an amino acid sequence that is different from the amino acid sequence of the native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may possess altered effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) compared to a native Fc region.

[0313] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the present invention, scFv may form a di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In certain embodiments of the present invention, scFv may form a (scFv)2, which refers to two or more single scFvs connected in parallel to form an antibody.

[0314] As used herein, the term "diabodies" means antibodies whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0315] As used herein, the term "multispecific molecule", also known as "multispecific antibody", refers to an antibody that has binding specificity for at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises multiple antigen-binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain comprised by the multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, a F(ab')2 fragment, or a scFv). In some cases, each antigen-binding domain is connected by a peptide linker.

[0316] In some embodiments, each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0317] Antigen-binding fragments of antibodies (e.g., the above-described antibody fragments) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and antigen-binding fragments can be screened in the same manner as screening intact antibodies.

[0318] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). In certain embodiments, the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In some embodiments, the donor antibody can be a mouse antibody with expected properties (e.g., antigen specificity, affinity and / or reactivity, etc.). To prepare a humanized antibody, the CDR region of the donor antibody can be inserted into a human framework sequence using methods known in the art. In some cases, the humanized framework sequence may include amino acid mutations that are replaced by corresponding non-human residues. In addition, the humanized antibody may also include residues that are not found in the original donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or human framework sequence to further improve or optimize the performance of the humanized antibody.

[0319] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but the antibody retains binding activity to the target antigen. In certain embodiments, the term "chimeric antibody" may include an antibody in which the heavy chain variable region and light chain variable region of the antibody are derived from a first antibody, and the heavy chain constant region and light chain constant region of the antibody are derived from a second antibody.

[0320] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0321] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0322] As used herein, the term "variant" refers to a polypeptide or peptide comprising an amino acid sequence that has been altered (by amino acid residue substitution, deletion, and / or addition) in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, variants have functions that are similar, identical, or improved to the polypeptide or peptide from which they are derived.

[0323] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The intensity or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Typically, an antibody binds to an epitope in an antigen or antigen with an equilibrium dissociation constant (KD) of about 1000 nM or less (e.g., about 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM or less).

[0324] The specific binding properties between two molecules can be measured using methods well known in the art. For example, the activity of an antibody binding to an antigen is determined by measuring the speed of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated by the actual rates of association and dissociation based on the concentration. The ratio of kdis / kon is equal to the dissociation constant, KD. KD, kon, and kdis values ​​can be measured using any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR).

[0325] The term "epitope" refers to an area or region on an antigen that is capable of specific binding to an antibody. An epitope can be formed by continuous amino acids (linear epitope) or non-continuous amino acids (conformational epitope), for example, by the folding of the antigen (i.e., by the tertiary folding of the proteinaceous antigen) so that the non-continuous amino acids are spatially accessible. For example, an epitope can comprise at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using methods known in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0326] When "competition" is used in relation to antigen-binding proteins (e.g., antibodies) that compete for the same epitope, it refers to competition between the antigen-binding proteins for binding to a common antigen, which can generally be determined by determining that the antigen-binding protein (e.g., antibody) to be tested inhibits (e.g., reduces) specific binding of a reference antigen-binding protein (e.g., reference antibody) to the antigen. Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another antigen binding protein, including, but not limited to, solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct labeled sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeled RIA with an I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15), etc. In some embodiments, binding of a reference antibody (e.g., an anti-CD161 antibody) to an antigen is inhibited by at least 40% (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 90%, 95%, 97%, or 98% or more) by an antibody that competitively binds to it.

[0327] As used herein, the detectable label of the present invention can be any substance detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), fluorescent proteins (e.g., phycoerythrin (PE)), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent reagents, such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin. In certain embodiments, the detectable label can be attached to the antibody of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0328] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0329] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0330] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., with similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0331] The term "post-translational modification" or "PTM" refers to structural modifications of proteins that occur after translation. Structural modifications can be covalent or non-covalent. Common PTMs include, but are not limited to, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, hydroxylation, acetylation, lipidation, biotinylation, glycosylation, prenylation, and sulfation. Certain PTMs can modulate protein conformation, activity, and physicochemical properties.

[0332] The compilation of the twenty conventional amino acids involved herein follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art (amino acid three-letter codes and single-letter codes are as described in J. biol. chem, 243, p3558 (1968)). For example, alanine can be represented by A or Ala.

[0333] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0334] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome for a subject who exhibits symptoms of the disease or is diagnosed with the disease. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to the expected survival (e.g., survival without treatment).

[0335] As used herein, the term "subject" refers to a mammal, such as a human or a non-human primate (eg, cynomolgus monkey). In certain embodiments, the subject has a solid tumor, a hematological tumor, or an autoimmune disease.

[0336] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactic effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered treatments.

[0337] In the present invention, unless otherwise specified, the "first" (e.g., the first antigen or the first antigen-binding domain) and the "second" (e.g., the second antigen or the second antigen-binding domain) are mainly for reference distinction and do not have a typical order meaning.

[0338] Advantageous Effects of the Invention

[0339] In some embodiments, the present invention provides anti-CD161 antibodies that can bind to human and cynomolgus monkey CD161 proteins with high affinity at the cellular level and protein level, and can effectively block the binding of CD161 to its receptor Clec2D at the cellular level. In in vitro activity evaluation, the antibodies of the present invention can effectively promote the activation of T cells, promote the killing ability of PBMCs to tumor cells, enhance NK cell degranulation and promote the killing effect of NK cells on tumor cells. The antibodies of the present invention can be widely used in the preparation of drugs such as solid tumors, hematological tumors and autoimmune diseases, and have important clinical significance for the treatment of the diseases. In addition, the antibodies of the present invention also have species cross-binding activity and activation activity to human and monkey CD161, which is beneficial for carrying out preclinical evaluation studies in animal models.

[0340] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only intended to illustrate the present invention and are not intended to limit the scope of the invention. Based on the following detailed description of the drawings and preferred embodiments, the various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0341] Figure 1: Flow cytometry analysis of CD161 antibody binding to CHOK1-HuCD161 cells. CHOK1 stable cells overexpressing human CD161 protein were incubated with different concentrations of CD161 chimeric and humanized antibodies (M01, B01-B10), and the amount of antibody on the cells was measured by flow cytometry and expressed as mean fluorescence intensity (Y-axis).

[0342] Figure 2: Binding of CD161 antibodies to CHOK1-cynoCD161 cells was determined by flow cytometry. CHOK1 stable cells overexpressing monkey CD161 protein were incubated with different concentrations of CD161 chimeric and humanized antibodies (M01, B01-B10), and the amount of antibody on the cells was determined by flow cytometry and expressed as mean fluorescence intensity (Y-axis).

[0343] Figure 3: Binding of CD161 antibodies to CHOK1-mCD161 cells was measured by flow cytometry. CHOK1 stable cells overexpressing mouse CD161 protein were incubated with different concentrations of CD161 chimeric and humanized antibodies (M01, B01-B10), and the amount of antibody on the cells was measured by flow cytometry and expressed as mean fluorescence intensity (Y-axis).

[0344] Figure 4: CD161 antibody blocking CLEC2D ligand binding to Jurkat-CD161 cells as determined by flow cytometry. Different concentrations of CD161 chimeric and humanized antibodies (M01, B01-B10) were co-incubated with Jurkat-CD161 cells and a fixed concentration of biotinylated bivalent CLEC2D ligand (BiCLEC2D-HuFc). The amount of BiCLEC2D-HuFc on the cells was determined by flow cytometry and expressed as mean fluorescence intensity (Y axis).

[0345] Figure 5: Luciferase reporter gene assay to determine whether CD161 antibodies activate CD161 downstream signaling pathways. Effector cells overexpressing the CD161-NFAT reporter gene (Jurkat-NFAT Luc-HuCD161) were co-cultured with target cells overexpressing the CLEC2D ligand (Daudi-HuClec2D). OKT3 antibody at a final concentration of 50 nM and various concentrations of CD161 chimeric or humanized antibodies (M01, B01-B10) were added to the culture system. After co-incubation, luciferase detection substrate was added, and luciferase activity under the conditions was recorded, expressed as chemiluminescence intensity (Y-axis).

[0346] Figure 6: Enzyme-linked immunosorbent assay (ELISA) testing the binding of CD161 antibodies to human CD161 protein. The chimeric antibody M01 was used as a positive control, and the human IgG1 (N297A) isotype antibody was used as a negative control. The binding ability of humanized antibodies (B03, B05, and B07) to the bivalent human CD161 protein (human CD161-Fc) was tested in an ELISA. The absorbance at a wavelength of 450 nm (OD450) was plotted on the Y-axis. Results are presented as the mean ± SD of two replicates.

[0347] Figure 7: Enzyme-linked immunosorbent assay (ELISA) testing the binding of CD161 antibodies to monkey CD161 protein. The chimeric antibody M01 was used as a positive control, and the human IgG1 (N297A) isotype antibody was used as a negative control to test the binding of humanized antibodies (B03, B05, and B07) to monkey CD161 protein (cyno CD161-His) at the ELISA level. The absorbance at 450 nm (OD450) was plotted on the Y-axis, and the results are presented as the mean ± SD of two replicates.

[0348] Figure 8: Enzyme-linked immunosorbent assay (ELISA) testing the binding of CD161 antibodies to mouse CD161 protein. Chimeric antibody M01 and human IgG1 (N297A) isotype antibody were used as negative controls to test the binding of humanized antibodies (B03, B05, and B07) to mouse CD161 protein (mouse CD161-His) in an ELISA. The absorbance at 450 nm (OD450) was plotted on the Y-axis. Results are presented as the mean ± SD of two replicates.

[0349] Figure 9: CD161 antibody enhances the cytotoxicity of human peripheral blood mononuclear cells (PBMC) against target cells expressing CLEC2D ligand. In the presence of different concentrations of CD161 antibody or control, the cell cytotoxicity was tested using human PBMC as effector cells and Daudi-HuClec2D stably transfected cell lines as target cells. The percentage of cytotoxicity (Y axis) was calculated using the EuTDA kit (PerkinElmer, AD0116) and the protocol described in Example 10. The results are expressed as the mean ± SD of two replicates.

[0350] Figure 10: CD161 antibody enhances human NK cell activation. CD56-positive cells were isolated from PBMCs according to the instructions of the supplier (Miltenyi Biotec, 130-097-042). NK cells were used as effector cells and the Daudi-HuClec2D stably transfected cell line was used as target cells in the presence of different concentrations of CD161 antibody or control. The CD107a expression ratio (Y-axis) of CD56-positive cells was measured by flow cytometry to represent the level of NK cell activation.

[0351] Figure 11: CD161 antibodies enhance the cytotoxic activity of human NK cells against target cells expressing CLEC2D ligands. CD56-positive cells were sorted from PBMCs according to the method described in the instructions of the supplier (Miltenyi Biotec, 130-097-042). In the presence of different concentrations of CD161 antibodies or controls, NK cells were used as effector cells and Daudi-HuClec2D stably transfected cell lines were co-incubated as target cells. Apoptosis of CD56-negative cells was detected by flow cytometry, and the percentage of apoptosis (Y-axis) was calculated according to the protocol described in Example 11 to represent the cytotoxic activity of NK cells against target cells.

[0352] Figure 12: Results of experiments to test the ability of CD161 lead antibodies (M01, M02, M03) to bind to human CD161 at the cellular level.

[0353] Figure 13: Results of experiments to test the ability of CD161 lead antibodies (M07, M09, M013, M14, M15) to bind to human CD161 at the cellular level.

[0354] Figure 14: Results of experiments to test the ability of CD161 lead antibodies (M01, M02, M03) to bind to monkey CD161 at the cellular level.

[0355] Figure 15: Results of experiments to detect the ability of CD161 lead antibodies (M09, M013, M14, M15) to bind to monkey CD161 at the cellular level.

[0356] Figure 16: Results of experiments in which the CD161 lead antibodies (M01, M02, M03) blocked the binding of CD161 to its receptor protein Clec2D.

[0357] Figure 17: Results of experiments in which CD161 lead antibodies (M07, M09, M013, M14, and M15) blocked the binding of CD161 to its receptor protein Clec2D;

[0358] Figure 18: NFAT reporter gene assay results for detecting activation of CD161 signaling pathway by CD161 lead antibodies (M02, M03);

[0359] Figure 19: NFAT reporter gene assay to detect activation of CD161 signaling pathway by CD161 lead antibody (M01);

[0360] Figure 20: NFAT reporter gene assay to detect the activation of CD161 signaling pathway by CD161 lead antibodies (M09, M013, M14, M15).

[0361] FIG21 : Results of experiments to test the ability of PTM-removed CD161 antibodies (B11, B12, B13) and B03 to bind to human CD161 at the cellular level.

[0362] FIG22 : Results of experiments to detect the ability of PTM-removed CD161 antibodies (B14, B15, B16) and B03 to bind to human CD161 at the cellular level.

[0363] FIG23 : Results of experiments to detect the ability of PTM-removed CD161 antibodies (B11, B12, B13) and B03 to bind to monkey CD161 at the cellular level.

[0364] FIG24 : Results of an experiment to detect the ability of PTM-removed CD161 antibodies (B14, B15, B16) and B03 to bind to monkey CD161 at the cellular level.

[0365] FIG25 : Results of experiments to test the ability of PTM-removed CD161 antibodies (B11, B12, B13) and B03 to bind to mouse CD161 at the cellular level.

[0366] FIG26 : Results of experiments to test the ability of PTM-removed CD161 antibodies (B14, B15, B16) and B03 to bind to mouse CD161 at the cellular level.

[0367] FIG27 : Results of experiments in which PTM-removed CD161 antibodies (B11, B12, B13) and B03 blocked the binding of CD161 to its receptor protein Clec2D.

[0368] FIG28 : Results of experiments in which PTM-removed CD161 antibodies (B14, B15, B16) and B03 blocked the binding of CD161 to its receptor protein Clec2D.

[0369] Figure 29: NFAT reporter gene assay to detect activation of the CD161 signaling pathway by PTM-removed CD161 antibodies (B14, B15, B16) and B03;

[0370] FIG30 : Results of an experiment to detect the ability of PTM-removed CD161 antibodies (B14, B15, B16) and B03 to bind to human CD161 at the protein level.

[0371] FIG31 : Results of an experiment to detect the ability of PTM-removed CD161 antibodies (B14, B15, B16) and B03 to bind to monkey CD161 at the protein level.

[0372] FIG32 : Results of an experiment to detect the binding ability of PTM-removed CD161 antibodies (B14, B15, B16) and B03 to mouse CD161 at the protein level.

[0373] Sequence information

[0374] Table 1: The information of the sequences involved in the present invention is described in the table below: DETAILED DESCRIPTION

[0375] The invention will now be described in the following non-limiting examples.

[0376] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0377] Example 1: Expression and purification of CD161 antigen and detection protein

[0378] Protein Expression

[0379] The DNA sequences of the extracellular domain of CD161 were designed and synthesized using the amino acid sequences at positions 67-225 of the human CD161 extracellular region (Gln 67-Ser 225) (UniProt No.: Q12918), the amino acid sequence at positions 67-227 of the cynomolgus macaque CD161 extracellular region (Gln 67-Leu 227) (NCBI No.: XP_005570142.2), and the amino acid sequence at positions 67-227 of the mouse CD161 extracellular region (Gln 67-His 227) (UniProt No.: P27811) as templates, and the His tag or the Fc domain of the human IgG1 wild-type heavy chain was fused to the N-terminus. The synthesized DNA sequence was cloned into the pcDNA3.1 plasmid (Invitrogen, V79020) to produce four recombinant fusion protein expression plasmids pcDNA3.1-HuCD161-His, pcDNA3.1-CynoCD161-His, pcDNA3.1-MouseCD161-His and pcDNA3.1-HuCD161-Fc. The plasmid expressing the target protein was stained with ExpiFectamine TM293 transfection kit (Invitrogen, A14525) was used to transiently transfect into Expi293F cells (Gibco, A14527) and Expi293 was used to TM Expression Medium (Gibco, A1435102) was shaken and cultured in a CO2 incubator at 37°C. After 5-7 days, the cell culture medium was collected and centrifuged to remove cellular components to obtain the culture supernatant containing the His- or Fc-tagged CD161 protein for subsequent experiments.

[0380] 1-2. Protein Purification

[0381] (1) Purification of recombinant proteins with His tags:

[0382] Collect the cell expression supernatant sample, centrifuge at high speed to remove impurities, and replace the buffer with PBS, adding imidazole to a final concentration of 5mM. Equilibrate the nickel column with a PBS solution containing 5mM imidazole and rinse 2-5 times the column volume. Load the replaced supernatant sample onto the IMAC column. Rinse the column with a PBS solution containing 5mM imidazole until the A280 reading drops to the baseline. Then rinse the chromatography column with PBS+10mM imidazole to remove non-specifically bound impurities and collect the effluent. Elute the target protein with a PBS solution containing 300mM imidazole and collect the elution peak. The collected eluate is concentrated and further purified by Superdex200 (Cytiva, 28-9893-35) gel chromatography with PBS as the mobile phase. Remove the aggregate peak and collect the elution peak. The obtained protein is identified as correct by electrophoresis and SEC-HPLC and then aliquoted for use. Obtain His-tagged HuCD161-His, MouseCD161-His, and CynoCD161-His (for subsequent detection of the antibodies disclosed in the present invention):

[0383] Human CD161 protein extracellular domain with His tag (HuCD161-His):

[0384] Mouse CD161 protein extracellular domain with His tag (MouseCD161-His):

[0385] CynoCD161-His cynomolgus monkey CD161 protein extracellular domain:

[0386] Note: The underlined part is the 8×His tag

[0387] (2) Purification of HuCD161-Fc fusion protein:

[0388] The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the supernatant was subjected to MabSelect PrismA (Cytiva, 17-5498-02) affinity chromatography. The MabSelect PrismA chromatography column was first regenerated with 0.1M NaOH, rinsed with pure water, and then balanced with PBS. After the supernatant was combined, it was washed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with 0.1M acetate buffer at pH = 3.5 and neutralized with 1M Tris-HCl. After the eluted sample was appropriately concentrated, it was further purified by gel chromatography Superdex200 (Cytiva, 28-9893-35) balanced with PBS. The target protein was collected and concentrated to an appropriate concentration in the receiving tube to obtain HuCD161-Fc (for subsequent detection of the immunogen and disclosed antibodies of the present invention). This method is used for the preparation and purification of hybridoma antibodies and immunogen HuCD161-Fc protein.

[0389] Human CD161 protein extracellular domain with Fc tag (HuCD161-Fc):

[0390] Note: The underlined portion represents the amino acid sequence 100-330 of human IgG-Fc (Pro 100-Lys 330) (UniProt No.: P01857-1).

[0391] Example 2: Preparation of monoclonal hybridoma clones and sequencing of clones

[0392] 2-1. Immunity and Fusion

[0393] Immunizations were performed using 6-8 week old Balb / c and SJL mice (purchased from Shanghai Slack) and housed under SPF conditions. For the primary immunization, 100 μg of HuCD161-Fc protein (0.20 mL) emulsified in Freund's complete adjuvant was injected intraperitoneally. For the booster immunization, 50 μg of HuCD161-Fc protein (0.20 mL) was injected intraperitoneally in Freund's incomplete adjuvant. A two-week interval separated the primary immunization and the first booster immunization, with three weeks between each subsequent booster immunization. Blood was collected seven days after each booster immunization, and serum antibody titer and specificity were assessed by ELISA. After two booster immunizations, mice with the highest titers were selected. For the final immunization, selected mice were intraperitoneally injected with 50 μg of purified HuCD161-hFc. Five days later, the mice were sacrificed, and splenocytes were harvested and electrofused with the murine myeloma cell line SP2 / 0 (ATCC) cells. The cells were then diluted and cultured.

[0394] 2-2. Screening of hybridoma clones

[0395] Hybridoma clones were screened on a large scale using human, cynomolgus monkey, and mouse CD161 proteins and overexpressing cell lines. Positive clones with an OD 450nm > 1.0 and an MFI value in iQue3 greater than 10 times the negative control value were selected and expanded into 24-well plates. After 3 days, the supernatant was collected to test the blocking activity of the hybridoma. Hybridoma clones with a blocking inhibition rate of more than 50% for CD161 receptor were subcloned by limiting dilution to ensure that they were monoclonal. Monoclonal positive hybridoma clones were inoculated into SFM serum-free medium (Invitrogen, 12045076) containing 2% (w / w) FBS and expanded at 37°C and 5% (v / v) CO2. Selected positive clone hybridoma cells were frozen in liquid nitrogen.

[0396] 2-3. Determination of hybridoma VH and VL sequences

[0397] Positive clones were cultured to an appropriate concentration and 5×10 7 hybridoma cells, using TRIzol TM Plus RNA purification kit (Invitrogen, 12183555) was used to extract RNA from hybridoma clones. The reverse transcription kit used was PrimeScript TM The first strand cDNA was synthesized using the II 1st Strand cDNA Synthesis Kit (Takara, 6210A) and the PCR kit was used as the Premix Taq TM (Takara, RR003A) for amplification. Take 5 μL of PCR product for agarose gel electrophoresis detection, and purify the positive samples using a column recovery kit, where the recovery kit is Gel & PCR Clean-up (MACHEREY-NAGEL, 740609). Perform ligation reaction: 50 ng sample, 50 ng T vector, 0.5 μL ligase, 1 μL buffer, 10 μL reaction system, react at 16°C for half an hour to obtain ligation product, wherein the T vector ligation kit is pMD TM19-T Vector Cloning Kit (Takara, 6013); 5 μL of the ligation product was added to 100 μL of F-DH5α Rapid Transformation Competent Cells (Weidi Biotech, DF1001) and incubated on ice for 5 minutes. The product was then plated onto LB medium preheated to 37°C and incubated overnight at 37°C. The next day, a 30 μL PCR system was prepared using primers M13F and M13R from the T vector. Colony PCR was performed. A pipette tip was used to pick up a colony and pipette it into the PCR reaction system. 0.5 μL was then aspirated and spotted onto another LB medium containing 100 μg / mL ampicillin to preserve the strain. After the PCR reaction, 5 μL was removed for agarose gel electrophoresis. Positive samples were sequenced to obtain the heavy and light chain variable region sequences of the antibodies (designated A01-A15, respectively). The variable region sequence compositions are shown in Tables 2 and 3 below.

[0398] Table 2: Heavy chain variable region sequence composition of anti-CD161 antibodies A01-A15

[0399] Note: The CDRs are defined according to the Kabat numbering system.

[0400] Table 3: Sequence composition of the light chain variable region of anti-CD161 antibodies A01-A15

[0401] Note: The CDRs are defined according to the Kabat numbering system.

[0402] Example 3: Acquisition and preparation of anti-human CD161 chimeric antibody M01

[0403] The heavy and light chain variable region sequences of antibodies A01-A15 were obtained according to Example 2. The heavy chain variable region was directionally cloned into an expression vector (Invitrogen, Catalog No. V79020) containing a signal peptide and the human antibody heavy chain IgG1 (N297A) constant region (SEQ ID NO: 81). The light chain variable region was directionally cloned into an expression vector (Invitrogen, Catalog No. V79020) containing a signal peptide and the human antibody light chain kappa constant region (SEQ ID NO: 82). The recombinant vectors were transfected into Expi293F cells (using the same method as in Example 1) and cultured. After the culture was completed, the cell culture supernatant was collected, the cells were removed by filtration, and the culture supernatant was clarified by filtration using a 0.45 μm filter membrane. The supernatant was then purified using MabSelect PrismA affinity chromatography and Superdex200 gel chromatography (the purification method was the same as in Example 1-2: Purification of HuCD161-Fc fusion protein) to obtain anti-human CD161 chimeric antibodies: lead antibodies M01-M15 (M01-M15 correspond to chimeric antibodies A01-A15, respectively, for example, the chimeric antibody corresponding to A01 is M01, the chimeric antibody corresponding to A02 is M02, the chimeric antibody corresponding to A03 is M03, and so on). The sequence composition of the exemplary anti-human CD161 chimeric antibody M01 is shown in Table 4 below and is used in subsequent examples.

[0404] Table 4: Sequence composition of anti-CD161 chimeric antibody M01

[0405] Example 4: Determination of affinity constants of anti-human CD161 chimeric antibodies

[0406] The affinity and binding kinetics of antibodies for human and monkey CD161-His were characterized by surface plasmon resonance (SPR) assay using Biacore 8K Plus (GE healthcare). Protein A chips (Cytiva, 29127555) were used to capture candidate antibodies at a concentration of 1 μg / mL, and gradient dilutions of HuCD161-His (starting concentration of 25 nM, 2-fold dilution, 5 concentration points) and CynoCD161-His (starting concentration of 50 nM, 2-fold dilution, 5 concentration points) were flowed through the sensor chip at a flow rate of 10 μL / min. The running buffer was 1× PBS-P+ (Cytiva, 50-105-5354). The detection conditions for HuCD161-His were: capture time 60 s, antigen binding time 120 s, dissociation time 600 s, and regeneration condition Gly-HCl (pH 1.5) 30 s. The detection conditions for CynoCD161-His were: capture time 60 s, antigen binding time 120 s, dissociation time 120 s, and regeneration condition Glycine 1.5 (Cytiva, BR100354) 30 s. After double subtraction (control channel and zero concentration), the association and dissociation curves were fitted to a "1:1 binding" model using Biacore 8K Plus Insight Evaluation Software. The affinity of the antibody for HuCD161-His and CynoCD161-His recombinant proteins was determined using Biacore 8K Plus. The results are shown in Table 5. As shown in Table 5, the M01 antibody can bind to human CD161 with an affinity of 0.25 nM. In addition, the M01 antibody has monkey species cross-reactivity, binding to monkey CD161 protein with an affinity of 6.11 nM.

[0407] Table 5: Affinity constants of M01 for HuCD161-His and CynoCD161-His

[0408] Example 5: Antibody humanization

[0409] Antibody humanization is primarily designed using a CDR grafting and back mutation approach. Briefly, the process begins with humanized sequence analysis and, through sequence alignment (NCBI-Igblast), selection of human IGHV and IGKV genes that are highly frequent in human antibody libraries and highly homologous to mouse antibodies as humanization templates. After selecting the human antibody framework, homology modeling is used to predict key amino acids in the mouse antibody variable regions that may determine structure, and back mutations are then designed for the grafted framework regions. According to the above principles, for the humanization of M01, the germline genes IGHV1-69*02 (67.3%) + IGHJ1*01 and IGKV1-33*01 (68.4%) + IGKJ2*01 were selected as the variable region transplantation framework for CDR transplantation and important amino acid back mutation design: 5 heavy chain variable region sequences (VH1, VH2, VH3, VH4, VH5) and 2 light chain variable region sequences (VL1, VL2) were designed respectively, and then cross-combined for expression, with a total of 10 expression combinations (see Table 6).

[0410] Table 6: Humanized antibody expression combinations

[0411] The gene of the humanized antibody sequence is cloned into the pcDNA 3.1 vector and transfected into Expi293F cells for the production of humanized antibodies (specific method is the same as in Example 3: acquisition and preparation of anti-human CD161 chimeric antibody M01). After the culture is completed, the cell culture supernatant is collected, the cells are removed by filtration, and the culture supernatant is clarified by filtration with a 0.45 μm filter membrane, and then purified using MabSelect PrismA affinity chromatography and Superdex200 gel chromatography (purification method is the same as in Example 1-2). The purified expressed humanized CD161 antibody is sterile filtered with a 0.22 μm filter and the protein concentration (A280 / 1.4) is detected. The results are shown in Table 7 below. The sequence composition of the purified expressed humanized CD161 antibody is shown in Table 8-Table 10 below, and is used in subsequent embodiments.

[0412] Table 7: Humanized CD161 antibody detection analysis

[0413] Table 8: Sequence composition of the heavy chain variable region of humanized CD161 antibody

[0414] Note: The CDRs are defined according to the Kabat numbering system.

[0415] Table 9: Sequence composition of the light chain variable region of the humanized CD161 antibody

[0416] Note: The CDRs are defined according to the Kabat numbering system.

[0417] Table 10: Sequence composition of humanized CD161 antibody

[0418] In addition, the HCDR2 regions of the M01 and B03 antibodies were modified to reduce the risk of post-translational modification of the antibodies. The light chain and heavy chain amino acid sequences of the modified antibodies B11-B13 and B14-16 are shown in Tables 11 and 12 below;

[0419] Table 11: Amino acid sequence list of modified antibody light chain

[0420] Note: The CDRs are defined according to the Kabat numbering system.

[0421] Table 12: Amino acid sequence of modified antibody heavy chain

[0422] Note: The CDRs are defined according to the Kabat numbering system.

[0423] Example 6: Detection of the binding ability of CD161 humanized antibodies to CD161 overexpressing cells

[0424] The nucleotide sequences encoding the V5 tag (amino acid sequence: GKPIPNPLLGLDST) and the full-length amino acid sequence of CD161 of human CD161 (Uniprot, Q12918-1), monkey CD161 (NCBI, XP_005570142.1), and mouse CD161 (Uniprot, P27811) were cloned into the pcDNA3.1 plasmid and transfected (Lipofectamine 3000, purchased from Invitrogen) with CHOK1 cell line (purchased from ATCC cell bank, CCL-61) and then incubated in 10% (v / v) fetal bovine serum (Biological) containing 0.5 μg / mL puromycin (Gibco, A1113803). Industries, 04-002-1A) were selectively cultured for two weeks in F12-K medium (Gibco, 21127022). Positively identified stable cell pools were subcloned using limiting dilution and incubated at 37°C in 5% (v / v) CO2. After approximately two weeks, a portion of monoclonal wells were selected and expanded into 6-well plates. The amplified clones were screened by flow cytometry using a known V5 tag antibody. Cell lines with good growth, high fluorescence intensity, and monoclonal activity were selected for further expansion and cryopreservation in liquid nitrogen. These cells, CHOK1-HuCD161, CHOK1-CynoCD161, and CHOK1-MouseCD161, were obtained, stably expressing human, monkey, and mouse CD161, respectively.

[0425] The CHOK1-HuCD161 stable cell line, CHOK1-CynoCD161 stable cell line and CHOK1-MouseCD161 stable cell line were expanded and cultured in T-75 cell culture flasks to 90% confluence, the culture medium was discarded, the cells were washed twice with PBS buffer, and then the cells were digested with Trypsin-EDTA (Gibco, 25200-072). The cells were collected and washed twice with PBS buffer. The collected cells were suspended to 2×10 6 Cells / mL, 100 μL per well was added to a 96-well V bottom plate (NEST, 701201), and an equal volume of the purified serially diluted anti-CD161 antibody (M01, B01-B10) obtained in Example 3 and Example 5 was added to the test sample. Incubate on ice for 1 hour, wash twice with FACS buffer by centrifugation, add 1:5000 diluted goat anti-human-IgG-AF488 secondary antibody (Invitrogen, A11013) per well, and incubate on ice for 1 hour. Wash twice with FACS buffer by centrifugation. Add 100 μL FACS buffer to resuspend the cells and analyze by flow cytometry (FACS Celesta TM, BD) detection and analysis results. Dose-response data were fitted to the fluorescence signal using a four-parameter logistic model using GraphPad Prism. The results are shown in Figures 1-3.

[0426] Figures 1 and 2 demonstrate that the test antibodies can dose-dependently bind to human and monkey CD161 proteins on the cell surface, respectively, demonstrating that the antibodies specifically bind to human CD161 and exhibit species cross-reactivity with monkey CD161. Figure 3 demonstrates that the test antibodies do not cross-react with mouse CD161 proteins.

[0427] Example 7: CD161 humanized antibody blocks the binding of CD161 to its receptor protein Clec2D

[0428] Expression and biotin labeling of BiClec2D-HuFc protein

[0429] To synthesize a bivalent Clec2D fusion protein in vitro, two copies encoding Clec2D (from NCBI, sequence number NM_013269.6) were concatenated via a flexible linker and linked to the amino acid sequence 109-330 (Cys 109-Lys 330) encoding human IgG-Fc (from UniProt, sequence number P01857-1) via a GSG flexible linker to construct the pcDNA3.1-BiClec2D-HuFc plasmid. This plasmid was transfected into Expi293F cells and expressed and purified (transfection and purification methods were the same as in Example 1). Proteins that passed the test were further biotinylated using the Biotin Labeling Kit-NH2 (Tongren Chemical, LK03) in a 37°C incubator (refer to the kit instructions for specific steps).

[0430] A Jurkat stably transfected cell line overexpressing human CD161 protein (Jurkat-CD161, using the same method as in Example 6) was constructed and incubated on ice with serially diluted biotinylated BiClec2D-HuFc. After 1 hour, the cells were washed and labeled with streptavidin-APC (Biolegend, 405207). The BiClec2D-HuFc labeling effect was assessed by flow cytometry, and the optimal concentration was determined for subsequent blocking experiments.

[0431] Jurkat-CD161 cells were collected, washed twice with PBS, and counted and counted at 3×10 5 / well was seeded into a 96-well V-bottom plate. Biotinylated BiClec2D-HuFc was diluted with FACS buffer to a final concentration of 2 μg / mL and added to a 96-well plate. An equal volume gradient dilution (starting concentration was 100 nM, 5-fold dilution) of the CD161 antibody to be tested (M01, B01-B10) was added at the same time. Incubate on ice for 1 hour, wash twice with FACS buffer by centrifugation, add 100 μL of diluted streptavidin-APC (Biolegend, 405207) to each well, and incubate on ice for 1 hour. Wash twice with FACS buffer by centrifugation. Add 100 μL of FACS buffer to resuspend the cells and analyze by flow cytometry ( iQue3, SARTORIUS) detection and analysis results are shown in Figure 4.

[0432] FIG4 illustrates that each tested CD161 antibody was able to dose-dependently block the binding of Jurkat-CD161 to the bivalent CLEC2D ligand (BiClec2D-HuFc).

[0433] Example 8: NFAT reporter gene assay to detect activation of CD161 signaling pathway by CD161 humanized antibody

[0434] 8-1. Construction of Jurkat-NFAT Luc-HuCD161 and Daudi-HuClec2D Stable Cell Lines

[0435] The pGL4.30[luc2P NFAT-RE Hygro] plasmid (purchased from Promega) was transfected into the Jurkat-CD161 cells in the aforementioned example (the method is the same as in Example 6), and the cells were cultured and screened with RPMI-1640 medium (Gibco, 11875119) containing hygromycin B (Gibco, 10687010), puromycin hydrochloride (Gibco, A1113803) and 10% fetal bovine serum to obtain a stable cell line stably expressing NFAT RE-Luciferase and human CD161 (herein referred to as Jurkat-NFAT Luc-HuCD161). The human Clec2D sequence was cloned into the pcDNA3.1 plasmid and transfected into the Daudi cell line (the method is the same as in Example 6). After screening with puromycin hydrochloride (Gibco, A1113803), a stable cell line stably expressing human Clec2D (herein referred to as Daudi-HuClec2D) was obtained. The obtained cell line was tested for mycoplasma and expression stability and then expanded for subsequent experiments.

[0436] 8-2. NFAT reporter gene experiment

[0437] After collecting Jurkat-NFAT Luc-HuCD161 and Daudi-HuClec2D stably transfected cells, Jurkat-NFAT Luc-HuCD161 were cultured at 1×10 5 Cells were seeded into 96-well plates at a density of 1000 cells / well. NFAT reporter gene activation and inhibition experiments were performed using serially diluted OKT3 antibodies (Invitrogen, 16-0037-85) and Daudi-HuClec2D cells. Appropriate OKT3 antibody and cell concentration ratios were selected for subsequent NFAT reporter gene experiments.

[0438] The cells were washed with PBS, and Jurkat-NFAT Luc-HuCD161 and Daudi-HuClec2D were seeded into 96-well opaque plates (Beyotime, FCP968) at a concentration ratio of 1:3, with the density of Jurkat-NFAT Luc-HuCD161 being 1×10 5 / well, the density of Daudi-HuClec2D is 3×10 5 / well. At the same time, add CD161 antibodies (M01, B01-B10) with a concentration gradient dilution (initial concentration is 100nM, 5-fold dilution) and OKT3 with a final concentration of 50ng / mL. Place in a cell culture incubator and continue to culture for 4 hours. The enhanced luciferase reporter gene assay kit (Vkey-Bio, A2000101N) was used to detect the activity of firefly luciferase in cells.

[0439] The results, as shown in Figure 5, show that all tested CD161 antibodies enhanced the chemiluminescent signal in a dose-dependent manner, indicating that the NFAT signaling pathway was activated in a dose-dependent manner. These results suggest that CD161 antibodies can further enhance the activation of the CD161 signaling pathway induced by OKT3 antibodies by blocking the inhibitory effect of CLEC2D.

[0440] Example 9: ELISA assay to detect the binding of CD161 humanized antibody to CD161 protein

[0441] The purified CD161 antibodies obtained in Examples 3 and 5 were tested for binding reaction with the CD161 protein. The HuCD161-Fc, CynoCD161-His, and MouseCD161-His proteins purified in Example 1 were diluted with PBS to a final concentration of 1 μg / mL, and then added to a 96-well high-binding ELISA plate (BEAVER, 40301) at 100 μL / well. The plates were sealed with plastic film and coated overnight at 4°C. The next day, the plates were washed twice with PBST (PBS + 0.05% Tween 20) and then blocked with PBST + 1% (w / w) BSA at room temperature for 2 hours. The blocking solution was subsequently discarded, the plate was washed twice with PBST, and the purified gradient diluted CD161 antibody and control IgG1 (starting concentration 100nM, 5-fold dilution) obtained in Example 3 and Example 5 were added to 100 μL per well, wherein the control IgG1 was a human IgG1 (N297A) isotype control (Thermo, 31154). After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST, and an anti-human Kappa light chain secondary antibody (Sigma, A7164) labeled with HRP (horseradish peroxidase) diluted 1:5000 was added. After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST, 100 μL TMB substrate (SURMODICS, TMBC-1000-01) was added to each well, and after incubation at room temperature for 15 minutes, 50 μL stop solution (Solarbio, C1058) was added to each well to terminate the reaction. OD 450 nm values ​​were read using an ELISA plate reader (SpectraMax 384plus, Molec μLar Device), and the dose-response data were fitted to the OD 450 nm readings using a four-parameter logistic model using GraphPad Prism.

[0442] The results are shown in Figure 6. The purified humanized antibodies B03, B05, and B07 were able to bind to the human CD161 bivalent recombinant protein in a dose-dependent manner in ELISA. They also retained cross-binding activity with monkey CD161 (Figure 7) and showed no cross-reactivity with mouse CD161 (Figure 8). The control IgG1 showed no specific binding to human, monkey, or mouse CD161.

[0443] Example 10: Anti-CD161 antibody-promoted PBMC killing ability on target cells

[0444] use EuTDA kit (Perkinelmer, AD0116) was used to detect the killing ability of PBMC promoted by CD161 antibody on Daudi-HuClec2D stably transfected cells. PBMC (purchased from Aoneng Bio) were cultured in vitro with RMPI-1640 medium containing 20ng / mL IL-2 for 24h. The next day, PBMC were collected and added to a 96-well V-bottom plate (NEST, 701201) with gradient dilution of antibodies (starting concentration 20nM, 5-fold dilution) and incubated for 15min. Target cells were collected by centrifugation and resuspended in complete culture medium to 1×10 6 / mL and incubated with BATDA in a 37°C incubator for 20 minutes. The cells were washed three times with PBS and resuspended, and added to a 96-well plate, where the density of target cells was 1×10 4 / well, the ratio of effector cells to target cells (E:T) was 30:1. Spontaneous release group (only target cells added), maximum release group (target cells added with 10μL lysis buffer) and blank group (no cells added) were set up respectively. After incubation in a 37℃ incubator for 2 hours, the culture plate was centrifuged, 20μL of supernatant was carefully transferred to a flat bottom plate and 200μL of europium solution was added. Before reading the values ​​using a mμLtimode microplate reader (Tecan, SPARK), the mixed solution was shaken at room temperature for 15 min and the killing rate was calculated using the following formula:

[0445] Killing rate = (experimental group reading - spontaneous release reading) / (maximum release group reading - spontaneous release group reading) × 100%.

[0446] The results are shown in Figure 9. The control IgG1 isotype control (Thermo, 31154) could not enhance the killing of Daudi-HuClec2D target cells by PBMC. The chimeric antibody M01 and humanized antibodies B03, B05, and B07 could enhance the killing activity of PBMC cells against Daudi-HuClec2D in a dose-dependent manner.

[0447] Example 11: Activation of primary NK cells promoted by anti-CD161 antibodies and their ability to kill target cells

[0448] NK cells were isolated from PBMC cells using human CD56 MicroBeads (Miltenyi Biotec, 130-097-042) (refer to the kit instructions for detailed steps). The isolated NK cells were cultured in vitro with RMPI-1640 medium containing 20 ng / mL IL-2 for 24 h. NK cells were collected and plated at 2×10 5The cells were seeded at a density of 100 μg / well in a 96-well V-bottom plate, and an equal volume of serially diluted anti-CD161 antibody (starting concentration 20 nM, 5-fold dilution) was added. The cells were incubated at 37°C for 30 min. Daudi-HuClec2D target cells were collected and counted, and target cells were added at an effector cell to target cell (E:T) ratio of 2:1. After 2 h of culture, the cells were washed twice with PBS and LIVE / DEAD were added at a concentration of 1:1000. TM Cell death dye (Invitrogen, L34964A) was added, and Anti-CD56-APC (Biolegend, 318310) and Anti-CD107a-FITC (Biolegend, 328606) were added at a concentration of 5 μL / well and incubated on ice for 1 hour. The cells were washed twice by centrifugation with FACS buffer. The cells were suspended in 100 μL FACS buffer and analyzed by flow cytometry (FACS Celesta TM , BD) detected and analyzed the percentage of CD107a expression on primary NK cells and the apoptosis rate of CD56-negative target cells.

[0449] The results are shown in Figures 10-11. The control IgG1 isotype control (Thermo, 31154) was unable to activate NK cells or enhance NK cell killing of Daudi-HuClec2D target cells. The chimeric antibody M01 and humanized antibodies B03, B05, and B07 all promoted NK cell degranulation and Daudi-HuClec2D killing in a dose-dependent manner.

[0450] In addition, the results of the half-effective concentration (EC50) test for promoting the degranulation effect of primary NK cells and the killing effect on Daudi-HuClec2D are shown in Table 12. The results show that the EC50 values ​​of the anti-CD161 antibodies of the present invention are all lower than those of the positive control antibody rezorstobart (rezorstobart is an anti-CD161 antibody, and its variable region sequence refers to the variable region of the "Ab9" antibody of the international patent application WO2023028501A1, and the constant region is the same as the constant region of B03 of the present invention).

[0451] Table 12 EC50 values ​​of anti-CD161 antibodies promoting primary NK activation and killing of target cells

[0452] Example 12: Detection of the Binding Ability of CD161 Lead Antibodies to CD161-Overexpressing Cells

[0453] The binding ability of the lead antibody was tested using CHOK1 cells overexpressing the full-length human and monkey CD161 proteins. The cell line was constructed in the same manner as in Example 6. The binding activity of the test samples of the successively diluted CD161 lead antibodies (M01, M02, M03, M07, M09, M13, M14, and M15) to the overexpressing cell lines was tested using the method described in Example 6. The results are shown in Figures 12-15.

[0454] Figures 12 and 13 respectively show that at the cellular level, the tested CD161 lead antibodies (M01, M02, M03, M07, M09, M13, M14 and M15) can bind to the human CD161 protein on the cell surface in a dose-dependent manner, indicating that these lead antibodies can specifically bind to the human CD161 protein.

[0455] Figures 14 and 15 respectively show that at the cellular level, the tested CD161 lead antibodies (M01, M02, M03, M09, M13, M14 and M15) can bind to the monkey CD161 protein on the cell surface in a dose-dependent manner, indicating that these antibodies can have species cross-reactivity with the monkey CD161 protein.

[0456] Example 13: CD161 lead antibody blocks the binding of CD161 to its receptor protein Clec2D

[0457] The same method as in Example 7 was used to detect the binding activity of CD161 to its receptor protein Clec2D by serial dilution of the CD161 lead antibodies (M01, M02, M03, M07, M09, M13, M14 and M15) in the detection volume (starting concentration of 100 nM, 5-fold dilution).

[0458] The results are shown in Figures 16 and 17. The CD161 lead antibodies (M01, M02, M03, M07, M09, M13, M14, and M15) were able to dose-dependently block the binding of Jurkat-CD161 to the bivalent CLEC2D ligand (BiClec2D-HuFc).

[0459] Example 14: NFAT reporter gene assay to detect activation of CD161 signaling pathway by CD161 lead antibody

[0460] The same cell lines and detection methods as in Example 8 were used to detect the activity of serially diluted CD161 lead antibodies (M01, M02, M03, M09, M13, M14, and M15) in activating the CD161 signaling pathway.

[0461] The results, as shown in Figures 18-20, show that the tested CD161 lead antibodies M01, M02, M03, M09, M13, M14, and M15 dose-dependently enhanced the chemiluminescent signal, indicating dose-dependent activation of the NFAT signaling pathway. These results demonstrate that CD161 antibodies can further enhance OKT3-induced CD161 signaling pathway activation by blocking the inhibitory effect of CLEC2D.

[0462] Example 15: Detection of the Binding Ability of PTM-Removed Anti-CD161 Antibodies to CD161-Overexpressing Cells

[0463] The same method as described in Example 6 of the present invention was used to perform cell-based antibody-antigen binding assays to determine the binding activity of PTM-removed anti-CD161 antibodies (B11, B12, B13, B14, B15, and B16) to overexpression cell lines.

[0464] The experimental results are shown in Figures 21 to 26. The results show that the PTM-removed anti-CD161 antibody retains the same binding ability to human and monkey CD161 proteins as the B03 humanized antibody at the cellular level, and also does not bind to mouse CD161.

[0465] Example 16: PTM-removed anti-CD161 antibodies block the binding of CD161 to its receptor protein Clec2D

[0466] Cell-based receptor ligand binding assays were performed using the same method described in Example 7 of the present invention to determine the ability of PTM-removed anti-CD161 antibody variants (B11, B12, B13, B14, B15 and B16) to block the binding of Jurkat-CD161 to the bivalent CLEC2D ligand (BiClec2D-HuFc).

[0467] The experimental results are shown in Figures 27-28, which demonstrate that the PTM-removed anti-CD161 antibody blocked the binding of Jurkat-CD161 to the bivalent CLEC2D ligand (BiClec2D-HuFc) at a level comparable to B03.

[0468] Example 17: NFAT reporter gene assay to detect activation of CD161 signaling pathway by PTM-removed anti-CD161 antibodies

[0469] The same cell lines and detection methods as in Example 8 of the present invention were used to detect the activity of the PTM-removed anti-CD161 antibody in activating the CD161 signaling pathway.

[0470] The experimental results are shown in FIG29 , which show that the anti-CD161 antibodies B14, B15, and B16 enhance the chemiluminescence signal in a dose-dependent manner comparable to that of B03.

[0471] Example 18: Detection of the binding ability of PTM-removed anti-CD161 antibodies to human CD161 bivalent recombinant protein

[0472] The same method as in Example 9 of the present invention was used to perform a protein-based antibody-antigen binding assay to determine the binding activity of the PTM-removed anti-CD161 antibody to the human CD161 bivalent recombinant protein.

[0473] The experimental results are shown in Figures 30-32, which show that the PTM-removed anti-CD161 antibodies B14, B15 and B16 retain the binding ability of the B03 humanized antibody to human and monkey CD161 proteins at the protein level, and have no cross-reaction with mouse CD161.

[0474] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-CD161 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the anti-CD161 antibody comprises: (1) three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in any one of SEQ ID NOs: 69-72; and three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO: 79; (2) three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in SEQ ID NO: 68; and three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO: 79 or 80; (3) three complementarity determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO:49; and three complementarity determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO:61; (4) the three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in any one of SEQ ID NOs: 69-72; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO: 80; (5) three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in any one of SEQ ID NOs:73-75; and three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO:61; (6) the three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in any one of SEQ ID NOs: 76-78; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO: 79; (7) three complementarity determining regions HCDR1-HCDR3 contained in the VH set forth in SEQ ID NO:49; and three complementarity determining regions LCDR1-LCDR3 contained in the VL set forth in SEQ ID NO:66; (8) three complementarity determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO:49; and three complementarity determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO:67; (9) three complementarity determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 50; and three complementarity determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 62; (10) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 51; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 63; (11) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 52; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 61; (12) three complementary determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 53; and three complementary determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 61; (13) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 54; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 64; (14) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 55; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 65; (15) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 54; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 61; (16) three complementary determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 56; and three complementary determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 61; (17) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 57; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 61; (18) three complementary determining regions HCDR1-HCDR3 contained in VH as shown in SEQ ID NO: 58; and three complementary determining regions LCDR1-LCDR3 contained in VL as shown in SEQ ID NO: 61; (19) the three complementarity determining regions HCDR1-HCDR3 contained in the VH of SEQ ID NO: 59; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL of SEQ ID NO: 61; or (20) The three complementarity determining regions HCDR1-HCDR3 contained in the VH shown in SEQ ID NO:60; and the three complementarity determining regions LCDR1-LCDR3 contained in the VL shown in SEQ ID NO:

61.

2. The anti-CD161 antibody according to claim 1, wherein: (1) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 5, HCDR2 comprises the sequence of SEQ ID NO: 13, and HCDR3 comprises the sequence of SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of SEQ ID NO: 32, LCDR2 comprises the sequence of SEQ ID NO: 37, and LCDR3 comprises the sequence of SEQ ID NO: 42; (2) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 6, and HCDR3 comprises the sequence shown in SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; (3) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 5, HCDR2 comprises the sequence of any one of SEQ ID NOs: 14-16, and HCDR3 comprises the sequence of SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of SEQ ID NO: 32, LCDR2 comprises the sequence of SEQ ID NO: 37, and LCDR3 comprises the sequence of SEQ ID NO: 42; (4) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 5, HCDR2 comprises the sequence of any one of SEQ ID NOs: 17-19, and HCDR3 comprises the sequence of SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence of SEQ ID NO: 32, LCDR2 comprises the sequence of SEQ ID NO: 37, and LCDR3 comprises the sequence of SEQ ID NO: 42; (5) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 6, and HCDR3 comprises the sequence shown in SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 41, and LCDR3 comprises the sequence shown in SEQ ID NO: 47; (6) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 6, and HCDR3 comprises the sequence shown in SEQ ID NO: 20; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 36, LCDR2 comprises the sequence shown in SEQ ID NO: 41, and LCDR3 comprises the sequence shown in SEQ ID NO: 48 (7) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 7, and HCDR3 comprises the sequence shown in SEQ ID NO: 21; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 33, LCDR2 comprises the sequence shown in SEQ ID NO: 38, and LCDR3 comprises the sequence shown in SEQ ID NO: 43; (8) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 8, and HCDR3 comprises the sequence shown in SEQ ID NO: 22; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 33, LCDR2 comprises the sequence shown in SEQ ID NO: 38, and LCDR3 comprises the sequence shown in SEQ ID NO: 44; (9) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 23; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; (10) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 24; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; (11) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 10, and HCDR3 comprises the sequence shown in SEQ ID NO: 25; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 34, LCDR2 comprises the sequence shown in SEQ ID NO: 39, and LCDR3 comprises the sequence shown in SEQ ID NO: 45; (12) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 8, and HCDR3 comprises the sequence shown in SEQ ID NO: 26; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 35, LCDR2 comprises the sequence shown in SEQ ID NO: 40, and LCDR3 comprises the sequence shown in SEQ ID NO: 46; (13) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 10, and HCDR3 comprises the sequence shown in SEQ ID NO: 25; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; (14) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 11, and HCDR3 comprises the sequence shown in SEQ ID NO: 27; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; (15) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 28; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; (16) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 29; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; (17) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 9, and HCDR3 comprises the sequence shown in SEQ ID NO: 30, and the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; or (18) The VH of the anti-CD161 antibody comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 5, HCDR2 comprises the sequence shown in SEQ ID NO: 12, and HCDR3 comprises the sequence shown in SEQ ID NO: 31; the VL of the anti-CD161 antibody comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO: 32, LCDR2 comprises the sequence shown in SEQ ID NO: 37, and LCDR3 comprises the sequence shown in SEQ ID NO: 42; Optionally, the heavy chain variable region of the anti-CD161 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 5, 13 and 20, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 32, 37 and 42, respectively.

3. The anti-CD161 antibody according to claim 1 or 2, wherein The antibody comprises a framework region sequence derived from a human immunoglobulin; Optionally, the antibody comprises: a heavy chain framework region of a human heavy chain germline sequence, and / or a light chain framework region of a human light chain germline sequence; Optionally, the heavy chain framework region of the human heavy chain germline sequence and / or the light chain framework region of the human light chain germline sequence contains one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murine residues.

4. An anti-CD161 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) The VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 69-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof; (2) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 68 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof; (3) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 68 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 80 or a variant thereof; (4) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 69-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 80 or a variant thereof; (5) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 73-75 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; (6) the VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 76-78 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 79 or a variant thereof; (7) The VH of the anti-CD161 antibody comprises any one of SEQ ID NOs: 68-72 or a variant thereof, and the VL of the anti-CD161 antibody comprises a sequence shown in SEQ ID NOs: 79, 80 or a variant thereof; (8) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; (9) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 66 or a variant thereof; (10) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 49 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 67 or a variant thereof; (11) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 50 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 62 or a variant thereof; (12) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 51 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 63 or a variant thereof; (13) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 52 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; (14) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 53 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; (15) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 54 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 64 or a variant thereof; (16) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 55 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 65 or a variant thereof; (17) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 54 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; (18) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 56 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; (19) The VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 57 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; (20) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 58 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; (21) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 59 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; or (22) the VH of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 60 or a variant thereof, and the VL of the anti-CD161 antibody comprises the sequence shown in SEQ ID NO: 61 or a variant thereof; wherein the variant is a sequence having at least 80% sequence identity with the sequence from which it is derived; Optionally, the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived; Optionally, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived; Optionally, the anti-CD161 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 69, and a light chain variable region as shown in SEQ ID NO:

79.

5. The anti-CD161 antibody according to any one of claims 1 to 4, wherein The antibody further comprises a constant region; Optionally, the heavy chain of the antibody comprises a heavy chain constant region derived from a human immunoglobulin, and / or the light chain of the antibody comprises a light chain constant region derived from a human immunoglobulin; Optionally, the antibody heavy chain comprises a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; and / or, the antibody light chain comprises a human κ or λ light chain constant region; Optionally, the heavy chain of the anti-CD161 antibody comprises any one of SEQ ID NOs: 85-89 or variants thereof, and the light chain of the anti-CD161 antibody comprises a sequence shown in SEQ ID NOs: 96, 97 or variants thereof; Optionally, the anti-CD161 antibody, wherein: (1) The heavy chain comprises any one of SEQ ID NOs: 86-89 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 96 or a variant thereof; (2) the heavy chain comprises the sequence shown in SEQ ID NO: 85 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 96 or a variant thereof; (3) the heavy chain comprises the sequence shown in SEQ ID NO: 85 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 97 or a variant thereof; (4) the heavy chain comprises the sequence shown in SEQ ID NO: 83 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 84 or a variant thereof; (5) the heavy chain comprises any one of SEQ ID NOs: 86-89 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 97 or a variant thereof; (6) the heavy chain comprises any one of SEQ ID NOs: 90-92 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 84 or a variant thereof; or (7) The heavy chain comprises any one of SEQ ID NOs: 93-95 or a variant thereof, and the light chain comprises the sequence shown in SEQ ID NO: 96 or a variant thereof; wherein the variant is a sequence having at least 80% sequence identity with the sequence from which it is derived; Optionally, the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived; Optionally, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived; Optionally, the anti-CD161 antibody comprises a heavy chain as shown in SEQ ID NO: 86 and a light chain as shown in SEQ ID NO:

96.

6. The anti-CD161 antibody according to any one of claims 1 to 5, wherein The antibody is a complete antibody or an antigen-binding fragment; Optionally, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2 and diabody; Optionally, the anti-CD161 antibody is a murine antibody, a humanized antibody, a chimeric antibody or a multispecific antibody; Optionally, the anti-CD161 antibody has at least one of the following functions: (1) specifically binds to human CD161 antigen and / or specifically binds to cynomolgus macaque CD161 antigen; (2) blocking the binding of CD161 to its receptor Clec2D; (3) Promote CD3 antibody-mediated T cell activation; (4) Promote the killing ability of PBMCs against tumor cells; (5) Promote NK cell killing of tumor cells and / or enhance NK cell degranulation. 7 . An anti-CD161 antibody that binds to the same epitope as the anti-CD161 antibody of any one of claims 1 to 6 or competes with the anti-CD161 antibody of any one of claims 1 to 6 for binding to human CD161 antigen.

8. An isolated nucleic acid molecule encoding the anti-CD161 antibody of any one of claims 1 to 7.

9. A vector comprising the nucleic acid molecule according to claim 8; optionally, the vector is a cloning vector or an expression vector.

10. A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 9.

11. A method for preparing the anti-CD161 antibody according to any one of claims 1 to 7, comprising culturing the host cell according to claim 10 under conditions allowing expression of the antibody, and recovering the antibody from the cultured host cell.

12. A multispecific molecule comprising the anti-CD161 antibody of any one of claims 1 to 7; Optionally, the multispecific molecule specifically binds CD161 and specifically binds one or more other antigens; Optionally, the multispecific molecule further comprises at least one molecule with a second binding specificity for a second target.

13. A conjugate comprising the anti-CD161 antibody of any one of claims 1 to 7 and a coupling moiety; Optionally, the coupling moiety is selected from a protein tag, such as a purification tag; a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance) or biotin; a therapeutic agent, such as an anti-tumor drug or an immunosuppressant; or, another biologically active polypeptide.

14. A pharmaceutical composition comprising the anti-CD161 antibody of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the vector of claim 9, the host cell of claim 10, the multispecific molecule of claim 12, or the conjugate of claim 13; and one or more pharmaceutically acceptable excipients; Optionally, the pharmaceutical composition further comprises additional anti-tumor drugs and / or immunosuppressants.

15. Use of the anti-CD161 antibody of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the vector of claim 9, the host cell of claim 10, the multispecific molecule of claim 12, the conjugate of claim 13, or the pharmaceutical composition of claim 14 for preparing a medicament for preventing and / or treating a disease associated with CD161 in a subject; Optionally, the disease associated with CD161 is a solid tumor, a hematological tumor or an autoimmune disease; Optionally, the disease associated with CD161 is selected from B cell-derived non-Hodgkin's lymphoma, glioma, head and neck skin squamous cell carcinoma, melanoma, lung cancer, colorectal cancer and liver cancer; Optionally, the anti-CD161 antibody, nucleic acid molecule, vector, host cell, multispecific molecule, conjugate or pharmaceutical composition is used alone or in combination with another anti-tumor drug and / or immunosuppressant; Optionally, the subject is a human.

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