New application of anti-NKG2A antibody drug combination

Abstract

The invention belongs to the field of pharmacy, and provides an application of an anti-NKG2A antibody in preparation of a medicine for preventing or treating diseases in combination with another therapeutic agent. The other therapeutic agent may be selected from the group consisting of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CD20 antibody, an anti-CD70 antibody, an anti-EGFR antibody, or an anti-HER2 antibody. After the anti-NKG2A antibody is combined with another antibody for use, a synergistic effect can be generated, and the anti-NKG2A antibody has more effective capability of killing tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, and in particular relates to a new use of a combined drug containing an anti-NKG2A antibody. Background Art

[0002] NKG2A, also known as natural killer cell lectin-like receptor, is an inhibitory receptor in the NKG2 receptor family and is mainly expressed in NK cells, CD8 + On the surface of T cells, Th2 cells and NKT cells. On the surface of human immune cells, NKG2A and CD94 molecules (NK cell surface membrane protein) are connected in the form of disulfide bonds to form a heterodimeric complex NKG2A-CD94, which is recognized by the non-classical tissue compatibility complex I (major histocompatibility complex class I, MHC I) class molecule HLA-E on the target cell. HLA-E is the only ligand of the heterodimeric receptor CD94 / NKG2A and is a non-classical MHC class Ib molecule. This molecule is low in expression under normal circumstances, but many tumor cells highly express HLA-E, and NK and CD8 in tumor infiltration sites are highly expressed. + T cells highly express NKG2A, allowing tumor cells to evade NK and CD8 + T cell immune recognition. In addition, after the virus infects the body, it can induce host cells to express NK and CD8 + T cell NKG2A inhibitory receptor binds to ligands, escaping clearance from the immune system (Zheng M et al. Cellular and Molecular Immunology, 2020, 17(5)). Therefore, anti-NKG2A antibodies can release NK's killing activity by blocking this inhibitory signal and play an anti-tumor role.

[0003] On the other hand, the combined use of multiple antibodies can often achieve a synergistic anti-tumor or disease treatment effect. Therefore, the combined use of anti-NKG2A antibodies and other antibodies has broad research and application potential in the treatment of diseases. Summary of the Invention

[0004] Based on the research on anti-NKG2A antibodies, the applicant discovered the utility of combining the antibodies with other therapeutic agents (such as antibodies).

[0005] Preventive or therapeutic use

[0006] In a first aspect, provided is a use of an anti-NKG2A antibody in the preparation of a medicament for use in combination with another therapeutic agent for preventing or treating a disease, wherein the anti-NKG2A antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 2, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 4.

[0007] In some embodiments, the heavy chain variable region of the anti-NKG2A antibody comprises HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:6, and HCDR3 shown in SEQ ID NO:7, and the light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:9, and LCDR3 shown in SEQ ID NO:10.

[0008] In some embodiments, the heavy chain variable region of the anti-NKG2A antibody comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:4.

[0009] In some embodiments, the heavy chain variable region of the anti-NKG2A antibody comprises the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0010] In some embodiments, the anti-NKG2A antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1, and the light chain comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:3.

[0011] In this context, "at least 85%" means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of the foregoing values, which can be an integer or a decimal.

[0012] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-NKG2A antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0013] In some embodiments, the anti-NKG2A antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 3.

[0014] In some embodiments, the anti-NKG2A antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 3.

[0015] In some embodiments, the use, wherein the another therapeutic agent is selected from PD-1 neutralizers, EGFR antagonists, HER2 antagonists, anti-CD20 antibodies, anti-CD70 antibodies and immune cells.

[0016] In some embodiments, the use, wherein the other therapeutic agent is a PD-1 neutralizing agent.

[0017] In some embodiments, the PD-1 neutralizing agent is an anti-PD-L1 monoclonal antibody that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 neutralizing agent is an anti-PD-1 monoclonal antibody that inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 neutralizing agent is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)).

[0018] In some embodiments, the PD-1 neutralizing agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0019] In some embodiments, the PD-1 neutralizing agent is an anti-PD-L1 antibody. Any anti-PD-L1 antibody known in the art can be used in the present invention.

[0020] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 22.

[0021] In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of: avelumab, atezolizumab, socazolimab, adebrelimab, sugemalimab, envafolimab, durvalumab, cosibelimab, and KL-A167. The above-mentioned anti-PD-L1 antibodies include their respective biosimilar products.

[0022] In some embodiments, the anti-PD-L1 antibody is Avelumab.

[0023] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain as shown in SEQ ID NO:11 and a light chain as shown in SEQ ID NO:12.

[0024] In some embodiments, the PD-1 neutralizing agent is an anti-PD-1 antibody. Anti-PD-1 antibodies known in the art can be used for the purposes of the present invention.

[0025] In some embodiments, the aforementioned use, wherein the anti-PD-1 antibody is selected from Pembrolizumab, Nivolumab, Retifanlimab, Cadonilimab, BMS-986213 (Relatlimab + Nivolumab), Serplulimab, Zimberelimab, Penpulimab, Dostar The above-mentioned anti-PD-1 antibodies include biosimilar products of their respective categories.

[0026] In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0027] In some embodiments, the aforementioned use, wherein the other therapeutic agent is selected from EGFR antagonists.

[0028] In some embodiments, the EGFR antagonist is an anti-EGFR antibody or a small molecule EGFR inhibitor.

[0029] In some embodiments, the EGFR antagonist is an anti-EGFR antibody. Any anti-EGFR antibody known in the art can be used in the present invention.

[0030] In some embodiments, the anti-EGFR antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:27 and a light chain variable region having the amino acid sequence of SEQ ID NO:28.

[0031] In some embodiments, the anti-EGFR antibody comprises a heavy chain as shown in SEQ ID NO:17 and a light chain as shown in SEQ ID NO:18.

[0032] In some embodiments, the anti-EGFR antibody is selected from cetuximab, panitumumab, necitumumab, matuzumab, and ado-trastuzumab. The above anti-EGFR antibodies include their respective biosimilar products.

[0033] In some embodiments, the anti-EGFR antibody is cetuximab.

[0034] In some embodiments, the EGFR antagonist is a small molecule EGFR inhibitor. Small molecule EGFR inhibitors known in the art can be used in the present invention.

[0035] In some embodiments, the small molecule EGFR inhibitors include but are not limited to erlotinib, gefitinib, lapatinib, lapatinib ditosylate, and erlotinib hydrochloride.

[0036] In some embodiments, the use, wherein the other therapeutic agent is a HER2 antagonist.

[0037] In some embodiments, the HER2 antagonist is an anti-HER2 antibody or a small molecule HER2 inhibitor.

[0038] In some embodiments, the HER2 antagonist is an anti-HER2 antibody. Any anti-HER2 antibody known in the art can be used for the purposes of the present invention.

[0039] In some embodiments, the anti-HER2 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0040] In some embodiments, the anti-HER2 antibody comprises a heavy chain as shown in SEQ ID NO: 19 and a light chain as shown in SEQ ID NO: 20.

[0041] In some embodiments, the anti-HER2 antibody is selected from trastuzumab, pertuzumab, disitamab vedotin, zanidatamab, Zenocutuzumab, Trastuzumab Deruxtecan, disitamab vedotin, and Ado-trastuzumab Emtansine. The above-mentioned anti-HER2 antibodies include their respective biosimilar products.

[0042] In some embodiments, the anti-HER2 antibody is Trastuzumab.

[0043] In some embodiments, the HER2 antagonist is a small molecule HER2 inhibitor. Small molecule HER2 inhibitors known in the art can be used in the treatment methods of the present invention. Including but not limited to neratinib, lapatinib or lapatinib ditosylate and canertinib dihydrochloride.

[0044] In some embodiments, the use, wherein the other therapeutic agent is an anti-CD20 antibody. Known anti-CD20 antibodies can be used for the use of the present invention.

[0045] In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 24.

[0046] In some embodiments, the anti-CD20 antibody comprises a heavy chain as shown in SEQ ID NO:13 and a light chain as shown in SEQ ID NO:14.

[0047] In some embodiments, the anti-CD20 antibody is selected from zuberitamab, obinutuzumab, ibritumomab, ofatumumab, tositumomab, ocrelizumab, ublituximab, and rituximab. The above anti-CD20 antibodies include their respective biosimilar products.

[0048] In some embodiments, the anti-CD20 antibody is zabetuzumab.

[0049] In some embodiments, the use described herein, wherein the other therapeutic agent is an anti-CD70 antibody. Known anti-CD70 antibodies can be used for the use of the present invention.

[0050] In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 25 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 26.

[0051] In some embodiments, the anti-CD70 antibody comprises a heavy chain as shown in SEQ ID NO:15 and a light chain as shown in SEQ ID NO:16.

[0052] In some embodiments, the use described herein, wherein the second therapeutic agent is an immune cell, including but not limited to cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβT cells, and γδT cells. Particularly suitable immune cells for use in the present invention are NK cells and T cells.

[0053] T cells or NK cells can be from any source known in the art. For example, T cells and NK cells can be differentiated in vitro from hematopoietic stem cell populations (e.g., iPSCs), or can be obtained from a subject. T cells and NK cells can be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors.

[0054] Specifically, the NK cells include but are not limited to NK cell lines (including but not limited to NK-92, NKG, YT, NK-YS, HANK-1 and NKL, etc.), autologous NK cells (such as NK cells obtained by directly isolating from the human body and then expanding and culturing in vitro), autologous chimeric receptor-NK cells, and allogeneic NK cells (including allogeneic chimeric receptor-NK cells).

[0055] In some embodiments, the immune cells are selected from NK cells and T cells. In some embodiments, the NK cells are selected from autologous NK cells, chimeric receptor-NK cells, NK cell lines, and allogeneic NK cells.

[0056] In some embodiments, the aforementioned use, wherein the anti-NKG2A antibody and another therapeutic agent are administered simultaneously, separately or sequentially.

[0057] In some embodiments, the aforementioned use, wherein the disease is cancer, autoimmune disease or inflammatory disease.

[0058] In some embodiments, the cancer is a solid tumor.

[0059] In some embodiments, the cancer is a hematological neoplasm.

[0060] In some embodiments, the cancer is selected from non-small cell lung cancer, small cell lung cancer, ovarian cancer, fallopian tube cancer, colon cancer, colorectal cancer, breast cancer, melanoma, lung cancer, liver cancer, pancreatic cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer, and lymphocytic leukemia.

[0061] In some embodiments, the cancer is breast cancer, lung cancer, or B-lymphoblastic blastoma.

[0062] In some embodiments, the autoimmune or inflammatory disease is selected from ankylosing spondylitis, rheumatoid arthritis, autoimmune hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Wegener's granulomatosis, autoimmune hepatitis, Behcet's disease, Crohn's disease, primary biliary cirrhosis, scleroderma, ulcerative colitis, Sjögren's syndrome, type I diabetes, uveitis, Graves' disease, thyroiditis, type II diabetes, myocarditis, rheumatic fever, scleroderma, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, Guillain-Barré syndrome, multiple sclerosis, alopecia areata, pemphigus / pemphigoid, psoriasis, and vitiligo.

[0063] In some embodiments, the cancer is one in which HLA-E or Qa1 is expressed on the surface of the cancer cells. b cancer.

[0064] Drug combinations

[0065] In a second aspect, a drug combination is provided, comprising an anti-NKG2A antibody and another therapeutic agent, wherein the anti-NKG2A antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 2, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 4.

[0066] In some embodiments, the heavy chain variable region of the anti-NKG2A antibody comprises HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:6, and HCDR3 shown in SEQ ID NO:7, and the light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:9, and LCDR3 shown in SEQ ID NO:10.

[0067] In some embodiments, the heavy chain variable region of the anti-NKG2A antibody comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:4.

[0068] In some embodiments, the heavy chain variable region of the anti-NKG2A antibody comprises the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.

[0069] In some embodiments, the anti-NKG2A antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1, and the light chain comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:3.

[0070] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-NKG2A antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0071] In some embodiments, the anti-NKG2A antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 3.

[0072] In some embodiments, the anti-NKG2A antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 3.

[0073] In some embodiments, the additional therapeutic agent is as described above under "Prophylactic or therapeutic uses."

[0074] In some embodiments, the pharmaceutical combination, wherein the anti-NKG2A antibody and another therapeutic agent are administered simultaneously, separately, or sequentially.

[0075] Pharmaceutical composition

[0076] In a third aspect, the present invention provides a combination pharmaceutical composition for treating a disease, wherein the active ingredients of the composition include an anti-NKG2A antibody and another therapeutic agent, wherein the anti-NKG2A antibody and another therapeutic agent are as described above in the "preventive or therapeutic use".

[0077] In one embodiment, the pharmaceutical composition is in the form of a kit or pack.

[0078] In one embodiment, in the pharmaceutical composition, the anti-NKG2A antibody and another therapeutic agent are prepared as separate administration units and then combined; or the anti-NKG2A antibody and another therapeutic agent are prepared as a single administration unit.

[0079] It has been verified that the anti-NKG2A antibody of the present invention can produce a synergistic effect when used in combination with another antibody, and has the ability to kill tumor cells more effectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1A Figure 2 is the result of binding of hu-43VL (N26S-G34A) antibody to CHOK1 cells overexpressing human NKG2A; Figure 1B The figure shows the binding results of hu-43VL (N26S-G34A) antibody to CHOK1 cells overexpressing monkey NKG2A.

[0081] Figure 2 This figure shows the results of hu-43VL (N26S-G34A) antibody-mediated NK92 cell killing of LCL721.221 tumor cells.

[0082] Figure 3 This figure shows the results of the hu-43VL (N26S-G34A) antibody-mediated killing experiment of LCL721.221 cells by primary NK cells.

[0083] Figure 4The figure shows the comparison of the enhancement of the anti-PD-L1 antibody-mediated NK92 cell killing rate against target cells by anti-NKG2A antibody (hu-43VL(N26S-G34A)) relative to the negative control IgG4.

[0084] Figure 5 This is the killing effect result of the combined use of anti-NKG2A antibody and anti-CD20 monoclonal antibody.

[0085] Figure 6 This is the killing effect result of the combined use of anti-NKG2A antibody and anti-CD70 monoclonal antibody.

[0086] Figure 7 This is the result of anti-NKG2A antibodies promoting the killing of tumor cells by NK cells, where E / T represents the ratio of effector cells (NK cells) to target cells (LCL721.221-AEH), referred to as the effector-target ratio.

[0087] Figure 8 This is the killing effect result of the combined use of anti-NKG2A antibody and anti-EGFR monoclonal antibody.

[0088] Figure 9 This is the killing effect result of the combined use of anti-NKG2A antibody and anti-HER2 monoclonal antibody.

[0089] Figure 10 shows the difference between anti-NKG2A antibody and anti-EGFR monoclonal antibody ( Figure 10A ) and anti-HER2 monoclonal antibodies ( Figure 10B )The killing effect results of combined use. DETAILED DESCRIPTION

[0090] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," and the like should be understood as meaning "including but not limited to." Unless otherwise specified, "comprising" includes "consisting of." For example, for an anti-NKG2A antibody comprising a heavy chain variable region comprising the HCDR1 set forth in SEQ ID NO: 5, it explicitly encompasses the HCDR1 having the amino acid sequence set forth in SEQ ID NO: 5.

[0091] “And / or”, for example, “A and / or B” should be understood to mean “A and B” or “A or B”.

[0092] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur. As used in the specification and claims, the singular forms "a", "an" include the corresponding plural referents unless the content clearly dictates otherwise.

[0093] NKG2A (OMIM 161555, the entire disclosure of which is incorporated herein by reference) is a member of the NKG2 transcriptome (Houchins et al. (1991) J. Exp. Med. 173: 1017-1020). NKG2A is encoded by 7 exons spanning 25 kb, exhibiting some differential splicing. NKG2A, together with CD94, forms the heterodimeric inhibitory receptor CD94 / NKG2A found on the surface of NK cells, α / β T cells, γ / δ T cells, and a subset of NKT cells. Similar to inhibitory KIR receptors, NKG2A has an ITIM in its cytoplasmic domain. As used herein, "NKG2A" refers to any variant, derivative, or isoform of the NKG2A gene or the encoded protein.

[0094] HLA-E (OMIM 143010, the entire disclosure of which is incorporated herein by reference) is a non-classical MHC molecule expressed on the cell surface and regulated by peptide binding, such as fragments of signal sequences derived from other MHC class I molecules. Soluble forms of HLA-E have also been identified. In addition to its T cell receptor binding properties, HLA-E binds to natural killer (NK) cells, natural killer T cells (NKT), and subsets of T cells (α / β and γ / δ) by specifically binding to CD94 / NKG2A, CD94 / NKG2B, and CD94 / NKG2C (see, e.g., Braud et al., (1998) Nature 391: 795-799, the entire disclosure of which is incorporated herein by reference). Surface expression of HLA-E protects target cells from lysis by CD94 / NKG2A+ NK, T, or NKT cell clones. As used herein, "HLA-E" refers to any variant, derivative, or isoform of the HLA-E gene or encoded protein.

[0095] "Antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and full-length antibodies, as long as they exhibit the desired antigen-binding activity. Typically, natural IgG antibodies are heterotetrameric proteins composed of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH) and three constant domains (CH1, CH2, and CH3). From N to C-terminus, each light chain has a variable region (VL) and a constant light domain (CL). Depending on the context, a technician can determine the specific meaning of "antibody".

[0096] "Complementarity determining regions" or "CDRs" refer to the regions within the variable region that primarily contribute to antigen binding. VH and VL each contain four framework regions (FRs) and three complementarity determining regions (CDRs). VHs contain three CDRs: HCDR1, HCDR2, and HCDR3; VLs contain three CDRs: LCDR1, LCDR2, and LCDR3. From N-terminus to C-terminus, each VH and VL sequence is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0097] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: the Kabat numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the Chothia numbering system, and the ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278).

[0098] The correspondence between various numbering systems is well known to those skilled in the art. In other words, when a CDR sequence and its position in an antibody are provided under one numbering system, a skilled person is capable of determining the corresponding CDR sequence and its position in an antibody under another numbering system. Technical solutions corresponding to different numbering systems will be considered equivalent technical solutions.

[0099] In the expression "...having at least 85% sequence identity...", identity refers to the degree (percentage) to which the amino acids / nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned. During the alignment, gaps are introduced, if necessary, to maximize sequence identity. Sequence identity is determined by techniques known in the art, such as computer software (BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign).

[0100] As an example, "the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 2" means that the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6, and HCDR3 shown in SEQ ID NO: 7, and amino acid mutations are allowed to be introduced into regions outside the CDRs, thereby having at least 85% sequence identity with SEQ ID NO: 2.

[0101] As used herein, "pharmaceutical composition" and "composition" are used interchangeably and may also include kits, packs, and the like, all of which fall within the scope of the pharmaceutical compositions of the present invention. As used herein, a "pharmaceutical composition" refers to a combination of two active ingredients and may include other pharmaceutically acceptable carriers or excipients.

[0102] Those skilled in the art will appreciate that, in the present invention, the two antibodies in the composition can be mixed together to form a single administration unit, or can be independently administered as administration units and used separately or in combination.

[0103] "PD-1 neutralizer" is an agent that neutralizes PD-1 or reduces the inhibitory activity of human PD-1. In the context of the present invention, "reducing the inhibitory activity of human PD-1", "neutralizing PD-1" or "neutralizing the inhibitory activity of human PD-1" refers to a process in which the signal transduction ability of PD-1 caused by the interaction of one or more of PD-1 and its binding partners (such as PD-L1 or PD-L2) is inhibited. The agent that neutralizes the inhibitory activity of PD-1 reduces, blocks, inhibits, eliminates or interferes with the signal transduction caused by the interaction of one or more of PD-1 and its binding partners (such as PD-L1, PD-L2). Therefore, this agent can reduce the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes, so as to enhance T cell effector functions, such as proliferation, cytokine production and / or cytotoxicity. PD-1 neutralizers can interact with PD-1 and / or with one or more of its binding partners (such as PD-L1 and PD-L2).

[0104] In some embodiments, the PD-1 neutralizing agent is an anti-PD-L1 antibody. In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; also known as MPDL3280A, RG7446; see US 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(Suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI™, MEDI-4736; see WO 2011 / 066389), avelumab (Pfizer; also known as MSB-0010718C; see WO 2013 / 079174), STI-1014 (Sorrento; see WO 2013 / 181634), CX-072 (Cytomx; see WO 2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017 / 034916), or CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (April 2016)).

[0105] In certain embodiments, the anti-PD-L1 antibody is avelumab. Avelumab is a human IgG1λ monoclonal anti-PD-L1 antibody.

[0106] In certain embodiments, the anti-PD-L1 antibody is atezolizumab, which is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0107] In certain embodiments, the anti-PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgG1κ monoclonal anti-PD-L1 antibody.

[0108] Any anti-PD-1 antibody known in the art can be used in combination with the anti-NKG2A antibody described herein in the methods described herein.

[0109] In some embodiments, the PD-1 neutralizing agent is an anti-PD-1 antibody that inhibits the binding of PD-1 to PD-L1. In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al. 2014 Cancer Immunol Res. [Cancer Immunology Research] 2(9):846-56). In another embodiment, the anti-PD-1 antibody or fragment thereof competes with nivolumab for binding to PD-1. In some embodiments, the anti-PD-1 antibody binds to the same epitope as nivolumab. In certain embodiments, the anti-PD-1 antibody has the same heavy chain and light chain CDRs as nivolumab.

[0110] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as pembrolizumab and MK-3475) is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor PD-1. Pembrolizumab is described, for example, in U.S. Patent No. 8,900,587. Pembrolizumab has been approved by the FDA for the treatment of recurrent or refractory melanoma and advanced NSCLC. In another embodiment, the anti-PD-1 antibody (or its antigen-binding fragment) competes with pembrolizumab for binding to PD-1. In some embodiments, the anti-PD-1 antibody binds to the same epitope as pembrolizumab. In certain embodiments, the anti-PD-1 antibody has the same heavy chain CDRs and light chain CDRs as pembrolizumab.

[0111] "EGFR antagonists" refer to compounds that specifically bind to EGFR and prevent or reduce its signaling activity, and do not specifically bind to HER2, HER3, or HER4. Examples of such agents include antibodies and small molecule inhibitors that bind to EGFR.

[0112] Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab) and remodeled human 225 (H225) (see, WO 96 / 40210, Imclone Systems Inc.); IMC-11F8, a fully human EGFR-targeting antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Patent 5,212,290); humanized and chimeric antibodies that bind to EGFR, as described in U.S. Patent 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody to EGFR that competes with both EGF and TGF-α for EGFR binding; (EMD / Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 and described in US 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or a humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies can be conjugated to cytotoxic agents to generate immunoconjugates (see, for example, EP 659,439 A2, Merck Patent GmbH).

[0113] EGFR antagonists include small molecule inhibitors such as the compounds described in U.S. Patents 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO 98 / 14451, WO 98 / 50038, WO 99 / 09016, and WO 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, Genentech / OSI Pharmaceuticals); PD 183805 (CI 1033, 2-acrylamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc. Inc.); ZD1839, gefitinib (Iressa, 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); lapatinib (GW-572016) (GlaxoSmithKline), lapatinib ditosylate (SmithKline Beecham), erlotinib hydrochloride (OSI-774) (OSI Pharma); ZM105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Sugen); and AG1571 (SU 5271; Sugen).

[0114] The terms "HER2 receptor antagonist" and "HER2 antagonist" refer to compounds that inhibit the expression or function of the HER2 protein or gene. For the purposes of the present invention, HER2 antagonists refer to, for example, receptor tyrosine kinase inhibitors, particularly HER2 receptor protein inhibitors. Examples include antibodies and small molecule inhibitors that bind to HER2.

[0115] An "anti-HER2 antibody" or "HER2 antibody" is an antibody that binds to the HER2 receptor. Optionally, the HER2 antibody further interferes with the activation or function of HER2. Various anti-HER2 antibodies are known in the art. Preferably, such antibodies are monoclonal antibodies. They may be so-called chimeric antibodies, humanized antibodies, or fully human antibodies. They may be full-length anti-HER2 antibodies; anti-HER2 antibody fragments having the same biological activity; and include amino acid sequence variants and / or glycosylation variants of such antibodies or fragments. Examples of known humanized anti-HER2 antibodies are known by the INN names trastuzumab and pertuzumab. Another suitable anti-HER2 antibody is T-DM1 (enmetuzumab), which is an antibody-toxin conjugate composed of huMAb4D5-8 (HERCEPIN™) and maytansinoide (i.e., DM1 = N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine; i.e., a highly potent anti-microtubule agent). The conjugate (with an MCC linker) is currently under development for metastatic breast cancer. Other suitable anti-HER2 antibodies are trastuzumab for injection (DS-8201), A166 (trastuzumab botidotin), and SHR-A1811.Other HER2 antibodies with various properties have been described in Tagliabue et al., Int. J. Cancer, 47:933-937 (1991); McKenzie et al., Oncogene, 4:543-548 (1989); Cancer Res., 51:5361-5369 (1991); Bacus et al., Molecular Carcinogenesis, 3:350-362 (1990); Stancovski et al., PNAS (USA), 88:8691-8695 (1991); Bacus et al., Cancer Research, 52:2580-2589 (1992); Xu et al., Int. J. Cancer, 53:401-408 (1993); WO 94 / 00136; Kasprzyk et al., Cancer Research, 52:2771-2776 (1992); Hancock et al., Cancer Res., 51:4575-4580 (1991); Shawver et al., Cancer Res., 54:1367-1373 (1994); Arteaga et al., Cancer Res., 54:3758-3765 (1994); Harwerth et al., J. Biol. Chem., 267:15160-15167 (1992); U.S. Patent No. 5,783,186; and Klapper et al., Oncogene, 14:2099-2109 (1997). The most successful therapeutic anti-HER2 antibody is trastuzumab, sold under the trade name HERCEPIN™ by Genentech Inc. and F. Hoffmann-La Roche Ltd. Further details regarding the HER2 antigen and antibodies directed thereto are described in patent and non-patent publications (for a suitable overview, see US Pat. No. 5,821,337 and WO 2006 / 044908).

[0116] The terms "trastuzumab," "pertuzumab," and "T-DM1" encompass all corresponding anti-HER2 antibodies that meet the requirements necessary to obtain marketing authorization as an identical or biosimilar product in a country or region selected from the United States, Europe, and Japan. Trastuzumab has the CDR regions defined in EP-B-590058. Pertuzumab has the CDR regions defined in WO 01 / 00245. Trastuzumab T-DM1 is described in WO 2005 / 117986.

[0117] Exemplary HER2 antagonists include, but are not limited to, Neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide, and described in PCT Publication No. WO 05 / 028443); Lapatinib or Lapatinib ditosylate (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazol-1-yl) ... (2E)-N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furyl]oxy]-6-quinzolinyl]-4-(dimethylamino)-2-butenamide (BIBW-2992, CAS 850140-72-6); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indole-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazolamide (BMS690514); (2E)-N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furyl]oxy]-6-quinzolinyl]-4-(dimethylamino)-2-butenamide (BIBW-2992, CAS 850140-72-6); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indole-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazolamide -6-yl]-carbamic acid, (3S)-3- Linalool methyl ester (BMS 599626, CAS 714971-09-2); canertinib dihydrochloride (PD183805 or CI-1033); and N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinzolinamine (XL647, CAS 781613-23-8).

[0118] The term "cell" includes the primary cell of interest and its progeny.

[0119] The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product. The term "non-fixed combination" means that the active ingredients (e.g., (i) anti-NKG2A antibody, and (ii) another therapeutic agent (e.g., anti-PD-L1 antibody)) are administered to a subject as separate entities simultaneously, without specific time limits, or sequentially at equal or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of the two antibodies in the subject. In some embodiments, the anti-NKG2A antibody and the another therapeutic agent (e.g., anti-PD-L1 antibody) used in the pharmaceutical combination are administered at levels no greater than when they are administered alone. The term "fixed combination" means that the anti-NKG2A antibody and the other therapeutic agent (e.g., anti-PD-L1 antibody) in the pharmaceutical combination of the present invention are administered to a subject simultaneously, without specific time limits, or sequentially at equal or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of the two antibodies in the subject. The other therapeutic agent (e.g., an anti-PD-L1 antibody) is administered to the subject simultaneously as a single entity. The dosage and / or time interval of the anti-NKG2A antibody and the other therapeutic agent (e.g., an anti-PD-L1 antibody) in the pharmaceutical combination of the present invention are preferably selected so that the combined use of the anti-NKG2A antibody and the other therapeutic agent (e.g., an anti-PD-L1 antibody) can produce an effect greater than that achieved by using either antibody alone in treating a disease or condition. The anti-NKG2A antibody or the other therapeutic agent (e.g., an anti-PD-L1 antibody) can each be in the form of a separate formulation, which can be the same or different.

[0120] The term "administering" refers to physically introducing the anti-NKG2A antibody of the present invention and another therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of the anti-NKG2A antibody of the present invention and another therapeutic agent include oral, intravenous (e.g., infusion (also known as drip) or injection), intramuscular, subcutaneous, intraperitoneal, spinal, topical, or other parenteral routes of administration. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, including, but not limited to, intramuscular, intraarterial, intravenous, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Accordingly, the anti-NKG2A antibody of the present invention and another therapeutic agent can be formulated into capsules, tablets, injections (including infusions or injections), syrups, sprays, lozenges, liposomes, or suppositories, etc.

[0121] As used herein, the term "about" or "approximately" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within or exceeding 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to ±20%. Unless otherwise indicated, when a particular value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value.

[0122] "Administering" and "treating" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ or a biological fluid, refers to the contacting of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid. "Administering" and "treating" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research and experimental procedures. Treatment of cells includes contacting an agent with a cell, and contacting an agent with a fluid, wherein the fluid is in contact with the cell. "Administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo, by an agent, a diagnostic, a binding composition or by another cell. "Treatment" as applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0123] "Treatment" means administering a therapeutic agent, either internally or externally, such as a composition comprising an anti-NKG2A antibody disclosed herein or another therapeutic agent, to a subject having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Generally, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical test commonly used by a physician or other health care professional to assess the severity or progression of the symptom. Although the embodiments disclosed herein (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0124] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0125] The present invention will be explained below through specific examples, but the scope of the present invention is not limited thereto.

[0126] Unless otherwise specified, the mice, proteins, and cells used in the following examples were provided by Shanghai Ruizhi Chemical Research Co., Ltd.

[0127] In the following examples, the positive antibody Z270 used is Monalizumab, which was jointly developed by Innate Pharma and AstraZeneca. The sequence is linked to:

[0128] https: / / www.imgt.org / 3Dstructure-DB / cgi / details.cgi?pdbcode=10113.

[0129] The sequences of the antibodies used in the following examples are as follows:

[0130] (1) Anti-NKG2A antibody: also known as hu-43VL (N26S-G34A), for details, see Example 1 below.

[0131] (2) Anti-PD-L1 antibody: Avelumab, described in PCT publication WO2013 / 079174A1, the disclosure of which is incorporated herein by reference in its entirety. Avelumab is the International Nonproprietary Name (INN) of the anti-PD-L1 monoclonal antibody MSB0010718C and is described in WO2013 / 079174A1 by its full-length heavy and light chain sequences, where it is referred to as A09-246-2.

[0132] Avelumab heavy chain (SEQ ID NO: 11):

[0133] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0134] Avelumab heavy chain variable region (SEQ ID NO:21):

[0135] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS

[0136] Avelumab light chain (SEQ ID NO:12):

[0137] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPT VTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS。

[0138] Avelumab light chain variable region (SEQ ID NO:22):

[0139] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRP SGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL

[0140] (3) Anti-CD20 antibody: HS006, zebetuzumab, prepared with reference to CN117402885, the sequence of which is as follows;

[0141] Zebetuzumab heavy chain (SEQ ID NO: 13):

[0142] EVQLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWG TGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0143] Zebetuzumab heavy chain variable region (SEQ ID NO: 23)

[0144] EVQLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSS

[0145] Zebetuzumab light chain (SEQ ID NO: 14):

[0146] DIELSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVP

[0147] ARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLEIKRTVAAPSVFIFPPS

[0148] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0149] Zebetuzumab light chain variable region (SEQ ID NO: 24):

[0150] DIELSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVP ARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLEIK

[0151] (4) Anti-CD70 antibody, prepared with reference to the humanized 1F6 antibody of WO2006113909:

[0152] 1F6 heavy chain (SEQ ID NO: 15), wherein the underlined portion is the variable region:

[0153] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0154] 1F6 heavy chain variable region (SEQ ID NO:25):

[0155] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSS

[0156] 1F6 light chain (SEQ ID NO:16):

[0157] DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0158] 1F6 light chain variable region (SEQ ID NO:26):

[0159] DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNL ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIK

[0160] (5) Anti-EGFR antibodies: Cetuximab, c225 antibody, is an anti-EGFR antibody that has been shown to inhibit EGF-mediated tumor cell growth in vitro and was approved by the FDA for the treatment of head and neck cancer in 2011. Cetuximab is believed to act by blocking oncogenic signaling through the EGF receptor pathway and by inducing Fcγ receptor-mediated antibody-dependent cellular cytotoxicity (ADCC).

[0161] Cetuximab heavy chain (SEQ ID NO: 17):

[0162] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0163] Cetuximab heavy chain variable region (SEQ ID NO: 27):

[0164] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA

[0165] Cetuximab light chain (SEQ ID NO: 18):

[0166] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLEL KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

[0167] Cetuximab light chain variable region (SEQ ID NO: 28):

[0168] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSR FSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK

[0169] (6) Anti-HER2 antibodies are antibodies that bind to the HER2 receptor. Optionally, the anti-HER2 antibodies further interfere with the activation or function of HER2. The anti-HER2 antibody used in the present invention is Enrico, a biosimilar to trastuzumab.

[0170] Trastuzumab heavy chain (SEQ ID NO: 19):

[0171] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;

[0172] Trastuzumab heavy chain variable region (SEQ ID NO:29):

[0173] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0174] Trastuzumab light chain (SEQ ID NO:20):

[0175] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0176] Trastuzumab light chain variable region (SEQ ID NO:30):

[0177] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK.

[0178] Example 1. Screening and humanization of anti-NKG2A antibodies

[0179] Hybridoma technology was used to generate monoclonal antibodies (mAbs) with specificity and high affinity for human and monkey NKG2A, which did not bind to cells overexpressing human NKG2C.

[0180] Fourteen groups of mice (including four groups of Balb / c mice, nine groups of SJL mice, and one group of NOD mice, all female mice aged 6 to 8 weeks) were immunized with the following immunogens: 293F-hNKG2A / CD94 stable cell line, recombinant hNKG2A / CD94-ECD-Fc protein, 293F-cynoNKG2A / CD94 stable cells + recombinant hNKG2A / CD94-ECD-Fc protein, and hNKG2A / CD94 expression plasmid + recombinant hNKG2A / CD94-ECD-Fc protein.

[0181] After immunization, serum titers were assessed by FACS and ELISA. FACS assays were performed using CHOK1-hNKG2A / CD94, CHOK1-cynoNKG2A / CD94, and CHOK1-hNKG2C / CD94 stably transfected cells, with CHOK1-blank cells serving as a control. ELISA assays were performed using hNKG2A / CD94-ECD-Fc and cynoNKG2A / CD94-ECD-Fc proteins.

[0182] Mice with high immune titers are selected, and spleen cells are fused with myeloma cells. Two rounds of screening are performed using FACS. After the primary screening, positive hybridoma clones are transferred to 24-well plates for culture and then undergo a secondary screening. Positive clones obtained from the screening are subcloned and tested again using the primary and secondary screening methods. Positive monoclones are selected for antibody production, cryopreservation, and sequencing.

[0183] After sequencing, eight sequence-specific monoclonal antibodies were obtained. Monoclonal antibody No. 43 was selected for humanization. The humanization design was completed by Baiying Bio. That is, humanization was performed by CDR transplantation. The CDR region of antibody No. 43 was transplanted to the matching human variable region gene framework sequence, and back mutations were designed. At the same time, potential post-translational modification sites (such as glycosylation sites, deamidation, etc.) in the first CDR region of the light chain were mutated. Then, it was combined with the heavy chain constant region of IgG4 and the light chain constant region of kappa to finally obtain the humanized antibody hu-43VL (N26S-G34A). The specific sequence is as follows:

[0184] Table 1: CDR sequences of hu-43VL (N26S-G34A)

[0185] name sequence SEQ ID NO: HCDR1 NTYIH 5 HCDR2 RIDPASGSTEYAPKFQG 6 HCDR3 YGNFLYYYSMDY 7 LCDR1 RSSKSLLHSNANTYLY 8 LCDR2 RMSNLAS 9 LCDR3 MQHLENPYT 10

[0186] hu-43VL(N26S-G34A) heavy chain:

[0187] QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYIHWVRQAPGQGLEWMGRIDPASGSTEYAPKFQGRV TMTADTSTNTGYMELSSLRSEDTAVYYCARYGNFLYYYSMDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:1).

[0188] hu-43VL(N26S-G34A) heavy chain variable region:

[0189] QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYIHWVRQAPGQGLEWMGRIDPAS GSTEYAPKFQGRVTMTADTSTNTGYMELSSLRSEDTAVYYCARYGNFLYYYSMDYWGQ GTLVTVSS (SEQ ID NO: 2).

[0190] hu-43VL(N26S-G34A) light chain:

[0191] DIVMTQSPLSLPVTLGQPASISCRSSKSLLHSNANTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3).

[0192] hu-43VL(N26S-G34A) light chain variable region:

[0193] DIVMTQSPLSLPVTLGQPASISCRSSKSLLHSNANTYLYWFQQRPGQSPRLLIYRMSN LASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK (SEQ ID NO: 4).

[0194] Example 2. Activity detection of anti-NKG2A humanized monoclonal antibody

[0195] 2.1 Antibody Binding Activity Detection

[0196] To prepare the hu-43VL (N26S-G34A) antibody, human NKG2A-overexpressing cells (CHOK1-hNKG2A / CD94) and monkey NKG2A-overexpressing cells (CHOK1-cynoNKG2A / CD94) were digested with TrypLE (purchased from Gibco). Cells were harvested by centrifugation and resuspended to 2E6 / mL in FACS buffer (PBS + 2% FBS). 100 μL of cells were added to each well of a 96-well plate and centrifuged. The supernatant was discarded. 100 μL of hu-43VL (N26S-G34A) antibody and control antibody Z270 were serially diluted in FACS buffer (starting at 200 nM, with 5-fold dilutions, including 0, for a total of 10 concentrations). The cells were added to the cells at 100 μL / well, mixed, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. Add secondary antibody: anti-human IgG fluorescent secondary antibody 488 (purchased from Thermofisher A-11013), incubate at 4℃ for one hour, wash twice with FACS buffer, resuspend in PBS and analyze with FACS instrument. Figure 1A and 1B .

[0197] The results showed that hu-43VL(N26S-G34A) antibody could bind to CHOK1-hNKG2A / CD94 and CHOK1-cynoNKG2A / CD94 cells with high activity.

[0198] 2.2 Antibody-mediated NK92 cell-mediated killing of target cells

[0199] Target B lymphocyte blasts (LCL721.221) were harvested by centrifugation and resuspended at 2E6 / mL in 1640 medium supplemented with 10% fetal bovine serum. 0.5 mL of the cell suspension was added to a 24-well plate, which was then incubated overnight at 27°C. NK92 cells (purchased from Enzyme Biotech) were harvested by centrifugation, washed twice with PBS, and counted using trypan blue staining to adjust the cell density to 4E5 / mL. 50 μL of the NK92 cell suspension was added to a 96-well plate. The test antibody was diluted in 1640 medium supplemented with 10% fetal bovine serum, and 50 μL of the diluted antibody was added to the 96-well plate (starting at 200 nM, diluted 5-fold, including the zero concentration, for a total of 10 concentrations). The plate was incubated at 37°C for 0.5 hour. Target cells were harvested by centrifugation, washed twice with PBS, and resuspended at 1E6 / mL in 1640 medium supplemented with 10% fetal bovine serum. Add the marker BATDA (purchased from PerkinElmer) (2 μL / mL) to the target cell suspension, and place the cells in a 37°C incubator for labeling for 20 minutes. Wash the labeled target cells 4 times by centrifugation, and resuspend the cells to 1E5 / mL in 1640 culture medium containing 10% fetal bovine serum. Add 100 μL of target cell suspension to a 96-well culture plate, and place the culture plate in a 37°C incubator for incubation for 2 hours. Centrifuge the culture plate at 300g for 5 minutes, take 25 μL of culture supernatant to another 96-well flat-bottom plate, and add 200 μL of Eu-solution (purchased from PerkinElmer). Read the data using an Envision instrument and analyze the data using Graphpad software. See the results. Figure 2 .

[0200] The results showed that the hu-43VL (N26S-G34A) antibody had a high killing effect on target cells, and the killing activity was comparable to that of the positive control Z270 antibody.

[0201] Antibody-mediated NK cell cytotoxicity against target cells

[0202] Primary NK cells were isolated from PBMC (purchased from Miaoshun Biotechnology) using an NK cell isolation kit and cultured for 5-7 days to a cell density of 2E6 / mL. LCL721.221 cells were harvested by centrifugation and resuspended in 1640 medium (containing 1 mM inducing peptide) supplemented with 10% fetal bovine serum to a cell density of 2E6 / mL. 0.5 mL of the cell suspension was added to a 24-well plate, which was then incubated overnight at 27°C. Primary NK cells were harvested by centrifugation, washed twice with PBS, and counted using trypan blue staining to adjust the cell density to 4E5 / mL. 50 μL of the cell suspension was added to a 96-well plate. The test antibody was diluted in 1640 medium supplemented with 10% fetal bovine serum, and 50 μL of the diluted antibody was added to the 96-well plate (starting at 200 nM and diluted 5-fold, including the zero concentration, for a total of 10 concentrations). The plate was incubated at 37°C for 0.5 hours. LCL721.221 cells were collected by centrifugation, washed twice with PBS, and resuspended in 1640 culture medium containing 10% fetal bovine serum to 1E6 / mL. The marker BATDA (purchased from PerkinElmer) (2μL / mL) was added to the LCL721.221 cell suspension, and the cells were placed in a 37°C incubator for labeling for 20 minutes. The LCL721.221 cells were washed 4 times by centrifugation, and the cells were resuspended in 1E5 / mL with 1640 culture medium containing 10% fetal bovine serum. 100μL of LCL721.221 cell suspension was added to a 96-well culture plate, and the culture plate was placed in a 37°C incubator for incubation for 2 hours. The culture plate was centrifuged at 300g for 5 minutes, 25μL of culture supernatant was taken to another 96-well flat-bottom plate, and 200μL of Eu-solution was added. The data were read using an Envision instrument and analyzed using Graphpad software. The results are shown in Figure 3 .

[0203] The results showed that primary NK cells mediated by hu-43VL (N26S-G34A) antibody had a killing effect on LCL721.221 cells, and the killing activity was significantly better than the control molecule Z270, indicating that it has good drug development prospects.

[0204] Example 3. Anti-NKG2A Antibody Enhances PD-L1 Antibody-Mediated Killing of NK92 Cells on Target Cells

[0205] NK92MI-hCD16a cells (purchased from Huabo) were harvested by centrifugation, washed twice with 1640 complete medium, and counted by trypan blue staining. The cell density was adjusted to 4E5 / mL. 50 μL of the NK92MI-hCD16a cell suspension was added to a 96-well round-bottom plate. Hu-43VL (N26S-G34A) antibody and control IgG4 antibody were diluted in 1640 medium supplemented with 10% fetal bovine serum. 25 μL of each diluted hu-43VL (N26S-G34A) antibody (10 μg / mL) and control IgG4 antibody (10 μg / mL) were added to the 96-well plate. The culture plate was pre-incubated at 37°C for 0.5 hours. HLA-E overexpressing target B lymphocyte blasts, LCL721.221-AEH cells (purchased from Ruizhi Chemical), were collected by centrifugation, washed twice with serum-free 1640 medium, and resuspended in serum-free 1640 medium. The cells were counted using trypan blue staining and adjusted to a cell density of 1E6 / mL. Calcein-AM (purchased from MedChemExpress) was added to the target cell suspension (final concentration 10 μM), and the cells were placed in a 37°C incubator for 30 minutes. After centrifugation and discarding the supernatant, the labeled target cells were washed three times with 1640 medium containing 10% fetal bovine serum and resuspended at 1E5 / mL in 1640 medium containing 10% fetal bovine serum. 100 μL of the target cell suspension was added to a 96-well culture plate. Anti-PD-L1 antibody Avelumab and control antibody IgG1 were diluted with 1640 medium containing 10% fetal bovine serum, and 25 μL of diluted Avelumab antibody (starting concentration was 2 μg / mL, 3-fold dilution, a total of 7 concentration points) and control antibody IgG1 (2 μg / mL) were added to the 96-well plate. The culture plate was placed in a 37°C incubator and incubated for 70 minutes. The culture plate was centrifuged at 500g for 3 minutes, 100 μL of culture supernatant was taken to a 96-well flat-bottom plate, the data was read with a Tecan microplate reader, and the data was analyzed with Graphpad software. The experimental results are shown in Figure 4 .

[0206] like Figure 4 As shown in the data, the killing effect of the Avelumab+hu-43VL(N26S-G34A) group was better than that of the Avelumab+IgG4 group, that is, the hu-43VL(N26S-G34A) antibody could enhance the killing effect of NK92 cells on target cells mediated by Avelumab.

[0207] Example 4. Combined Effect of Anti-NKG2A Antibody and Anti-CD20 Monoclonal Antibody

[0208] The killing effect of the anti-NKG2A antibody hu-43VL (N26S-G34A) in combination with increasing doses of the anti-CD20 monoclonal antibody HS006 was evaluated. NK92MI-hCD16a cells (purchased from Huabo) were collected and washed with experimental medium and resuspended to 5×10 5 40 μL was added to each well of a 96-well cell culture plate; LCL721.221-HLA-E cells (purchased from Ruizhi Chemical) were collected by centrifugation, washed with experimental culture medium, and resuspended to 2.5×10 5 / mL, 40μL per well was added to the above 96-well cell culture plate. HS006 was diluted to an effective concentration of 8pg / mL and 40pg / mL, hu-43VL (N26S-G34A) was diluted to an effective concentration of 10μg / mL, and 20μL / well of each was added to the plate; at the same time, a hu-43VL (N26S-G34A) monoclonal antibody group and an IgG1 control group were set up, and the 96-well plate was placed in a 37°C incubator for 4 hours; the LDH kit (manufacturer: Roche; catalog number: 04744934001) was used for color development, and the plate was placed on an enzyme reader to read the plate and calculate the killing rate. The results are shown in Figure 5 .

[0209] The results showed that the killing effect of the anti-NKG2A monoclonal antibody hu-43VL (N26S-G34A) combined with the anti-CD20 monoclonal antibody was stronger than that of hu-43VL (N26S-G34A) or HS006 alone.

[0210] Example 5. Combined Effect of Anti-NKG2A Antibody and Anti-CD70 Monoclonal Antibody

[0211] Beijing Tianguangshi's fucose-knockout Chinese hamster ovary cells (CHOK1-AF) were used to express the anti-CD70 antibody 1F6. The cell culture supernatant was collected and purified to obtain the ADCC-enhanced 1F6 antibody.

[0212] LCL721.221-HLA-E cells (purchased from Ruizhi Chemical) were collected and washed with 2% BSA-PBS solution. The cell density was adjusted to 5×10 5 Each tube was added to a 1.5 mL EP tube; the 1F6 antibody and IgG1 control were diluted to 10 μg / mL and added to the cells, incubating at 4°C for 30 minutes. After washing the cells, PE-conjugated goat anti-human IgG (Invitrogen; Catalog No. 12-4998-82) secondary antibody was added and incubated at 4°C in the dark for 30 minutes. After washing and fixation, the cells were analyzed using a flow cytometer (BD Accuri C6 Plus). The results are shown in Table 2.

[0213] The results showed that 1F6 antibody could bind to LCL721.221-HLA-E cells.

[0214] Table 2. Binding activity of anti-CD70 monoclonal antibody 1F6 to cells

[0215] Antibody Mean fluorescence intensity 1F6 antibody 107712 IgG1 control 3586

[0216] NK92 cells (purchased from Huabo) were collected, washed with experimental medium and resuspended to 5×10 5 40 μL was added to each well of a 96-well cell culture plate; LCL721.221-HLA-E cells were collected by centrifugation, washed with experimental culture medium, and resuspended to 2.5×10 5 / mL, 40μL per well was added to the above 96-well cell culture plate; 1F6 antibody was diluted to an effective concentration of 6ng / mL, hu-43VL (N26S-G34A) was diluted to an effective concentration of 60μg / mL, 20μl / well of each was added to the plate, and a hu-43VL (N26S-G34A) control group and an IgG1 control group were set up at the same time, and incubated at 37°C for 4 hours; LDH kit (manufacturer: Roche; catalog number: 04744934001) was used for color development, and the plate was read on a microplate reader and the killing rate was calculated. The results are shown in Table 3 and Figure 6 shown.

[0217] The results showed that the killing effect of the anti-NKG2A monoclonal antibody hu-43VL (N26S-G34A) combined with the anti-CD70 monoclonal antibody was stronger than that of hu-43VL (N26S-G34A) or 1F6 antibody alone.

[0218] Table 3. Killing effect of anti-NKG2A mAb combined with anti-CD70 mAb

[0219] Grouping Kill rate (%) IgG1 control 0.28 1F6 9.79 hu-43VL(N26S-G34A) 21.77 hu-43VL(N26S-G34A)+1F6 53.07

[0220] Example 6. Anti-NKG2A antibody promotes NK cell killing of tumor cells

[0221] The target cells, B lymphocyte blasts LCL721.221-AEH cells (purchased from Ruizhi Chemical) that overexpress HLA-E, were collected by centrifugation, washed twice with serum-free 1640 medium, and resuspended in serum-free 1640 medium (purchased from Hyclone). The cells were stained with trypan blue and counted, and the cell density was adjusted to 1E6 / mL. The marker Calcein-AM (purchased from MedChemExpress, final concentration 10 μM) was added to the target cell suspension and the cells were placed in a 37°C incubator for 30 minutes. After incubation, 96-well plates were plated with 2×10 cells per well. 4 cells / 100μL. Grouped as follows:

[0222] (1) Spontaneous release: Inoculate 100 μL of target cells and 100 μL of 1640 culture medium.

[0223] (2) Maximum release: Inoculate 100 μL of target cells and 50 μL of 1640 culture medium, and add 50 μL of Triton X-100 after the co-culture.

[0224] (3)Experimental release:

[0225] NK group: 100 μL of target cells and 100 μL of NK cells (APE-NK cells, Jingda Bio) were inoculated (the number of NK cells added was calculated and diluted according to different effector-target ratios of 1:1, 2:1, 5:1, and 10:1);

[0226] NK + hu-43VL (N26S-G34A) group: 100 μL of target cells and 100 μL of NK cells were inoculated (the number of NK cells added was calculated and diluted according to different effector-target ratios of 1:1, 2:1, 5:1, and 10:1), and hu-43VL (N26S-G34A) antibody was added at a final concentration of 10 μg / mL;

[0227] NK + hu-43VL (N26S-G34A) pre-incubation group: 100 μL of target cells was inoculated, and 100 μL of NK cells (the number of NK cells added was calculated according to different effector-target ratios of 1:1, 2:1, 5:1, and 10:1, and diluted) and 10 μg / mL final concentration of hu-43VL (N26S-G34A) antibody were mixed and incubated for 30 minutes, and then transferred to a 96-well plate.

[0228] hu-43VL(N26S-G34A) control group: inoculate 100 μL of target cells, add hu-43VL(N26S-G34A) antibody at a final concentration of 10 μg / mL, and add 100 μL of 1640 culture medium.

[0229] Set up 3-6 replicate wells for each group. After inoculation, mix thoroughly and incubate in a 37°C, 5% CO2 incubator for 4 hours. For the Maximum Release group, add Triton X-100, gently shake to mix, and centrifuge at 400g for 5 minutes. Transfer 150 μL of supernatant from each well to a microplate. Fluorescence is measured using a microplate reader at excitation and emission wavelengths of 494 nm (Ex) / 517 nm (Em). Calculate the mean of the data for each replicate well.

[0230] Killing rate (%) = [(Experimental release-spontaneous release) / (maximum release-spontaneous release)] × 100%.

[0231] The results are as follows Figure 7 shown.

[0232] The results showed that hu-43VL (N26S-G34A) could significantly increase the cytotoxic activity of NK cells against target cells. Adding hu-43VL (N26S-G34A) after incubation with NK cells or adding them simultaneously had no significant effect on the cytotoxic activity.

[0233] Example 7. Combination of anti-NKG2A antibody and anti-EGFR monoclonal antibody to kill tumor cells

[0234] NK92 cells (purchased from Enzyme Biotechnology) were harvested by centrifugation, washed with PBS, and counted by trypan blue staining. The cell density was adjusted to 2E5 / mL. 50 μL of the NK92 cell suspension was added to a 96-well plate. hu-43VL (N26S-G34A) was diluted to a final concentration of 12.5 μg / mL in 1640 medium supplemented with 10% fetal bovine serum. Isotype control antibody IgG4 was diluted to a final concentration of 50 μg / mL. Cetuximab (c225 antibody, purchased from Selleck, catalog number A200003) was diluted to a final concentration of 1 μg / mL in 1640 medium supplemented with 10% fetal bovine serum. hu-43VL (N26S-G34A), cetuximab, and IgG4 were added to a 96-well plate and incubated with NK92 cells at 37°C for 30 minutes. After washing with PBS, A549 cells were digested, resuspended and centrifuged in 1640 medium containing 10% fetal bovine serum, and counted with trypan blue staining. The A549 cell density was adjusted to 2.5E5 / mL. 40 μL of A549 cell suspension was added to a 96-well plate, and the culture plate was placed in a 37°C incubator for 48 hours. After that, 100 μL of CellTiter-Glo reagent was added to each well, and the culture plate was placed on an orbital shaker for 3 minutes. The culture plate was then incubated at room temperature for 15 minutes, and the luminescent signal was detected with a microplate reader. The killing effect of hu-43VL (N26S-G34A) combined with cetuximab on A549 cells was calculated. The results are shown in Figure 8 .

[0235] The experimental results showed that both hu-43VL (N26S-G34A) and cetuximab alone significantly promoted the killing of A549 cells by NK92 cells. The combined administration group could enhance the killing effect on A549 cells compared with the hu-43VL (N26S-G34A) and cetuximab alone groups.

[0236] Example 8. Combination of anti-NKG2A antibody and anti-Her2 monoclonal antibody to kill tumor cells

[0237] NK92MI-hCD16a cells (purchased from Huabo) were harvested by centrifugation, washed twice with 1640 complete medium, and counted by trypan blue staining. The cell density was adjusted to 1E6 / mL. 50 μL of the NK92MI-hCD16a cell suspension was added to a 96-well round-bottom plate. hu-43VL (N26S-G34A) antibody and control IgG4 antibody were diluted in 1640 medium supplemented with 10% fetal bovine serum. 25 μL of each diluted hu-43VL (N26S-G34A) antibody (10 μg / mL) and control IgG4 antibody (10 μg / mL) were added to the 96-well plate. The culture plate was pre-incubated at 37°C for 0.5 hours. The day before the experiment, A431 cells were trypsinized and harvested by centrifugation. They were then resuspended at 1.5E6 / mL in 1640 medium supplemented with 10% fetal bovine serum (containing 1 mM inducible peptide). 1 mL of cell suspension was added to a 6-well plate and incubated overnight at 37°C. On the day of the experiment, A431 cells were trypsinized and harvested by centrifugation. After washing twice with serum-free 1640 medium, the cells were resuspended in serum-free 1640 medium and counted using trypan blue staining. The cell density was adjusted to 1E6 / mL. Calcein-AM (MedChemExpress) was added to the A431 cell suspension (final concentration 10 μM) and the cells were incubated at 37°C for 30 minutes. After centrifugation and discarding the supernatant, the labeled A431 cells were washed three times with 1640 medium supplemented with 10% fetal bovine serum and resuspended at 1E5 / mL in 1640 medium supplemented with 10% fetal bovine serum. 100 μL of target cell suspension was added to a 96-well plate. Trastuzumab and control antibody IgG1 were diluted in 1640 medium containing 10% fetal bovine serum, and 25 μL of diluted trastuzumab (starting concentration was 1 μg / mL, 3-fold dilution, a total of 7 concentration points) and control antibody IgG1 (1 μg / mL) were added to the 96-well plate. The culture plate was placed in a 37°C incubator and incubated for 105 minutes. The culture plate was centrifuged at 500g for 3 minutes, and 100 μL of culture supernatant was transferred to a 96-well flat-bottom plate. The data were read using a Tecan microplate reader and analyzed using Graphpad software. The experimental results are shown in Figure 9 .

[0238] The results showed that in the experiment of NK92MI-hCD16a cells killing A431 cells, the killing effect of the trastuzumab + hu-43VL (N26S-G34A) group was better than that of the trastuzumab + IgG4 group, that is, the hu-43VL (N26S-G34A) antibody can enhance the trastuzumab-mediated NK92 cell killing effect on target cells A431.

[0239] Example 9. Combination of anti-NKG2A antibody and anti-Her2 monoclonal antibody / EGFR monoclonal antibody to kill tumor cells

[0240] NK92MI-hCD16a cells (purchased from Huabo) were harvested by centrifugation, washed twice with 1640 complete medium, and counted by trypan blue staining. The cell density was adjusted to 1E6 / mL (E:T = 5:1), and 50 μL of the NK92MI-hCD16a cell suspension was added to a 96-well round-bottom plate. hu-43VL (N26S-G34A) antibody and control IgG4 antibody were diluted in 1640 medium supplemented with 10% fetal bovine serum, and 25 μL of the diluted hu-43VL (N26S-G34A) antibody (final concentration 3 μg / mL) and control IgG4 antibody (final concentration 3 μg / mL) were added to the 96-well plate. The culture plate was pre-incubated at 37°C for 0.5 hours.

[0241] The day before the experiment, Calu1-AEH cells (Calu1 cells overexpressing AHE, AHE reference NCBI Gene ID: 3133) were trypsinized and centrifuged to a density of 3.6E6 cells / 4mL in complete culture medium (McCoy's 5a + 10% FBS; containing 1 mM VL9 inducible peptide). 4 mL of the cell suspension was evenly distributed between two 60 mm culture dishes and incubated at 37°C overnight. On the day of the experiment, Calu1-AEH cells were trypsinized and centrifuged to a density of 4E6 cells / 4mL. All subsequent operations were performed in the dark. Calcein-AM (final concentration 10 μM) was added to the Calu1-AEH cell suspension, mixed thoroughly, and the cells were incubated at 37°C for 30 minutes. After centrifugation and discarding the supernatant, the labeled Calu1-AEH cells were washed three times with 1640 medium supplemented with 10% fetal bovine serum. The cells were resuspended at 1E5 / mL in 1640 medium supplemented with 10% fetal bovine serum. 100 μL of the Calu1-AEH cell suspension was added to a 96-well plate. Trastuzumab, cetuximab, or control IgG4 were diluted to varying concentrations in 1640 medium supplemented with 10% fetal bovine serum (7 steps with 3-fold dilutions for trastuzumab, 1 μg / mL for trastuzumab, and 7 steps with 3-fold dilutions for cetuximab, up to 200 ng / mL for cetuximab). 25 μL of each diluted antibody was added to the 96-well plate. After mixing thoroughly, the plate was incubated at 37°C for 2–3 hours. The plate was centrifuged at 500 g for 3 minutes, and 100 μL of the culture supernatant was transferred to a 96-well flat-bottom plate. Data were read using a Tecan microplate reader and analyzed using GraphPad software. The experimental results are shown in Figure 10A and Figure 10B .

[0242] The results showed that in the NK92MI-hCD16a cell-mediated killing of Calu1-AEH cells, the trastuzumab + hu-43VL (N26S-G34A) group showed superior killing efficacy compared to the trastuzumab + IgG4 group, while the cetuximab + hu-43VL (N26S-G34A) group showed superior killing efficacy compared to the cetuximab + IgG4 group. This indicates that the hu-43VL (N26S-G34A) antibody can enhance the trastuzumab / cetuximab-mediated killing of Calu1-AEH cells by NK92MI-hCD16a cells.

Claims

1. Use of an anti-NKG2A antibody in the preparation of a medicament for use in combination with another therapeutic agent for preventing or treating a disease, wherein the anti-NKG2A antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the sequence of SEQ ID NO: 2, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the sequence of SEQ ID NO: 4; Preferably, the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6, and HCDR3 shown in SEQ ID NO: 7, and the light chain variable region comprises LCDR1 shown in SEQ ID NO: 8, LCDR2 shown in SEQ ID NO: 9, and LCDR3 shown in SEQ ID NO: 10; More preferably, the heavy chain variable region of the anti-NKG2A antibody comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 2, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 4; More preferably, the anti-NKG2A antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

3.

2. The use according to claim 1, wherein the other therapeutic agent is selected from PD-1 neutralizers, EGFR antagonists, HER2 antagonists, anti-CD20 antibodies, anti-CD70 antibodies and immune cells.

3. The use according to claim 2, wherein the PD-1 neutralizing agent is an anti-PD-1 antibody or an anti-PD-L1 antibody; preferably, the anti-PD-L1 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22; more preferably, the anti-PD-L1 antibody is selected from the group consisting of: avelumab, atezolizumab, socazolimab, adebrelimab, sugemalimab, envafolimab, durvalumab, cosibelimab, and KL-A167; The anti-PD-1 antibody is selected from pembrolizumab, nivolumab, retifanlimab, cadonilimab, BMS-986213 (relatlimab + nivolumab), serplulimab, zimberelimab, penpulimab, dostarlimab, tirizumab, Tislelizumab, camrelizumab, toripalimab, sintilimab, cemiplimab, ivonescimab, prolgolimab, geptanolimab, QL-1604 (iparomlimab), and HX-008 (pucotenlimab).

4. The use according to claim 2, wherein the EGFR antagonist is an anti-EGFR antibody or a small molecule EGFR inhibitor; preferably, the anti-EGFR antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 28; more preferably, the anti-EGFR antibody is selected from cetuximab, panitumumab, necitumumab, matuzumab and ado-trastuzumab emtansine.

5. The use according to claim 2, wherein the HER2 antagonist is an anti-HER2 antibody or a small molecule HER2 inhibitor; preferably, the anti-HER2 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30; more preferably, the anti-HER2 antibody is selected from trastuzumab, pertuzumab, disitamab vedotin, zanidatamab, Zenocutuzumab, Trastuzumab Deruxtecan, vedicizumab and Ado-trastuzumab.

6. The use according to claim 2, wherein the anti-CD20 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 24; more preferably, the anti-CD20 antibody is selected from zuberitamab, obinutuzumab, ibritumomab, ofatumumab, tositumomab, ocrelizumab, ublituximab and rituximab.

7. The use according to claim 2, wherein the anti-CD70 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; more preferably, the anti-CD70 antibody comprises a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO:

16.

8. The use according to claim 2, wherein the immune cells are selected from NK cells and T cells; more preferably, the NK cells are selected from autologous NK cells, chimeric receptor-NK cells, NK cell lines and allogeneic NK cells.

9. The use according to any one of claims 1 to 8, wherein the anti-NKG2A antibody and another therapeutic agent are administered simultaneously, separately or sequentially.

10. The use according to any one of claims 1 to 9, wherein the disease is cancer, autoimmune disease or inflammatory disease; preferably, the cancer is selected from non-small cell lung cancer, small cell lung cancer, ovarian cancer, fallopian tube cancer, colon cancer, colorectal cancer, breast cancer, melanoma, lung cancer, liver cancer, pancreatic cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer and lymphocytic leukemia; More preferably, the cancer is one in which HLA-E or Qa1 is expressed on the surface of the cancer cells. b cancer.

Images