Antibodies or antigen-binding fragments binding to nkg2a and uses thereof

Abstract

The present disclosure relates to antibodies or antigen-binding fragments that bind to NKG2A and uses thereof. Antibodies or antigen binding fragments that bind to NKG2A can improve immune responses and lysis of HLA-E expressing tumor cells.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480040596.X (22) Application Date 2024.06.12 (66) Domestic Priority Data PCT / CN2023 / 101950 2023.06.21 CN PCT / CN2023 / 128764 2023.10.31 CN (85) PCT International Application Entering National Phase Date 2025.12.17 (86) PCT International Application Application Data PCT / CN2024 / 098762 2024.06.12 (87) PCT International Application Publication Data WO2024 / 260280 EN 2024.12.26 (71) Applicant Kewang (Suzhou) Biomedical Technology Co., Ltd. Address 215127 Unit E729, 5th Floor, Lecheng Plaza, Phase II, Biomedical Industrial Park, No. 218 Sangtian Street, Suzhou Area, China (Jiangsu) Pilot Free Trade Zone, Suzhou City, Jiangsu Province. Applicant: Kewang (Shanghai) Biomedical Technology Co., Ltd. (72) Inventors: Gao Jing, Gao Rui, Sun Dawei, Liu Zeyu, Qiu Quan, Yu Jingfeng, Zhou Hongping, Shi Mei, Sun Jun, Wang Chunian, Lu Hongtao (74) Patent Agency: Beijing Hankun Law Firm, 11602 Patent Attorneys: Peng Kezheng, Li Ying (51) Int.Cl. C07K 16 / 28 (2006.01) A61K 39 / 395 (2006.01) A61P 35 / 00 (2006.01) A61P 37 / 00 (2006.01) A61P 31 / 12 (2006.01) (54) Title of Invention: Antibody or Antigen-Binding Fragment that Binds to NKG2A and Its Use (57) Abstract: This disclosure relates to antibody or antigen-binding fragment that binds to NKG2A and its use. Antibody or antigen-binding fragment that binds to NKG2A can improve immune responses and lysis of tumor cells expressing HLA-E. Claims (5 pages), Description (25 pages), Sequence Listing (electronic publication), Figures (12 pages), CN 121358774 A 2026.01.16 CN 1 21 35 87 74 A 1. An antibody or antigen-binding fragment thereof that binds to NKG2A, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 36, RIDPANXNTKYAPNFQG (SEQ ID NO: 37), and SEQ ID NO: 38, respectively; X is G or A; and said VL comprises regions having amino acid sequences as shown in SEQ ID NO:1. The complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in 39, 40, and 41. 2. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in claim 1, wherein X is G. 3. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in claim 1, wherein X is A. 4. The antibody or antigen-binding fragment thereof that binds to NKG2A as claimed in claim 1, wherein the VH and the VL are selected from the following: a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11; b) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13 ... a) VL containing amino acid sequences having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14, and VH containing amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15; d) VH containing amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16, and VL containing amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15. NO: 17 A VL having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18; e) A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18, and a VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18.NO: 19 A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20; f) A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21; g) A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity with SEQ ID NO: 22. VH containing an amino acid sequence with 98%, 99%, or 100% sequence identity, and VL containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23; h) VH containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24, and VL containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25; i) VH containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25; 26. A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27; j) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 28, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29. (Claims 1 / 5 page 2 CN 121358774 A)VL containing amino acid sequences with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 30; k) VH containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31; l) VH containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32; and VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32. 33. A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 34; m) A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 35, and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 42; n) A VL containing at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity with SEQ ID NO: 42. VH containing an amino acid sequence with 98%, 99%, or 100% sequence identity, and VL containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13; o) VH containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 43, and VL containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29.5. The antibody or antigen-binding fragment thereof that binds to NKG2A as claimed in claim 1, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 10, and the VL comprises the amino acid sequence shown in SEQ ID NO: 11; the VH comprises the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises the amino acid sequence shown in SEQ ID NO: 13; the VH comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises the amino acid sequence shown in SEQ ID NO: 15; the VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence shown in SEQ ID NO: 17; the VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the VL comprises the amino acid sequence shown in SEQ ID NO: 19; the VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO: 21; the VH comprises the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises the amino acid sequence shown in SEQ ID NO: 23. The amino acid sequence shown in claim 23; the VH contains the amino acid sequence shown in SEQ ID NO: 24, and the VL contains the amino acid sequence shown in SEQ ID NO: 25; the VH contains the amino acid sequence shown in SEQ ID NO: 26, and the VL contains the amino acid sequence shown in SEQ ID NO: 27; the VH contains the amino acid sequence shown in SEQ ID NO: 28, and the VL contains the amino acid sequence shown in SEQ ID NO: 29; the VH contains the amino acid sequence shown in SEQ ID NO: 30, and the VL contains the amino acid sequence shown in SEQ ID NO: 31; the VH contains the amino acid sequence shown in SEQ ID NO: 32, and the VL contains the amino acid sequence shown in SEQ ID NO: 33; the VH contains the amino acid sequence shown in SEQ ID NO: 34, and the VL contains the amino acid sequence shown in SEQ ID NO: 3 ...36, and the VL contains the amino acid sequence shown in SEQ ID NO: 37; the VH contains the amino acid sequence shown in SEQ ID NO: 38, and the VL contains the amino acid sequence shown in SEQ The amino acid sequence shown in SEQ ID NO: 42, and the VL containing the amino acid sequence shown in SEQ ID NO: 13; or the VH containing the amino acid sequence shown in SEQ ID NO: 43, and the VL containing the amino acid sequence shown in SEQ ID NO: 29.6. The antibody or antigen-binding fragment of the NKG2A binding method as described in any one of claims 1-5, wherein the antibody or antigen-binding fragment of the NKG2A binding method does not bind to NKG2E or binds very little to NKG2E. 7. The antibody or antigen-binding fragment of the NKG2A binding method as described in any one of claims 1-6, wherein the antibody or antigen-binding fragment of the NKG2A binding method improves an immune response, optionally an innate immune response. 8. The antibody or antigen-binding fragment of the NKG2A binding method as described in any one of claims 1-7, wherein the antibody or antigen-binding fragment of the NKG2A binding method blocks the interaction between NKG2A / CD94 and an MHC-1 molecule, optionally, wherein the MHC-1 molecule is HLA-E. 9. The antibody or antigen-binding fragment of the NKG2A binding method as described in any one of claims 1-8, wherein the antibody or antigen-binding fragment improves the lysis of HLA-E-expressing cells, optionally, the HLA-E-expressing cells include HLA-E-expressing tumor cells. 10. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A increases the response of NK cells, optionally, the NK cells express NKG2A. 11. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A exerts NK-mediated cytotoxicity against HLA-E-expressing tumor cells. 12. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A exerts NK-mediated cytotoxicity against HLA-E-expressing tumor cells, wherein the EC50 does not exceed 1 nM, 0.8 nM, 0.5 nM, or 0.3 nM. 13. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A upregulates CD107a (lysosome-associated membrane protein 1). 14. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A reverses the inhibition of T cell responses, optionally, the antibody or antigen-binding fragment thereof that binds to NKG2A reverses NKG2A / HLA-E-mediated inhibition of T cell responses. 15. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-14, wherein...An antibody or antigen-binding fragment thereof that binds to NKG2A activates T cells or increases the response of T cells, optionally, wherein the T cells express NKG2A or NKG2C. 16. The antibody or antigen-binding fragment thereof that binds to NKG2A as claimed in any one of claims 1-15, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A activates NFAT signaling. 17. The antibody or antigen-binding fragment thereof that binds to NKG2A as claimed in any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A binds to NKG2A, wherein the EC50 does not exceed 1 nM, 0.8 nM, or 0.6 nM, or the KD (affinity constant) does not exceed 9.9E-8 nM, 9.9E-9 nM, or 9.9E-10 nM. 18. An antibody or antigen-binding fragment thereof that binds to NKG2A as claimed in any one of claims 1-17, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A blocks the interaction between NKG2A and HLA-E, wherein the IC50 is not greater than 1 nM, 0.8 nM, or 0.6 nM. 19. An antibody or antigen-binding fragment thereof that binds to NKG2A as claimed in any one of claims 1-18, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A is chimeric or humanized. 20. An antibody or antigen-binding fragment thereof that binds to NKG2A as claimed in any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A comprises an Fc fragment, optionally, the Fc fragment being wild-type human IgG or human IgG having one or more mutations. 21. An antibody or antigen-binding fragment thereof that binds to NKG2A, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A binds to a peptide as shown in SEQ ID NO: 8. 22. A binding site comprising an amino acid sequence as shown in SEQ ID NO: 8. 23. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-21. 24. An isolated vector comprising the isolated polynucleotide as described in claim 23. 25. A host cell comprising the isolated polynucleotide as described in claim 23 or the isolated vector as described in claim 24. 26. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-21, the isolated polynucleotide as described in claim 23, the isolated vector as described in claim 24, or the host cell as described in claim 25, and a pharmaceutically acceptable carrier.27. A kit comprising an antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-21, a binding site as described in claim 22, an isolated polynucleotide as described in claim 23, an isolated vector as described in claim 24, or a host cell as described in claim 25. 28. Use of an antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-21, an isolated polynucleotide as described in claim 23, an isolated vector as described in claim 24, a host cell as described in claim 25, or a pharmaceutical composition as described in claim 26, or a kit as described in claim 27, in the manufacture of a therapeutic agent for diagnosing, preventing, or treating a disease. 29. The use as described in claim 28, wherein the disease comprises oncology-related diseases, inflammatory or autoimmune diseases, or infectious diseases. 30. The use as described in claim 28 or 29, wherein the oncology-related disease includes breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies; the infectious disease includes viral infectious diseases and bacterial infectious diseases; the inflammatory or autoimmune disease includes arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory diseases. 31. A method for diagnosing, preventing, or treating a subject suffering from an oncology-related disease, an inflammatory or autoimmune disease, or an infectious disease, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-21, an isolated polynucleotide as described in claim 23, an isolated vector as described in claim 24, a host cell as described in claim 25, a pharmaceutical composition as described in claim 26, or a kit as described in claim 27. Claims 4 / 5, page 5, CN 121358774, A 32. The method of claim 31, wherein the method comprises enhancing the immune response to the disease, optionally, the method comprises enhancing the innate immune response. 33. The method of claim 31 or 32, wherein the method comprises reducing adverse effects in the diagnosis, prevention, or treatment of the disease. 34. The method of any one of claims 31-33, wherein the oncology-related disease includes breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies;The infectious diseases include viral infectious diseases and bacterial infectious diseases; the inflammatory or autoimmune diseases include arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory diseases. 35. The method of any one of claims 31-34, wherein the method improves the lysis of HLA-E-expressing cells; optionally, the method improves the lysis of HLA-E-expressing tumor cells. 36. The method of any one of claims 31-35, wherein the method comprises (1) increasing the NK cell response; (2) upregulating CD107a; (3) activating T cells or increasing the T cell response; or (4) activating NFAT signaling. Claims 5 / 5 Page 6 CN 121358774 A Antibody or antigen-binding fragment binding to NKG2A and its use Technical Field

[0001] This disclosure relates to antibodies or antigen-binding fragments binding to NKG2A, polynucleotides, vectors, host cells, and pharmaceutical compositions thereof. This disclosure also relates to the use of antibodies or antigen-binding fragments. Background Art

[0002] The statements in this section are provided only as background information in relation to this disclosure and do not necessarily constitute prior art.

[0003] Immuno-oncology has become a revolution in cancer treatment. Tumor control has been improved to an unprecedented degree by releasing therapeutic blocking antibodies against immunosuppressive 'checkpoints' (immune checkpoint inhibitors, ICIs). However, only a small number of patients respond to these immunotherapies. Currently, the identification of predictive biomarkers for treatment response is an important research topic and a number of factors have been identified. These factors include the number of T cells in the tumor and the total mutational burden of tumor cells, suggesting that ICI depends on natural immunity to neoantigens presented by HLA molecules (van Hall T, Andr é P et al., J Immunother Cancer, 2019 Oct 17 7(1):263.) NKG2A, also known as KLRC1 (killer cell lectin-like receptor C1) in humans, belongs to the NKG2 family and is a group of transmembrane proteins expressed on both T cells and NK cells. This protein family is characterized by a type II membrane orientation and the presence of a type C lectin domain. NKG2A is expressed as a heterodimer with CD94 (cytokine-like receptor D1, also known as KLRD1) on the surface of human and mouse cells, and recognizes the non-classical class I major histocompatibility complex (MHC-I) molecule human leukocyte antigen (HLA)-E in humans and Qa-1b in mice. The binding of NKG2A / CD94 to its homologous ligands inhibits T cell effector function and NK cell effector function (Le Dréan et al., 1998; Rapaport et al., 2015). Summary of the Invention

[0004] For the purposes described above, this disclosure relates to antibodies or antigen-binding fragments thereof that bind to NKG2A. Antibodies or antigen-binding fragments thereof that bind to NKG2A can improve immune responses (e.g., innate immune responses). In addition, antibodies or antigen-binding fragments thereof that bind to NKG2A can be used for the diagnosis, prevention, or treatment of oncology-related diseases (e.g., cancer treatment), inflammatory or autoimmune diseases, and infectious diseases.

[0005] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to NKG2A, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises complementarity-determining regions HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 36, RIDPANXNTKYAPNFQG (SEQ ID NO: 37) and SEQ ID NO: 38, respectively; X is G or A; and the VL comprises complementarity-determining regions LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 39, 40 and 41, respectively.

[0006] In one embodiment, VH and VL are selected from the following: a) VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11; b) VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12, and VL ... 13. A VL having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14; c) A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15;The VL containing the sequence; d) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16, and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17; e) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18, and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. a) VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20; f) VH containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21; g) VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21; 23. A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24; h) A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24; and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25; i) A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 26.The following are listed as VH and VL: a) VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27; b) VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 28; c) VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29; d) VL containing an amino acid sequence having at least 80%, 85%, 90 ... VH containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31; VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32; VH containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 33; VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 33; 34. A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 35; and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 42; and a VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13.VL;o) comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 43, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof that binds to NKG2A does not bind to NKG2E (cytotoxic lectin-like receptor C3, KLRC3) or hardly binds to NKG2E. Specification 2 / 25 pages 8 CN 121358774 A

[0008] In some embodiments, the antibody or antigen-binding fragment thereof that binds to NKG2A improves the immune response (e.g., innate immune response).

[0009] In some embodiments, an antibody or antigen-binding fragment thereof that binds to NKG2A blocks the interaction between the NKG2A / CD94 heterodimer and MHC-1 (major histocompatibility complex, class I) molecules (e.g., HLA-E (major histocompatibility complex, class I, E)).

[0010] In some embodiments, an antibody or antigen-binding fragment thereof that binds to NKG2A improves the lysis of HLA-E-expressing cells (e.g., HLA-E-expressing tumor cells).

[0011] In some embodiments, an antibody or antigen-binding fragment thereof that binds to NKG2A increases the response of natural killer (NK) cells (e.g., NK cells expressing NKG2A).

[0012] In some embodiments, an antibody or antigen-binding fragment thereof that binds to NKG2A reverses the inhibition of T cell responses, such as NKG2A / HLA-E-mediated inhibition of T cell responses. In some embodiments, the antibody or antigen-binding fragment includes, but is not limited to, activating T cells by activating NFAT (activated T cell nuclear factor) signaling.

[0013] In another aspect, this disclosure provides an antibody or antigen-binding fragment thereof that binds to NKG2A, the antibody or antigen-binding fragment thereof binding to a peptide as shown in SEQ ID NO: 8.

[0014] In another aspect, this disclosure provides a binding site comprising an amino acid sequence as shown in SEQ ID NO: 8.

[0015] In another aspect, this disclosure relates to an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof provided above that binds to NKG2A.

[0016] In another aspect, this disclosure relates to an isolated vector comprising the polynucleotide described above.

[0017] In another aspect, this disclosure relates to a host cell comprising the isolated polynucleotide or isolated carrier described above.

[0018] In another aspect, this disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to NKG2A, an isolated polynucleotide, an isolated carrier, or a host cell, and a pharmaceutically acceptable carrier.

[0019] In another aspect, this document provides the use of an antibody or antigen-binding fragment thereof that binds to NKG2A, an isolated polynucleotide, an isolated carrier, a host cell, or a pharmaceutical composition in the manufacture of a therapeutic agent for the diagnosis, prevention, or treatment of a disease.

[0020] In another aspect, this document provides a method for diagnosing, preventing, or treating a subject suffering from an oncology-related disease, an inflammatory or autoimmune disease, or an infectious disease, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof that binds to NKG2A, an isolated polynucleotide, an isolated carrier, a host cell, or a pharmaceutical composition provided above.

[0021] In some embodiments, the method includes enhancing an immune response or reducing adverse effects. In some embodiments, the disease is improved by stimulation of immune cells.

[0022] The following is a brief description of the accompanying drawings, which are presented for the purpose of illustrating the exemplary embodiments disclosed herein and are not intended to limit them.

[0023] Figure 1 shows the binding activity of cAb017, Tab1, Tab2, Tab3 and allotype hIgG4 with CHOK1-cynoNKG2A / CD94 cells as detected by FACS.

[0024] Figure 2 shows the binding activity of cAb017, Tab1, Tab2, Tab3 and allotype hIgG4 with CHOK1-cynoNKG2A / CD94 cells as detected by FACS.

[0025] Figure 3 shows the binding activity of cAb017, Tab1, Tab2, Tab3 and allotype hIgG4 with NK92 cells as detected by FACS.

[0026] Figure 4A shows the binding specificity of cAb017, Tab1, Tab2, Tab3, and isotype hIgG4 with CHOK1-hNKG2A / CD94 cells as detected by FACS.

[0027] Figure 4B shows the binding of cAb017, Tab1, Tab2, Tab3, positive control (antibody recognized by NKG2E), and human IgG4 isotype with NKG2E as detected by ELISA. The concentrations of each antibody tested, from left to right, are 200 nM, 20 nM, 2 nM, and 0 nM, respectively.

[0028] Figure 5 shows the blocking activity of cAb017, Tab1, Tab2, Tab3, and isotype hIgG4 bound to CHOK1-NKG2A / CD94 cells.

[0029] Figure 6 shows the effect of cAb017, Tab1, Tab2, Tab3, and isotype hIgG4 on NK92-mediated cytotoxicity as detected by FACS.

[0030] Figures 7A and 7B show the effect of cAb017, Tab1, Tab2, Tab3, and isotype hIgG4 on CD107a degranulation in NKG2A+NK cells or NKG2A-NK cells, respectively.

[0031] Figure 8 shows the effect of cAb017, Tab1, Tab2, Tab3, and isotype hIgG4 on primary NK-mediated cytotoxicity.

[0032] Figure 9 shows the functional activities of cAb017, Tab1, Tab2, Tab3, and isotype hIgG4 in reversing the inhibition of NFAT signaling in Jurkat cell lines expressing NKG2A / CD94 stimulated by CHO / OKT3 / HLA-E.

[0033] Figure 10 shows the functional activities of cAb017 and Tab1 in activating NFAT signaling in Jurkat cell lines expressing NKG2C / CD94 / DAP12. hIgG4 is an isotype of human IgG4.

[0034] Figure 11 shows the binding activities of cAb017, H17.H28, H17.H29, H17.H30, and isotype hIgG4 with 293T-hNKG2A / CD94 cells as detected by FACS.

[0035] Figure 12 shows the activity of cAb017 and its humanized variants H17.H28, H17.H29, and H17.H30 in blocking the interaction between NKG2A / CD94 and HLA-E, as assessed by competitive FACS.

[0036] Figure 13 shows the binding activity of cAb017 and its humanized variants H17.H68, H17.H69, H17.H70, H17.H71, H17.H72, H17.H73, H17.H74, H17.H75, H17.H76 and isotype hIgG4 to 293T / NKG2A / CD94 cells, as detected by FACS.

[0037] Figures 14A and 14B show how cAb017 and its humanized variants H17.H28, H17.H68, H17.H69, H17.H70, H17.H71, H17.H72, H17.H73, H17.H74, H17.H75, H17.H76 and isotype hIgG4 reversed the inhibition of NFAT signaling in Jurkat cell lines expressing NKG2A / CD94 stimulated by CHO / OKT3 / HLA-E.Functional activity.

[0038] Figure 15 shows the binding activity of cAb017 and cAb017-derived humanized variants H17.H28, H17.H68, H17.H69, H17.H70, H17.H71, H17.H72, H17.H73, H17.H74, H17.H75, H17.H76 and isotype hIgG4 to 293T / NKG2A / CD94 cells as detected by FACS.

[0039] Figure 16 illustrates the functional activity of cAb017, humanized variants derived from cAb017 (H17.H28, H17.H68, H17.H69, H17.H70, H17.H71, H17.H72), hIgG4 and cells-only (from left to right) in NKG2C / CD94 / DAP12-expressing Jurkat cell lines in activating NFAT signaling.

[0040] Figure 17 shows the functional activity of cAb017, humanized variants derived from cAb017 (H17.H28, H17.H73, H17.H74, H17.H75, H17.H76), hIgG4, and Jurkat cells only (from left to right) in activating NFAT signaling in Jurkat cell lines expressing NKG2C / CD94 / DAP12.

[0041] Figure 18 shows the functional activity of cAb017, H17.H73, hotspot removal variant H17.H73.DR02, H17.H28, hotspot removal variant H17.H28.DR02, hIgG4, and Jurkat cells only (from left to right) in activating NFAT signaling in Jurkat cell lines expressing NKG2C / CD94 / DAP12.

[0042] Figures 19A and 19B illustrate the functional activities of cAb017 and Tab1 in activating NK cell activity in the NKG2C+ population. hIgG4 is a human IgG4 isotype.

[0043] Figure 20 illustrates the effects of cAb017, H73.DR02, and H28.DR02 in NK92-mediated cytotoxicity assays. Detailed Description

[0044] The present disclosure will now be explained in more detail. This specification is not intended to be a detailed list of all different ways in which the invention can be implemented or all features that can be added to the invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be removed from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention and not to exhaustively specify all permutations, combinations, and variations thereof.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described and used herein may be used in the testing practices of this disclosure, preferred materials and methods are described herein. The following terminology will be used in describing and claiming protection for this disclosure.

[0046] Antibodies or antigen-binding fragments thereof that bind to NKG2A This document provides novel antibodies or antigen-binding fragments thereof that bind to NKG2A, wherein the antibody or antigen-binding fragment contains a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region containing complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region containing complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 contain amino acid sequences as shown in SEQ ID NO: 36, RIDPANXNTKYAPNFQG (SEQ ID NO: 37), and SEQ ID NO: 38, respectively; and LCDR1, LCDR2, and LCDR3 contain amino acid sequences as shown in SEQ ID NO: 39, 40, and 41, respectively.

[0047] In some embodiments, X is G or A. In some embodiments, X is G. In some embodiments, X is A.

[0048] As used herein, “antibody” refers to a polypeptide of the immunoglobulin (Ig) family that binds to an antigen. For example, naturally occurring IgG-type "antibodies" are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of both the heavy and light chains contain binding domains that interact with antigens. See Fundamental Immunology, Chapter 7 (edited by Paul W., 2nd ed., Raven Press, New York (1989)).

[0049] The term "antigen-binding fragment" refers to an antibody fragment, including Fab, F(ab)2, Fab', F(ab')2, Fv, domain antibodies (dAb), other monovalent and bivalent fragments, complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv, scFab, and scFabAC), chimeric antibodies, biantibodies, triantibodies, microantibodies, nanobodies, and peptides (containing at least a portion of an antibody sufficient to confer specific antigen binding to the peptide), as well as fusions and derivatives thereof. Antigen-binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. See, for example, Holliger and Hudson, Nature Biotechnology 23: 1126–1136 (2005) and Hust et al., BMC Biotech 7: 14 (2007).

[0050] In some embodiments, in order to generate humanized antibodies using the CDR transplantation method, the CDR is defined using the Kabat definition, in addition to the heavy chain CDR1 defined using the combination of the Kabat and Chothia systems.

[0051] The Kabat and Chothia systems are known to those skilled in the art, see, for example, Kabat EA, Wu TT, Perry HM et al., Sequence of Proteins of Immunological Interest [J], 1991; Chothia, C. et al., (1987) J. Mol. Biol. [Journal of Molecular Biology], 196:901-917; Al lazikani et al., (1997) J. Molec. Biol. [Journal of Molecular Biology], 273:927-948.

[0052] In some embodiments, HC DR1, HC DR2, and HC DR3 respectively contain amino acid sequences as shown in SEQ ID NO: 36, RIDPANXNTKYAPNFQG (SEQ ID NO: 37), and 38, where X is G; LCDR1, LCDR2, and LCDR3 respectively contain amino acid sequences as shown in SEQ ID NO: 39, 40, and 41.

[0053] In some embodiments, HC DR1, HC DR2, and HC DR3 respectively contain amino acid sequences as shown in SEQ ID NO: 36, RIDPANXNTKYAPNFQG (SEQ ID NO: 37), and 38, where X is G.The amino acid sequences shown in 37) and 38, where X is A; LCDR1, LCDR2 and LCDR3 contain the amino acid sequences shown in SEQ ID NO: 39, 40 and 41, respectively.

[0054] The amino acids in this disclosure are shown in standard single-letter codes, as is well known to those skilled in the art.

[0055] In some embodiments, VH and VL are selected from the following: a) VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11; b) VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91 13. A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14; c) A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15; d) A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16. VH containing an amino acid sequence with 97%, 98%, 99%, or 100% sequence identity, and VL containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17; e) VH containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18, and VL containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. (See page 6 / 25 of the specification for more details.)CN 121358774 A VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20; f) VH containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21; g) VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 22. VH containing amino acid sequences with 97%, 98%, 99%, or 100% sequence identity, and VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23; h) VH containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24, and VL containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25; i) VH containing amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25; 26. A VH having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27; and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27; j) A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 28; and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29;k) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 30, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31; l) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 30, and a VL ... 33. A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 34; m) A VH containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 35, and a VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 42; n) A VL containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% sequence identity with SEQ ID NO: 42. VH containing an amino acid sequence with 97%, 98%, 99% or 100% sequence identity, and VL containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity with SEQ ID NO: 13; o) VH containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity with SEQ ID NO: 43, and VL containing an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity with SEQ ID NO: 29.

[0056] The percentage of identity between two amino acid sequences is calculated by dividing the number of identical residues by the total number of amino acid residues.The percentage is determined by multiplying the quotient by 100. Spare parts are not included in the assessment of identity. Therefore, two copies of an identical sequence have 100% identity, but sequences with deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several computer programs can be used to determine sequence identity, such as those using algorithms such as BLAST. BLAST nucleotide searches are performed using the NBLAST program, and BLAST protein searches are performed using the BLASTP program, with the default parameters of each program used.

[0057] In this disclosure, antibodies or antigen-binding fragments that bind to NKG2A include conserved substitutions within their sequences, preferably not significantly affecting the desired activity of the polypeptide, antibody, or antigen-binding fragment. Substitutions may be naturally occurring or may be introduced, for example, using mutagenesis (e.g., Hutchinson et al., 1978, J. Biol. Chem. 253:6551). For example, the amino acids glycine, alanine, valine, leucine, and isoleucine (amino acids with aliphatic side chains) can often be substituted for each other. Among these possible substitutions, glycine and alanine are preferred for substitution (because they have relatively short side chains), while valine, leucine, and isoleucine are preferred for substitution (because they have larger, hydrophobic aliphatic side chains). Other amino acids that can typically be substituted for each other include, but are not limited to, phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); and asparagine and glutamine (amino acids with amide side chains).

[0058] In some embodiments, VH contains or has the amino acid sequence shown in SEQ ID NO: 10, and VL contains or has the amino acid sequence shown in SEQ ID NO: 11; VH contains or has the amino acid sequence shown in SEQ ID NO: 12, and VL contains or has the amino acid sequence shown in SEQ ID NO: 13; VH contains or has the amino acid sequence shown in SEQ ID NO: 14, and VL contains or has the amino acid sequence shown in SEQ ID NO: 15; VH contains or has the amino acid sequence shown in SEQ ID NO: 16, and VL contains or has the amino acid sequence shown in SEQ ID NO: 17; VH contains or has the amino acid sequence shown in SEQ ID NO: 18, and VL contains or has the amino acid sequence shown in SEQ ID NO: 19; VH contains or has the amino acid sequence shown in SEQ ID NO: 20, and VL contains or has the amino acid sequence shown in SEQ ID NO: 21.The amino acid sequences shown are as follows: VH contains or has the amino acid sequence shown in SEQ ID NO: 22, VL contains or has the amino acid sequence shown in SEQ ID NO: 23; VH contains the amino acid sequence shown in SEQ ID NO: 24, VL contains or has the amino acid sequence shown in SEQ ID NO: 25; VH contains or has the amino acid sequence shown in SEQ ID NO: 26, VL contains or has the amino acid sequence shown in SEQ ID NO: 27; VH contains or has the amino acid sequence shown in SEQ ID NO: 28, VL contains or has the amino acid sequence shown in SEQ ID NO: 29; VH contains or has the amino acid sequence shown in SEQ ID NO: 30, VL contains or has the amino acid sequence shown in SEQ ID NO: 31; VH contains or has the amino acid sequence shown in SEQ ID NO: 32, VL contains or has the amino acid sequence shown in SEQ ID NO: 33; VH contains or has the amino acid sequence shown in SEQ ID NO: 34, VL contains or has the amino acid sequence shown in SEQ ID NO: 35. The amino acid sequence shown; VH contains or has the amino acid sequence shown in SEQ ID NO: 42, and VL contains or has the amino acid sequence shown in SEQ ID NO: 13; or VH contains or has the amino acid sequence shown in SEQ ID NO: 43, and VL contains or has the amino acid sequence shown in SEQ ID NO: 29, or their conserved substitutions.

[0059] In some embodiments, the antibody or antigen-binding fragment encompasses (unless otherwise indicated or suggested by the context) monoclonal antibodies, polyclonal antibodies, mouse antibodies, hamster antibodies, goat antibodies, rabbit antibodies, chimeric antibodies, primate antibodies, humanized antibodies, (fully) human antibodies, multimeric antibodies, heterodimeric antibodies, hemidimeric antibodies, bivalent antibodies, trivalent or tetravalent antibodies, bispecific antibodies, single-chain antibodies (e.g., scFv, scFab, and scFabAC), biscFv, biantibodies, triantibodies or tetraantibodies, single-domain antibodies, and modified Fab fragments. In some embodiments, the antibody or antigen-binding fragment is monovalent.

[0060] In some embodiments, the antibody or antigen-binding fragment contains an Fc fragment that communicates with the immune system when the antibody binds to its target. The Fc fragment can be any class (e.g., IgG, IgE, IgM, IgD, or IgA) or subclass of immunoglobulin molecules. Preferably, the Fc fragment is an IgG molecule. In some embodiments, the Fc fragment is a human wild-type IgG Fc fragment.In some embodiments, the Fc fragment is human IgG, such as IgG1, IgG2, IgG3, or IgG4, optionally having one or more mutations compared to a wild-type human IgG molecule. An exemplary Fc fragment is human IgG4 with the S228P mutation, meaning that amino acid S (standard single-letter code) at position 228 (defined by Kabat) is mutated to amino acid P.

[0061] The antibody or antigen-binding fragment is chimeric or humanized.

[0062] Typically, chimeric antibodies comprise a heavy chain variable region and / or light chain variable region (including CDR and framework residues) of one species (typically mouse) fused to a constant region of another species (typically human). Humanized antibodies typically comprise a heavy chain and / or light chain CDR from a mouse antibody transplanted into a non-human primate antibody variable region framework or a human antibody variable region framework, typically further containing a human constant region. See, for example, Riechmann et al. (1988) Nature, 332: 323-327.

[0063] The methods for manufacturing all the antibodies or antigen-binding fragments described above are well known to those skilled in the art. See, for example, U.S. Patent No. 5,807,715; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 81(21):6851-5; Sharon et al. (1984) Nature [Nature] 309(5966):364-7; Takeda et al. (1985) Nature [Nature] 314(6010):452-4.

[0064] In some embodiments, antibodies or antigen-binding fragments are generated via a selective lymphocyte antibody method (SLAM) (Babcook et al., 1996, Proc. Natl. Acad. Sci, 93, 7843-7848; de Wildt et al., 1997, J. Immunol. Methods, 207:61-67; and Lagerkvist et al., 1995, BioTechniques, 18:862-869), which allows for isolation from any type of cell that produces high-affinity antibodies during an in vivo immune response. The above methods rely on the isolation of individual cells that produce antibodies, followed by clonal amplification of the cells, screening for clones that produce anti-NKG2A antibodies, and subsequent identification of the sequences of their variable heavy chain (VH) and light chain (VL) genes. Therefore, B cells that are positive for antibodies against NKG2A are isolated. B cells can be derived from humans, mice, rats, hamsters, rabbits, goats, or other mammalian species. This can be achieved, for example, through...Traditional recombinant DNA technology clones antibody genes from these B cells and expresses them in host cells (e.g., E. coli). Cells expressing antibodies can be purified using conventional methods. If the antibodies are of non-human origin, they can be humanized using conventional methods (e.g., by mutagenesis of their genes). Humanized antibodies can then be expressed in host cells and purified.

[0065] Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler and Milstein, Nature, 1975, 256:495-497), three-source hybridoma technology, human B cell hybridoma technology (Kozbor et al., Immunology Today, 1983, 4, 72) and EBV-hybridoma technology (Cole et al., “Monoclonal Antibodies and Cancer Therapy”, pp. 77-96, Alan R. Liss, Inc., 1985). Methods for generating and manufacturing recombinant antibodies are well known in the art (see, for example, Simmons et al., 2002, Journal of Immunological Methods, 263, 133-147). Specification 9 / 25 pages 15 CN 121358774 A

[0066] The antibodies or antigen-binding fragments disclosed herein can also be generated using various phage display methods known in the art, including those disclosed in the following literature: Brinkman et al., 1995, J. Immunol. Methods, 182:41-50; Ames et al., 1995, J. Immunol. Methods, 184, 177-186; Kettleborough et al., 1994, Eur. J. Immunol., 24, 952-958.

[0067] Furthermore, transgenic (e.g., genetically engineered) mice, or other organisms (including other mammals), can be used to generate antibodies or antigen-binding fragments (see, for example, US 6,300,129). For example, it is known that mice can be used to generate large quantities of high-affinity antibodies having human variable sequences (see, for example, US 6,586,251), wherein the mice are engineered to replace only the variable regions (heavy chain V, D, and J segments, and light chain V and J segments) of the mouse immune loci with the corresponding human variable sequences.

[0068] In some embodiments, the antibody or antigen-binding fragment provided herein binds to a peptide as shown in SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment provided herein binds to a peptide as shown in SEQ ID NO: 9.

[0069] This disclosure provides a binding site containing an amino acid sequence as shown in SEQ ID NO: 8 or 9. In some embodiments, the binding site contains an amino acid sequence as shown in SEQ ID NO: 8.

[0070] NKG2A is an inhibitory receptor selectively expressed on cytotoxic lymphocytes, including natural killer (NK) cells and CD8-positive (CD8+) T cells. It is understood that blocking NKG2A and one or more of its ligands, MHC-1 molecules (such as HLA-E (human), Qa-1 (mouse)), is beneficial for improving natural killer (NK) and CD8+ T cell-mediated immune responses in subjects in need (e.g., humans, mice). NKG2A and CD94 molecules (e.g., co-expressed on NK cells or CD8+ T cells) form a repressive isotype NKG2A / CD94 heterodimer. This disclosure provides examples of antibodies or antigen-binding fragments that bind to NKG2A, thereby blocking the interaction between the heterodimer NKG2A / CD94 and MHC-1 molecules (preferably human MHC-1 protein HLA-E).

[0071] Antibodies or antigen-binding fragments that bind to NKG2A provided in this disclosure improve immune responses. In some embodiments, antibodies or antigen-binding fragments that bind to NKG2A provided herein improve innate immune responses.

[0072] In some embodiments, the antibody or antigen-binding fragment blocks the interaction between NKG2A and HLA-E, wherein the IC50 is no more than 1 nM, 0.8 nM, or 0.6 nM.

[0073] In some embodiments, the antibody or antigen-binding fragment reverses the suppression of T cell responses, optionally NKG2A / HLA-E mediated suppression of T cell responses.

[0074] In some embodiments, the antibody or antigen-binding fragment activates T cells or increases T cell responses, such as by activating NFAT signaling.

[0075] In some embodiments, the antibody or antigen-binding fragment activates NFAT signaling in T cells expressing NKG2C (killer cell lectin-like receptor C2, KLRC2). In some embodiments, the antibody or antigen-binding fragment activates NFAT signaling in T cells expressing NKG2A.

[0076] In some embodiments, the antibody or antigen-binding fragment activates T cells expressing NKG2C. In some embodiments, the antibody or antigen-binding fragment activates T cells expressing NKG2A.

[0077] In some embodiments, the antibody or antigen-binding fragment increases NK cell responses; optionally, NK cells express...NKG2A. In some embodiments, the antibody or antigen-binding fragment upregulates CD107a (lysosome-associated membrane protein 1). In some embodiments, the antibody or antigen-binding fragment increases NK cell responses by upregulating CD107a.

[0078] In some embodiments, the antibody or antigen-binding fragment improves the lysis of cells expressing MHC-1 molecules. In some embodiments, the antibody or antigen-binding fragment improves the lysis of cells expressing HLA-E (such as tumor cells expressing HLA-E). Specification 10 / 25 pages 16 CN 121358774 A

[0079] The antibody or antigen-binding fragment binds to NKG2A with an EC50 not exceeding 1 nM, 0.8 nM, or 0.6 nM, or a KD (affinity constant, KD = koff / kon, or KD = Kd / Ka) not exceeding 9.9E-8 nM, 9.9E-9 nM, or 9.9E-10 nM.

[0080] In some embodiments, the antibody or antigen-binding fragment exerts NK-mediated cytotoxicity against HLA-E-expressing tumor cells.

[0081] The antibody or antigen-binding fragment exerts NK-mediated cytotoxicity against HLA-E-expressing tumor cells, wherein the EC50 is not greater than 1 nM, 0.8 nM, 0.5 nM, or 0.3 nM.

[0082] EC50 or IC50 can be measured by methods well known in the art, such as FACS assay, competitive FACS (fluorescence-activated cell sorting). KD can be measured by methods well known in the art, such as octet.

[0083] In some embodiments, the antibody or antigen-binding fragment does not bind to NKG2E or binds very little to NKG2E, so the antibody or antigen-binding fragment hardly reduces or diminishes the activation activity mediated by NKG2E or NKG2E / CD94 heterodimer.

[0084] In some embodiments, the antibody or antigen-binding fragment binds weakly to NKG2C.

[0085] In some embodiments, the antibody or antigen-binding fragment is cross-reactive; optionally, the antibody or antigen-binding fragment binds to cynomolgus monkey NKG2A and human NKG2A.

[0086] Polynucleotides, Vectors, and Host Cells This disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment that binds to NKG2A, the antibody or antigen-binding fragment containing at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence shown in SEQ ID NO: 10-35, 42, and 43.

[0087] In some embodiments, the isolated polynucleotide encodes an antibody or antigen-binding fragment that binds to NKG2A as shown in SEQ ID NO: 10-35, 42, and 43.An antibody or antigen-binding fragment thereof that binds to NKG2A.

[0088] A polynucleotide is a nucleic acid sequence of DNA, RNA, a DNA / RNA hybrid, or a modified thereof. In some embodiments, the polynucleotide is a nucleic acid sequence of DNA. The encoding polynucleotide (DNA or RNA) may be a recombinant molecule or a synthetic molecule.

[0089] This disclosure also relates to sequence variants of the polynucleotides described above. For example, this disclosure includes nucleic acid sequences that are about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, 99.5%, 99.9%, or 100% identical to any of the polynucleotide sequences provided herein, including fragments thereof and their complementary sequences. This disclosure also includes polynucleotides that differ from the polynucleotide sequences specifically provided herein due to the degeneracy of the genetic code.

[0090] The polynucleotide may further include regulatory sequences (e.g., promoter sequences, untranslated 5' regions, and untranslated 3' regions) and / or vector sequences. For example, the polynucleotide constitutes a vector.

[0091] As used herein, the term "vector" refers to a polynucleotide that can be engineered to contain one or more clonal polynucleotides capable of replicating in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers) regulating the expression of a target polypeptide, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase).

[0092] In some embodiments, a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses) is used to introduce the polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein). This may involve the use of a non-pathogenic (deficient), replicative virus, or a replication-deficient virus may be used.

[0093] In one aspect, this disclosure also provides an isolated vector containing the polynucleotides as described herein. The provided isolated polynucleotides can be inserted into the vector using recombination techniques known in the art for further cloning (DNA amplification) or for expression.

[0094] Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0095] In some embodiments, the vector provided herein, at least one promoter operatively linked to a nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including...).Includes lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, multivacuolar papillomaviruses (e.g., SV40), λ phage, and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM. pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0096] This disclosure provides host cells containing isolated polynucleotides or isolated vectors as described herein.

[0097] As used herein, the term "host cell" refers to a cell that may be or is a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate cells.

[0098] Vectors containing polynucleotide sequences encoding antibody or antigen-binding fragments may be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the above-mentioned polynucleotides (DNA, RNA, or DNA / RNA hybrid nucleic acid sequences) in the vectors described herein are, for example, prokaryotic cells of *Escherichia coli*, or other microbial cells, or eukaryotic cells (including, but not limited to, mammalian cells such as human cells, mouse cells, monkey cells, rabbit cells, goat cells, hamster cells, or rat cells, insect cells, avian cells, plant cells, and eukaryotic cells).

[0099] In some embodiments, the host cell may be, for example, (1) bacterial cells such as *Escherichia coli*; (2) fungal cells and *Aspergillus* cells, yeast cells such as *Saccharomyces cerevisiae* and *Kluyveromyces lactis*; (3) insect cell lines such as EXPRESSF® (a cell line from *Spodoptera frugiperda*) cells (Protein Sciences Corp.).Meriden, Conn., USA; (4) mammalian cells; or (5) plant cells.

[0100] Typical mammalian cells include COS1 and COS7 cells, Chinese hamster ovary (CHO) cells, NSO myeloma cells, NIH 3T3 cells, 293 cells, HEPG2 cells, HeLa cells, C127, 3T3, BHK, Bowes melanoma cells, L cells, MDCK, HEK293, WI38, mouse ES cell lines (e.g., derived from strains 129 / SV, C57 / BL6, DBA-1, 129 / SVJ), K562, Jurkat cells, and BW5147. The present invention therefore provides cells expressing the antibodies of the present invention, including but not limited to hybridoma cells, B cells, plasma cells, and mammalian and human host cells (e.g., adult embryonic stem cells) recombinantly modified to express the antibodies of the present invention. Other useful mammalian cell lines are well known and readily available from the American Type Culture Collection (“ATCC”) (Manassas, Va., USA) and the Coriell Cell Bank (Camden, NJ, USA) of the National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository. These cell types are only representative and this list is not intended to be exhaustive.

[0101] In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NSO, 293, and derivatives thereof, as described on pages 12 / 25 of CN 121358774 A.

[0102] Vectors can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection, and infection and / or transduction (e.g., using recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0103] Kits This disclosure provides a kit containing an antibody or antigen-binding fragment thereof that binds to NKG2A as described above, isolated polynucleotides, isolated vectors, or host cells. If desired, such a kit may further include one or more of a variety of conventional pharmaceutical kit components, such as those having one or more pharmaceutically acceptable vectors.The containers, other containers, etc., of the agent are obvious to those skilled in the art. Instructions for use, instructions for use, and / or instructions for mixing components, such as inserts or labels indicating the amount of components to be applied, may also be included in the kit.

[0104] Pharmaceutical Composition This disclosure provides a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that binds to NKG2A as described above, isolated polynucleotides, isolated carriers, or host cells, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated in any manner known in the art.

[0105] The pharmaceutical composition can be formulated for parenteral administration (e.g., oral, intranasal, or by inhalation, ophthalmic artery, rectum, vein, artery, intramuscular, intradermal, subcutaneous, or intraperitoneal) in unit dosage forms (i.e., physically discrete units containing a predetermined amount of the active compound for ease of administration and dosage uniformity). The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0106] Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, sterile diluents (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methylparaben, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate or phosphate), and isotonic agents (e.g., sugars (e.g., dextran), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers.

[0107] Formulations of the compositions may be formulated and encapsulated in ampoules, disposable syringes or multi-dose vials. Where necessary (e.g., in injectable formulations), appropriate flowability may be maintained by, for example, the use of a coating (e.g., lecithin or a surfactant). The absorption of an antibody or its antigen-binding fragment can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microcapsule delivery systems that may include biodegradable biocompatible polymers (e.g., ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0108] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solutions, suspensions, emulsions, or solid forms suitable for producing liquid solutions, suspensions, or emulsions. Injectable formulations may include sterile and / or non-febrile solutions prepared for injection, sterile dried soluble products (e.g., lyophilized powders, including tablets for subcutaneous injection) prepared for combination with a solvent prior to use, and formulations prepared for use with...Sterile suspensions for injection, sterile dried insoluble products ready for combination with a medium before use, and sterile and / or non-fever emulsions. Solutions may be aqueous or non-aqueous.

[0109] In some embodiments, sterile lyophilized powders are prepared by dissolving antibody or antigen-binding fragments as disclosed herein in a suitable solvent. The solvent may contain other pharmacological components that improve the stability of the powder or excipients for preparing reconstituted solutions from powders. Excipients that may be used include, but are not limited to, water, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable reagents.

[0110] Antibodies or antigen-binding fragments, or pharmaceutical compositions containing them, may be included individually in a container, package, or dispenser, or as part of a kit along with a label and instructions for use.

[0111] Treatment Methods This disclosure provides the use of the antibodies or antigen-binding fragments, isolated polynucleotides, isolated carriers, host cells, or pharmaceutical compositions described above in the manufacture of therapeutic agents for diagnosing, preventing, or treating diseases. In some embodiments, the therapeutic agent may reverse NGK2A-mediated immunosuppression or improve the immune response.

[0112] This disclosure provides the use of the antibodies or antigen-binding fragments, isolated polynucleotides, isolated carriers, host cells, or pharmaceutical compositions described above in the manufacture of therapeutic agents for improving the immune response to a disease.

[0113] This disclosure provides a method for diagnosing, preventing, or treating a subject suffering from an oncology-related disease, an inflammatory or autoimmune disease, or an infectious disease, the method comprising administering to the subject a therapeutically effective amount of the antibodies or antigen-binding fragments, isolated polynucleotides, isolated carriers, host cells, or pharmaceutical compositions described above.

[0114] The term “therapeutically effective amount” refers to the amount of the composition or active agent disclosed herein that effectively “treats” the disease of the subject.

[0115] As used herein, the term “treat, treating, or treatment” means to reduce or improve a disease (i.e., to slow or stop the development of the disease or at least one of its clinical symptoms); or to reduce or improve at least one physical parameter or biomarker associated with the disease, including those physical parameters or biomarkers that the patient may not be able to identify. For example, in the case of cancer, “treat, treating, or treatment” can mean to inhibit or slow the growth, proliferation, or metastasis of a tumor or malignant cells, to remove all or part of a tumor, to inhibit or slow tumor growth and metastasis, to delay the development of a tumor, or some combination thereof.

[0116] As used herein, the term “prevent, preventing, or prevention” means preventive treatment of a disease; or to delay the onset or progression of a disease.

[0117] In some embodiments, the therapeutic agents described above (i.e., antibodies or antigen-binding fragments thereof that bind to NKG2A, isolated polynucleotides, isolated carriers, host cells, or pharmaceutical compositions) or active agents (e.g., antibodies or antigen-binding fragments thereof that bind to NKG2A) can enhance the immune response to a disease or reduce adverse effects on the diagnosis, prevention, or treatment of a disease.

[0118] In some embodiments, the method may optionally enhance the immune response (e.g., the innate immune response) by blocking the NKG2A signaling pathway, which helps treat a wide range of diseases.

[0119] In some embodiments, the method may increase the response of NK cells and / or T cells. In some embodiments, the disease is improved by stimulation of immune cells (e.g., NK cells, T cells). In some embodiments, the method may activate T cells. In some embodiments, the method may activate NK cells, thereby upregulating CD107a.

[0120] In some embodiments, the method may activate NFAT signaling, thereby upregulating the response of T cells.

[0121] In some embodiments, the method improves the lysis of HLA-E-expressing cells (such as HLA-E-expressing tumor cells), wherein the tumor herein may be a malignant tumor or a benign tumor.

[0122] In some embodiments, the disease includes oncology-related diseases, inflammatory or autoimmune diseases, or infectious diseases.

[0123] In some embodiments, oncology-related diseases include solid tumors or liquid tumors. In some embodiments, oncology-related diseases include malignant tumors and benign tumors. In some embodiments, oncology-related diseases include breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, stomach cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), and colorectal cancer.

[0124] In some embodiments, the infectious disease includes viral infectious diseases and bacterial infectious diseases.

[0125] In some embodiments, inflammation or autoimmune disease is characterized by redness, swelling, fever, and pain as a result of capillary dilation and edema and the migration of phagocytic leukocytes. Some examples of inflammatory responses include: arthritis, conjunctivitis, and other skin conditions.Touch dermatitis, hyperIgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory diseases. Idiopathic inflammatory diseases include, for example, psoriasis and lupus (e.g., systemic lupus erythematosus (SLE), drug-induced lupus erythematosus, and lupus nephritis).

[0126] In some embodiments, the disease contains a tumor. In some embodiments, the disease contains cancer such as B-cell lymphoma. Non-limitingly, the treatment reduces the rate of increase in tumor volume over time in the subject, reduces the risk of metastasis, or reduces the risk of additional metastasis in the subject. In some embodiments, the treatment may halt, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment may result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in the subject.

[0127] The subjects include mammals including primates, rodents, canines, and pigs, such as mice, rats, rabbits, cats, dogs, pigs, monkeys, chimpanzees, gorillas, etc. In some embodiments, the subjects include mice, cynomolgus monkeys, and humans. Unless otherwise stated, the terms “patient” or “subject” are used interchangeably herein.

[0128] The daily dose of the therapeutic agent may be obtained from cell culture assays, animal studies, or clinical studies. A therapeutically effective amount of the therapeutic agent or active agent (e.g., antibody or antigen-binding fragment) will be the amount by which the disease is treated in the subject, reducing the severity, frequency, and / or duration of one or more symptoms of the disease in the subject. Efficacy and administration may be determined by a healthcare professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in the subject. Additionally, it should be understood that a particular dose level for any particular subject will depend on a variety of factors, including the activity of the particular compound used, the subject’s age, weight, general health condition, sex, and diet, the time of administration, the route of administration, the excretion rate, and the half-life of the antibody or antigen-binding fragment in vivo.

[0129] Examples The invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0130] Standard references that elucidate the general principles of recombinant DNA technology known to those skilled in the art include Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (supplements in 1998 and 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.Plainview, NY (1989); Kaufman et al., eds., Handbook of Molecular and Cellular Methods in Biology and Medicine, CRC Press, Boca Raton (1995); McPherson, ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). The specification is on pages 15 / 25, 21 CN 121358774 A

[0131] Standard references that set forth general principles of immunology known to those skilled in the art include Harlow and Lane, Antibodies: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), and Roitt et al., Immunology, 3rd edition, Mosby-Year Book Europe Limited, London (1993). Standard references that set forth general principles of medical physiology and pharmacology known to those skilled in the art include Fauci et al., editors, Harrison's Principles of Internal Medicine, 14th edition, McGraw-Hill Companies, Inc. (1998).

[0132] The materials used in the embodiments of this disclosure are known and commercially available.

[0133] Example 1: Reagent Generation 1.1 Reference Antibodies Reference antibodies Tab1, Tab2, and Tab3 have been prepared according to patents WO 2020102501 A1 (SEQ ID NO: 107 and 118), WO 2019126514 A2 (SEQ ID NO: 13 and 14), and CN 111153995 A (SEQ ID NO: 1 and 14), respectively.5) Generation. Table 1 shows the heavy chain variable region (VH) sequence and light chain variable region (VL) sequence of Tab1, Tab2 and Tab3 using standard single-letter codes.

[0134] Table 1. Variable region sequences of Tab1, Tab2 and Tab3

[0135] 1.2 Stable cell lines Stable cell lines expressing human, cynomolgus monkey and mouse NKG2A / CD94, CHOK1 / hNKG2A, 293T / hNKG2A, CHOK1 / cynoNKG2A and CHOK1 / mNKG2A have been generated for hybridoma screening and in vitro assays. CHOK1 cells or 293T cells were transfected with human (or "h"), cynomolgus monkey (or "cyno") or mouse (or "m") NKG2A / CD94 expression plasmids and selectively cultured for 2 weeks in a medium containing 0.2 μg / mL puromycin. Single-cell clones were then isolated by limiting dilution and selected by FACS to obtain monoclonal cell lines stably expressing human, cynomolgus monkey, or mouse NKG2A / CD94. To generate a stable Jurkat-NFAT-human NKG2A / CD94 library, Jurkat-NFAT cells were transfected with human NKG2A and CD94 expression plasmids and selectively cultured in medium containing 400 μg / mL hygromycin and 0.2 μg / mL puromycin. To generate a stable Jurkat-NFAT-human NKG2C / CD94 library, Jurkat-NFAT cells were transfected with human NKG2C / CD94 and DAP12 (transmembrane immune signaling linker, TYROBP) expression plasmids and selectively cultured in medium containing 800 μg / mL hygromycin and 0.2 μg / mL puromycin.

[0136] 1.3 Recombinant protein with human Fc tag human NKG2A / CD94 extracellular domain (ECD, UNIPROT_P26715, Pro94-Leu233, accession number #Q1 3241-1, Lys 32-Ile179) was purchased from ChemPartner for immunoassay and hybridoma screening.

[0137] Recombinant protein with 6×his tag human CD94 extracellular domain (ECD, accession number #Q13241-1, Lys32-Ile179) was purchased from Acro Biosystems.

[0138] The recombinant protein (rhNKG2E-ECD-Fc protein) with the human Fc tag containing the human NKG2E extracellular domain (ECD, accession number #Q07444-1, Glu98-Ser240) was purchased from R&D Systems for specific characterization.

[0139] The recombinant protein HLA-E*01:03 HLA-A leader 3-11 tetramer-VMAPRTLVL-PE (HLA-E-PE) was purchased from MBL and used for cell-based blocking assays.

[0140] Example 2: Hybridoma Development and Screening 2.1 Immunization and Fusion A rapid immunization strategy was adopted, and 40 mice from two strains (SJL, NOD) were immunized with Fc-tagged human NKG2A / CD94 ECD-Fc recombinant protein or human NKG2A / CD94 overexpressing cell lines. Human and cynomolgus monkey NKG2A / CD94 overexpressing cell lines were used as antigens, and the serum titers of immunized mice were detected by FACS. When the serum titers reached a high level, a final booster was performed. Three days after the final booster, the confluent spleen cells were harvested and fused with SP2 / 0 mouse myeloma cells. The fused cells were then seeded into 384-well plates for screening.

[0141] 2.2 Ten to twelve days after the initial and secondary screenings, the supernatant harvested from each well of hybridoma cells from the initial screening was measured by ACUMEN using CHOK1 / hNKG2A / CD94 and CHOK1 / hNKG2C / CD94 as antigens. Hybridoma cells in positive wells were amplified and confirmed by secondary screening using the same ELISA assay as the initial screening. FACS binding using CHOK1 / hNKG2A / CD94, CHOK1 / cynoNKG2A / CD94, and CHOK1 was also included in the secondary screening. Hybridoma cells secreting antibodies with the highest human NKG2A binding and cross-reactivity with cynomolgus monkey activity were then subcloned.

[0142] 2.3 Hybridoma subcloning and screening The selected hybridoma cells were limitedly diluted to a density of 1 cell / well in 96-well plates to obtain monoclonal hybridoma cells. The supernatant harvested from these monoclonal cells was screened by the same FACS assay as in Example 2.2. The antibodies secreted by the positive clone were quantified by biolayer interference method, and then the binding affinity of human NKG2A / CD94 and the activity of blocking the interaction between NKG2A / CD94 and its ligand HLA-E were measured. As shown in Table 2, the monoclonal antibody secreted from clone 147B6-2E10 showed nanomolar binding affinity of human NKG2A / CD94 and good blocking activity.

[0143] Table 2. Summary of mouse antibody characterization Instructions 17 / 25 pages 23 CN 121358774 A

[0144] Example 3: Generation and characterization of chimeric antibodies 3.1 Generation of chimeric antibodies The heavy chain variable region and light chain variable region of the monoclonal antibody secreted by clone 147B6-2E10 were sequenced. According to the sequencing results, a human IgG4 chimeric antibody with S228P mutation in the Fc region was generated and named cAb017, where the suffix "c" is used.Represents chimerism. The variable region of cAb017 is shown below with a standard single-letter code, and its CDR is underlined. Except for the heavy chain CDR1, which is defined using a combination of the Kabat and Chothia systems, the CDR is defined using the Kabat definition.

[0145] VH: EVQLQQSVAELVRPGASVKLSCTGSGFNIQNTYMHWVKQRPEQGLEWIGRI DPANGNTKYAPNF QGKATILAETSSNTAYLQLSSLTSEDTAIYYCAREGGDYYGSSYALAYWGQGASVTVSS (SEQ ID NO: 10) VL: DVQIIQSPSYLAASPGETITINCRASKSISKFLAWYQEKPGKTNKLLIYSGSTLQ SGIPSRFSG SGSGTDFTLTISSLEPEDFAMYYCQQHNEFPWTFGGGTKVEIK (SEQ ID NO: 11) 3.2 Characterization of Chimeric Antibodies 3.2.1 Binding Activity Using CHOK1-hNKG2A / CD94 or 293T-hNKG2A / CD94, CHOK1-cynoNKG2A / CD94, and NK92 cells as antigens, the binding activity of cAb017, three benchmark antibodies Tab1, 2, and 3, and the human IgG4 isotype (hIgG4) human NKG2A / CD94 was detected by FACS assay. All tested monoclonal antibodies (mAbs) bound strongly to CHOK1-hNKG2A / CD94 (Figure 1), CHOK1-cynoNKG2A / CD94 (Figure 2), and NK92 (Figure 3) with similar affinities. Table 3 summarizes the EC50 and maximum geometric mean fluorescence intensity (MFI) values ​​obtained using a four-parameter nonlinear fitting analysis with GraphPad Prism 9.0.

[0146] Table 3. Binding of cAb017 and Tab1, 2, 3 to membrane NKG2A / CD94

[0147] 3.2.2 Affinity Assay The binding affinity of cAb017 and Tab1, 2, 3 to human NKG2A / CD94 ECD recombinant protein was determined using the octet method. The association and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​for each antibody were calculated and summarized in Table 4. cAb017 (KD, 0.028 nM) showed a higher antigen-binding affinity than all reference antibodies tested.

[0148] Table 4. Binding affinity of cAb017, Tab1, Tab2, and Tab3 to human NKG2A / CD94

[0149] 3.2.3 Binding SpecificityUsing CHOK1-hNKG2C / CD94 cells (Figure 4A) as antigens, the binding of cAb017, Tab1, Tab2, and Tab3 to NKG2C was detected by FACS assay (refer to the method described in Example 3.2.1). As shown in Figure 4A, cAb017 and Tab1 could bind weakly to NKG2C, while Tab2 and Tab3 did not bind to NKG2C.

[0150] Using rhNKG2E-ECD-Fc protein (Figure 4B) as antigens, the binding of cAb017, Tab1, Tab2, Tab3, antibodies recognized by NKG2E (positive control), and human IgG4 isotype to NKG2E was detected by ELISA assay. As shown in Figure 4B, all tested antibodies could not recognize the NKG2E protein.

[0151] 3.2.4 Blocking Activity The blocking activity of cAb017 and Tab1, 2, and 3 against the interaction between NKG2A / CD94 and HLA-E was assessed by competitive FACS. In short, CHOK1-NKG2A / CD94 cells were pre-incubated for 30 minutes with 10 µg / mL of the test antibody or IgG4 isotype. Then, recombinant protein of the HLA-E-PE complex tetramer was added and incubated for another hour. The blocking activity was determined by quantifying the blocking effect of the ligands on CHOK1-NKG2A / CD94 cells. As shown in Figure 5, cAb017 and Tab1, 2 completely blocked the interaction between NKG2A / CD94 and HLA-E, while Tab3 only partially blocked the interaction. Table 5 summarizes the IC50 values ​​and highest blocking rates using a four-parameter nonlinear fitting analysis with GraphPad Prism 9.0.

[0152] Table 5. Blocking activities of cAb017, Tab1, Tab2 and Tab3

[0153] 3.2.5 NK92-mediated cytotoxicity assay The effects of cAb017, Tab1, Tab2 and Tab3 in NK92-mediated cytotoxicity were investigated. Briefly, serially diluted antibody and 100 μl / well (three replicates) of NK-92 cells were added to a U-bottom 96-well microtiter plate for pre-incubation. Target cells (tumor cell line LCL721.221) were loaded with specific peptides to induce HLA-E expression. Target cells were labeled with the fluorescence-enhancing ligand specification (page 19 / 25, CN 121358774 A, DELFIA BATDA reagent) and 100 μl (1 × 10⁴) of target cells were added to each well. After short-term centrifugation, the co-cultures were incubated at 37°C in a 95% humidified chamber containing 5% CO2 for 2 h. They were then centrifuged for 5 min, and 20 μl of supernatant was extracted from each well and added to 100 mL of water.μl of europium solution (Eu). The percentage of specific release (lysis) was calculated using the following formula: % Specific release = × 100 As shown in Figure 6, all tested antibodies improved the specific lysis of tumor cells expressing HLA-E. And cAb017 and Tab3 showed the strongest efficacy. EC50 values ​​were analyzed using a four-parameter nonlinear fitting of GraphPad Prism 9.0 as shown below.

[0154]

[0155] Span = Maximum specific release - Minimum specific release 3.3.6 CD107a degranulation assay The effects of cAb017, Tab1, Tab2 and Tab3 on NK cell degranulation were studied using this assay. Briefly, NK cells were isolated from human PBMCs using a Ficoll gradient and a human NK cell isolation kit (Miltenyi Biotech). NK cells were cultured overnight with recombinant human (rh) IL-2 (400 IU / ml). After activation, cell viability was greater than 90%, as indicated by cell counting. NK cells were then co-cultured for four hours with 5 × 10⁴ cells of the HLA-E-expressing human B lymphoblast target cell line 721.221. Cells were then collected and stained against the following markers: anti-human CD3, anti-human CD56, live / dead, anti-human CD107a, and anti-NKG2A antibody. Fresh cells were obtained using BD Canto II. The percentage of degranulation (%CD10⁷) in NKG2A+ or NKG2A-NK cells was subsequently analyzed using FlowJo (flow cytometry software). NK cell degranulation increased in the presence of cAb017, Tab1, Tab2, and Tab3, as measured by CD107a expression by flow cytometry (Figure 7A). No increased NK cell response was observed on NKG2A-negative (-) NK cells, indicating that the antibody effect is specific to NK cells expressing NKG2A (Fig. 7B).

[0156] 3.2.7 Primary NK Cell Cytotoxicity Assay This assay was used to investigate the effects of cAb017, Tab1, Tab2, and Tab3 on primary NK-mediated cytotoxicity. Briefly, NK cells were isolated from human PBMCs using a Ficoll gradient and a human NK cell isolation kit (Mitentech Biotechnology Co., Ltd.). NK cells were cultured for 4–5 days with recombinant human IL-2 (Peprotech, catalog number 200-02) and recombinant human IL-12 (Peprotech, catalog number 200-12). After NK activation, serially diluted antibody and NK cells were added to a U-bottom 96-well microtiter plate for pre-incubation. Target cells (tumor cell line LCL721.221) were loaded with a specific peptide to induce HLA-E expression. The expression was enhanced with fluorescence.Target cells were labeled with ligands (DELFIA BATDA reagent), and 100 μL (1 × 10⁴) of target cells were added to each well. After short-term centrifugation, the co-cultures were incubated at 37°C in a 95% humidified chamber containing 5% CO₂ for 2 h. They were then centrifuged for 5 min, and 20 μL of supernatant was extracted from each well and added to 100 μL of europium solution (Eu). The percentage of specific release (specific cytotoxicity) was calculated using the following formula: % specific release = (Cab017 / Cab3) × 100. As shown in Figure 8, cAb017 and Tab3 significantly improved primary NK-mediated specific lysis against HLA-E-expressing tumor cells.

[0157] 3.2.8 Jurkat-NFAT NKG2A / CD94 Reporter Gene Assay In Jurkat cell lines expressing NKG2A / CD94 stimulated with CHO / OKT3 / HLA-E (engineered Chinese hamster ovary cells expressing single-chain OKT3 and HLA-E), the functional activities of cAb017, Tab1, Tab2, and Tab3 in reversing the inhibition of NFAT signaling were tested. Jurkat effector cells expressing NKG2A were co-cultured with CHO / scOKT3 / HLA-E target cells at an effector cell:target cell ratio (E:T) of 6:1. Anti-NKG2A antibody or hIgG4 isotype antibody was added to the coculture at titrated concentrations. After incubation at 37°C for five hours, luciferase activity was quantified using One-Glo reagent. The relative luciferase unit (RLU) data were plotted using GraphPad software. The NKG2A antibody reversed the NKG2A / HLA-E-mediated inhibition of T cell responses. Specifically, as shown in Figure 9, cAb017 reversed the inhibition of NFAT signaling in CHO / OKT3 / HLA-E-stimulated Jurkat T cell lines expressing NKG2A, with an EC50 value of 0.31 nM.

[0158] 3.2.9 Jurkat-NFAT NKG2C / CD94 reporter gene assay The functional activity of cAb017 and Tab1 in NFAT signaling activation was tested in Jurkat cell lines expressing NKG2C / CD94 / DAP12. The test antibody or allotype antibody was added to the co-culture at titrated concentrations. After incubation at 37°C for five hours, luciferase activity was quantified using One-Glo reagent. The relative luciferase unit (RLU) data were plotted using GraphPad software. As shown in Figure 10, in the Jurkat T cell line expressing NKG2C, cAb017 can activate NFAT signaling; however, Tab1 does not induce NFAT activation.

[0159] 3.2.10 Epitope Analysis: Epitope binning of cAb017, Tab1, Tab2, and Tab3 was performed using a competitive ELISA method. In short, Ab1 was fixed overnight on ELISA plates at 4°C. After blocking with PBS-2% BSA to reduce nonspecific binding, the plates were washed with PBS-0.05% Tween 20 and incubated at 37°C for 1 hour with a mixture of 50 μl / well of Ab2 and 50 μl / well of biotinylated NKG2A / CD94 ECD recombinant protein. After washing, horseradish peroxidase (HRP)-labeled detection antibody was added, and the plates were incubated at 37°C for 1 hour. Color development was initiated by adding 100 μl / well of TMB solution. After incubation at room temperature (RT) for 10–15 minutes, the reaction was stopped by adding 100 μl of 1N HCl. The absorbance of these plates at 450 nm was then immediately read using a microplate reader.

[0160] Finally, the relative binding signals of the tandem Ab2 were calculated by normalization. A high relative binding signal indicates low competition for the binding antigen between Ab2 and Ab1. Similarly, a low relative signal indicates high competition between Ab2 and Ab1. As shown in Table 6, all antibodies are in the same bin, although Tab2 does not fully compete with other antibodies, which may be due to its relatively weak affinity for NKG2A / CD94.

[0161] Table 6. Epitope identification of cAb017, Tab1, Tab2 and Tab3

[0162] The binding epitopes of cAb017, Tab1, Tab2 and Tab3 on NKG2A / CD94 were further plotted using deuterium-hydrogen exchange mass spectrometry (HDX-MS). Key peptides “IDNEEEMKF” (SEQ ID NO: 7), “FKHEIKDSDNAEL” (SEQ ID NO: 8), and “LQVNRL” (SEQ ID NO: 9) were identified and named peptide-1, peptide-2, and peptide-3, respectively.

[0163] Table 7. Epitope mapping of cAb017, Tab1, Tab2, and Tab3

[0164] Binding of Tab1 and cAb017 resulted in a reduction in hydrogen-deuterium exchange in peptide-3, suggesting that antibody coverage of this region on NKG2A / CD94 may be key to binding to NKG2C.

[0165] 3.2.11 NK Activation Assay This assay investigated the effects of cAb017 and Tab1 on NKG2C-positive NK cells. In short, NK cells were isolated from human PBMCs using a Ficoll gradient and a human NK cell isolation kit (Mitentech Biotechnology Co., Ltd.). NK cells were combined with recombinant human (rh) IL-2 (400 mg / L)Cells were cultured overnight together with IU / ml. After activation, cell viability was greater than 90%, as indicated by cell counting. The test antibody and isotype control were absorbed into 96-well plates, and NK cells were then incubated for 6 hours. The percentages of %CD107a and %IFNγ in NKG2C+NK cells were then analyzed on FlowJo (flow cytometry software). NK cell activity increased in the presence of cAb017 and Tab1, as measured by CD107a (Fig. 19A) and %IFNγ (Fig. 19B) expression obtained by flow cytometry. The data show that the antibody can specifically activate NKG2C-positive NK cells and shows greater potency than the baseline antibody (Tab1).

[0166] Example 4: Antibody Humanization 4.1 Humanization Design cAb017 was humanized using complementarity-determining region (CDR) transplantation.

[0167] In short, IGH1-2*06 and IGKV1-33*01 were initially selected as humanization templates for the heavy and light chains, respectively, based on their homology with the original mouse antibody sequences. Except for the heavy chain CDR1, which was defined using a combination of the Kabat and Chothia systems, the CDR was defined using the Kabat definition. For transplantation, different combinations of the CDR and classical residues from cAb017 were transplanted onto the templates. The resulting variants were generated and designated as H17.H28 to H17.H30 and H17.H68 to H17.H76, where the prefix "hu" indicates "humanization" and the numbers in the suffix indicate the sequence number. All variants were tested in multiple in vitro assays to select the variant that best preserved the characteristics of the parent antibody.

[0168] The sequences of H17.H01 to H17.H24 are shown below. Instructions for Use, Pages 22 / 25, 28, CN 121358774 A

[0169]

[0170] 4.2 Characterization of Humanized Variants 4.2.1 Binding Activity The binding of cAb017 and cAb017-derived humanized variants to human NKG2A / CD94 on the cell membrane was detected by FACS assay using 293T / h NKG2A / CD94 cells. As shown in Figure 11, H17.H28, H17.H29, and H17.H30 retained the human NKG2A / CD94 binding activity of the parental antibody cAb017. Table 8 summarizes the EC50 and highest MFI values ​​using a four-parameter nonlinear fitting analysis with GraphPad Prism 9.0.

[0171] Table 8. Binding of cAb017-derived humanized variants to 293T / hNKG2A / CD94

[0172] 4.2.2 Affinity Detection The binding affinity of the cAb017-derived humanized variants to human NKG2A / CD94 was determined using the Octet method. The association and dissociation curves were plotted using a 1:1 ratio.1. The model was fitted and the Ka / Kd / KD values ​​of each antibody were calculated and summarized in Table 9. H17.H28, H17.H29, and H17.H30 retained the human NKG2A / CD94 binding affinity of the parent antibody cAb017.

[0173] Table 9. Human NKG2A / CD94 binding affinity of humanized variants derived from cAb017 Specification 23 / 25 pages 29 CN 121358774 A

[0174] 4.2.3 Blocking activity The blocking activity of cAb017 and humanized variants derived from cAb017 H17.H28, H17.H29, and H17.H30 against the interaction between NKG2A / CD94 and HLA-E was evaluated by competitive FACS (Figure 12, refer to the method described in Example 3.2.4). Table 10 summarizes the IC50 values ​​and highest blocking rates using a four-parameter nonlinear fitting analysis with GraphPad Prism 9.0.

[0175] Table 10. Blocking activity of cAb017 and humanized variants derived from cAb017

[0176] 4.2.4 Functional validation 4.2.4.1 Binding activity Using 293T / hNKG2A / CD94 cells as antigens, the binding activity of cAb017 and humanized variants of human NKG2A / CD94 was detected by FACS assay. All tested mAbs: H17.H28, H17.H68, H17.H69, H17.H70, H17.H71, H17.H72, H17.H73, H17.H74, H17.H75, H17.H76 bound strongly to 293T / hNKG2A / CD94 with similar affinity (Figure 13).

[0177] 4.2.4.2 Jurkat-NFAT NKG2A / CD94 Reporter Gene Assay In Jurkat cell lines expressing NKG2A / CD94 stimulated by CHO / OKT3 / HLA-E, the functional activity of cAb017 and humanized variants in reversing the inhibition of NFAT signaling was tested. Jurkat effector cells expressing NKG2A were co-cultured with CHO / scOKT3 / HLA-E target cells at an effector cell:target cell ratio (E:T) of 6:1. Anti-NKG2A antibody or hIgG4 isotype antibody was added to the coculture at titrated concentrations. After incubation at 37°C for five hours, luciferase activity was quantified using One-Glo reagent. The relative luciferase units (RLU) data were plotted using GraphPad software. The NKG2A antibody reversed the inhibition of T cell responses mediated by NKG2A / HLA-E. Specifically, as shown in Figures 14A and 14B, Jurkat expressing NKG2A under CHO / OKT3 / HLA-E stimulation.In T cell lines, cAb017 and all humanized variants reversed the inhibition of NFAT signaling with similar potency.

[0178] 4.2.4.3 Binding Specificity: Using Jurkat-NFAT-NKG2C / CD94 cells (Fig. 15) as antigens, the binding of cAb017 and cAb017-derived humanized variants to NKG2C was detected by FACS assay. As shown in Fig. 15, all variants could bind to NKG2C.

[0179] 4.2.4.4 Jurkat-NFAT NKG2C / CD94 Reporter Gene Assay: The functional activity of cAb017 and humanized variants in NFAT signaling activation was tested in Jurkat cell lines expressing NKG2C / CD94 / DAP12. The test antibody or allotype antibody was mixed with anti-human IgG4 Fc antibody and added to the coculture at titrated concentrations. After incubation at 37°C for five hours, luciferase activity was quantified using One-Glo reagent. Using GraphPad software, the relative luciferase unit (RLU) data were plotted. As shown in Figures 16 and 17, all variants were able to activate NFAT signaling in the Jurkat T cell line expressing NKG2C.

[0180] 4.2.5 Hotspot Removal Manual 24 / 25 pages 30 CN 121358774 A The CDR regions of the light and heavy chains of H17.H28 and H17.H73 were analyzed, wherein the amino acid sequence NG in the heavy chain CDR2 was mutated to NA to obtain hotspot removal variants H17.H28.DR02 and H17.H73.DR02. The VH and VL of H17.H28.DR02 are shown in SEQ ID NO: 42 and 13, respectively. The VH and VL of H17.H73.DR02 are shown in SEQ ID NO: 43 and 29, respectively.

[0181] 4.2.5.1 Affinity Assay The binding affinity of hotspot-derived humanized variants to human NKG2A / CD94 was determined using the Octet method. The association and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​for each antibody were calculated and summarized in Table 11. H17.H28.DR02 and H17.H73.DR02 retained the binding affinity of their parent antibodies to human NKG2A / CD94.

[0182] Table 11. Binding affinity of human NKG2A / CD94 for cAb017-derived humanized variants

[0183] 4.2.5.2 Jurkat-NFAT NKG2C / CD94 reporter gene assay The functional activity of H17.H28.DR02 and H17.H73.DR02 in activating NFAT signaling was tested in Jurkat cell lines expressing NKG2C / CD94 / DAP12. The test antibody or allotype antibody was mixed with anti-human IgG4.Fc antibodies were mixed and added to the co-culture at titrated concentrations. After incubation at 37°C for five hours, luciferase activity was quantified using One-Glo reagent. The relative luciferase unit (RLU) data were plotted using GraphPad software. As shown in Figure 18, all variants were able to activate NFAT signaling in the Jurkat T cell line expressing NKG2C.

[0184] 4.2.5.3 NK92-mediated cytotoxicity assay The effects of cAb017, H73.DR02, and H28.DR02 on NK92-mediated cytotoxicity were investigated in assay 3.2.5. As shown in Figure 20, all tested antibodies were able to improve the specific lysis of HLA-E-expressing tumor cells. H73.DR02 and H28.DR02 showed similar potency to cAb017. EC50 values ​​were analyzed using a four-parameter nonlinear fitting method with GraphPad Prism 9.0 as follows:

[0185] Span = Maximum Specific Release - Minimum Specific Release. While specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that may not be foreseen or currently foreseen by the applicant or other persons skilled in the art are possible. Therefore, the appended claims, as filed and as may be amended, are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents. Instruction Manual Page 25 / 25 31 CN 121358774 A Figure 1 Figure 2 Instruction Manual Figure 1 / 12 Page 32 CN 121358774 A Figure 3 Figure 4A Instruction Manual Figure 2 / 12 Page 33 CN 121358774 A Figure 4B Figure 5 Instruction Manual Figure 3 / 12 Page 34 CN 121358774 A Figure 6 Figure 7A Instruction Manual Figure 4 / 12 Page 35 CN 121358774 A Figure 7B Figure 8 Instruction Manual Figure 5 / 12 Page 36 CN 121358774 A Figure 9 Figure 10 Instruction Manual Figure 6 / 12 Page 37 CN 121358774 A Figure 11 Figure 12 Instruction Manual Figure 7 / 12 Page 38 CN 121358774 A Figure 13 Figure 14A Instruction Manual Figure 8 / 12 Page 39 CN 121358774 A Figure 14B Figure 15 Appendix to the Instruction Manual, Page 9 / 12, 40 CN 121358774 A Figure 16 Figure 17 Appendix to the Instruction Manual, Page 10 / 12, 41 CN 121358774 A Figure 18 Figure 19A Appendix to the Instruction Manual, Page 11 / 12, 42CN 121358774 A Figure 19B Figure 20 Instruction Manual Drawings 12 / 12 Page 43 CN 121358774 A

Claims

1. An antibody or antigen-binding fragment thereof that binds to NKG2A, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 36, RIDPANXNTKYAPNFQG (SEQ ID NO: 37), and SEQ ID NO: 38, respectively; X is G or A; and the VL comprises complementarity determining regions LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 39, 40, and 41, respectively.

2. The antibody or antigen-binding fragment thereof that binds to NKG2A of claim 1, wherein X is G.

3. The antibody or antigen-binding fragment thereof that binds to NKG2A of claim 1, wherein X is A.

4. The antibody or antigen-binding fragment thereof that binds to NKG2A of claim 1, wherein the VH and the VL are selected from the following: a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 11; b) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 13; c) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 15; ​ ​ ​ ​ d) a VHcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and a VLcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 17; e) a VHcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18, and a VLcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 19; f) a VHcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20, and a VLcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 21; g) a VHcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, and a VLcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 23; h) a VHcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, and a VLcomprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 25; i) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 27; j) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 29; k) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 30, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 31; l) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 33; m) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 35; n) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 42, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 13; o) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO:

29.

5. The antibody or antigen-binding fragment thereof that binds to NKG2A of claim 1, wherein the VH comprises an amino acid sequence as set forth in SEQ ID NO: 10, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 13; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 14, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 15; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 16, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 17; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 18, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 19; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 20, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 21; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 22, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 23; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 24, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 25; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 26, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 27; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 29; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 30, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 31; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 34, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 35; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 42, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 13; or the VH comprises an amino acid sequence as set forth in SEQ ID NO: 43, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 29, or a conservative substitution thereof.

6. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A does not bind to NKG2E or does not substantially bind to NKG2E.

7. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A improves an immune response, optionally, an innate immune response.

8. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A blocks the interaction between NKG2A / CD94 and an MHC-1 molecule, optionally, the MHC-1 molecule is HLA-E.

9. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-8, wherein the antibody or antigen-binding fragment improves lysis of a cell expressing HLA-E, optionally, the cell expressing HLA-E comprises a tumor cell expressing HLA-E.

10. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A increases a response of an NK cell, optionally, the NK cell expresses NKG2A.

11. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A exerts an NK-mediated cytotoxic effect on a tumor cell expressing HLA-E.

12. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A exerts NK-mediated cytotoxicity effects on tumor cells expressing HLA-E, wherein EC 50 no more than 1 nM, 0.8 nM, 0.5 nM, or 0.3 nM.

13. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A upregulates CD 107a (lysosome-associated membrane protein 1).

14. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A reverses inhibition of T cell responses, optionally, the antibody or antigen-binding fragment thereof that binds to NKG2A reverses NKG2A / HLA-E-mediated inhibition of T cell responses.

15. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A activates or increases the response of T cells, optionally, the T cells express NKG2A or NKG2C.

16. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-15, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A activates NFAT signaling.

17. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A binds to NKG2A with an EC 50 not more than 1 nM, 0.8 nM, or 0.6 nM, or a KD (affinity constant) of not more than 9.9E-8 nM, 9.9E-9 nM, or 9.9E-10 nM.

18. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-17, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A blocks the interaction between NKG2A and HLA-E, wherein IC 50 no more than 1 nM, 0.8 nM, or 0.6 nM.

19. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-18, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A is chimeric or humanized.

20. The antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A comprises an Fc fragment, optionally, the Fc fragment is wild-type human IgG or human IgG with one or more mutations.

21. An antibody or antigen-binding fragment thereof that binds to NKG2A, the antibody or antigen-binding fragment thereof that binds to NKG2A binds to a peptide as set forth in SEQ ID NO:

8.

22. A binding site comprising an amino acid sequence as set forth in SEQ ID NO:

8.

23. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-21.

24. An isolated vector comprising the isolated polynucleotide of claim 23.

25. A host cell comprising the isolated polynucleotide of claim 23 or the isolated vector of claim 24.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-21, the isolated polynucleotide of claim 23, the isolated vector of claim 24, or the host cell of claim 25, and a pharmaceutically acceptable carrier.

27. A kit comprising the antibody or antigen-binding fragment thereof that binds to NKG2A of any one of claims 1-21, the binding site of claim 22, the isolated polynucleotide of claim 23, the isolated vector of claim 24, or the host cell of claim 25.

28. Use of an antibody or antigen-binding fragment thereof that binds to NKG2A according to any one of claims 1-21, an isolated polynucleotide according to claim 23, an isolated vector according to claim 24, a host cell according to claim 25, or a pharmaceutical composition according to claim 26, or a kit according to claim 27 in the manufacture of a therapeutic agent for the diagnosis, prevention or treatment of a disease.

29. The use of claim 28, wherein the disease comprises an oncology-related disease, an inflammatory or autoimmune disease, or an infectious disease.

30. The use of claim 28 or 29, wherein the oncology-related disease comprises breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a hematological malignancy; the infectious disease comprises a viral infectious disease and a bacterial infectious disease; the inflammatory or autoimmune disease comprises arthritis, contact dermatitis, hyper IgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory disease.

31. A method of diagnosing, preventing or treating a subject having an oncology-related disease, an inflammatory or autoimmune disease, or an infectious disease, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to NKG2A according to any one of claims 1-21, an isolated polynucleotide according to claim 23, an isolated vector according to claim 24, a host cell according to claim 25, a pharmaceutical composition according to claim 26, or a kit according to claim 27.

32. The method of claim 31, wherein the method comprises enhancing an immune response to the disease, optionally, the method comprises enhancing an innate immune response.

33. The method of claim 31 or 32, wherein the method comprises reducing adverse effects in the diagnosis, prevention or treatment of the disease.

34. The method of any one of claims 31-33, wherein the oncology-related disease comprises breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a hematological malignancy; the infectious disease comprises a viral infectious disease and a bacterial infectious disease; the inflammatory or autoimmune disease comprises arthritis, contact dermatitis, hyper IgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory disease.

35. The method of any one of claims 31-34, wherein the method improves lysis of cells expressing HLA-E; optionally, the method improves lysis of tumor cells expressing HLA-E.

36. The method of any one of claims 31-35, wherein the method comprises ​ ​ ​ ​ (1) increasing NK cell responses; (2) upregulating CD 107a; (3) activating T cells or increasing T cell responses; or (4) activating NFAT signaling.

Images