Nucleic acid molecules encoding anti-NKG2A antibodies and uses thereof

Abstract

The present invention belongs to the field of biology, and relates to a nucleic acid molecule encoding an anti-NKG2A antibody, a recombinant vector comprising the nucleic acid molecule, and an expression method wherein the nucleic acid molecule comprises codons optimized for expression cells. By utilizing the nucleic acid molecule disclosed by the invention, the expression quantity of the anti-NKG2A antibody in cells can be increased, the yield of the anti-NKG2A antibody is increased, and the production cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biology and relates to nucleic acid molecules encoding anti-NKG2A antibodies and applications thereof. Background Art

[0002] NKG2A, also known as natural killer cell lectin-like receptor, is an inhibitory receptor in the NKG2 receptor family and is mainly expressed in NK cells, CD8 + On the surface of T cells, Th2 cells and NKT cells. On the surface of human immune cells, NKG2A and CD94 molecules (NK cell surface membrane protein) are connected in the form of disulfide bonds to form a heterodimeric complex NKG2A-CD94, which is recognized by the non-classical histocompatibility complex class I (major histocompatibility complex class I, MHC I) HLA-E on the target cell. HLA-E is the only ligand for the heterodimeric receptor CD94 / NKG2A and is a non-classical MHC class Ib molecule. This molecule is expressed at low levels under normal circumstances, but many tumor cells highly express HLA-E, and NK and CD8 + T cells highly express NKG2A, allowing tumor cells to evade NK and CD8 + T cell immune recognition. In addition, after the virus infects the body, it can induce host cells to express NK and CD8 + T cell NKG2A inhibitory receptor binds to the ligand, escaping the clearance of the immune system (Zheng M et al. Cellular and Molecular Immunology, 2020, 17(5)). Therefore, anti-NKG2A antibodies can release the killing activity of NK by blocking this inhibitory signal and play an anti-tumor role.

[0003] Existing cell lines for producing anti-NKG2A antibodies have limited expression, significantly impacting production. Therefore, increasing the production of anti-NKG2A antibodies is a major issue that needs to be addressed in this field and is a key step in achieving mass production of antibodies. Summary of the Invention

[0004] The purpose of the present invention is to optimize and analyze the monoclonal antibody against NKG2A antibody (also known as hu-43VL (N26S-G34A)), screen out codons that can be highly expressed in cells, and thus increase the expression level of the monoclonal antibody in the cells.

[0005] Herein, the amino acid sequences of the heavy chain and light chain of the anti-NKG2A antibody (hu-43VL(N26S-G34A)) are shown in SEQ ID NOs: 1 and 3, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 2 and 4, respectively; the three CDR region sequences of the heavy chain variable region are shown in SEQ ID NOs: 5, 6 and 7, respectively, and the three CDR region sequences of the light chain variable region are shown in SEQ ID NOs: 8, 9 and 10, respectively.

[0006] In a first aspect, a nucleic acid molecule is provided, comprising nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of an anti-NKG2A antibody, and nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3 of the light chain variable region of an anti-NKG2A antibody, wherein:

[0007] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 15, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 16, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 17; or

[0008] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 26, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 27; or

[0009] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 35, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 36, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 37; and

[0010] The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 18, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 19, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 20; or

[0011] The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:28, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:29, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:30; or

[0012] The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:38, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:39, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:40.

[0013] In some preferred embodiments, the nucleic acid molecule comprises nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of an anti-NKG2A antibody, and nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3 of the light chain variable region of an anti-NKG2A antibody, wherein:

[0014] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 15, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 16, the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 17, the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 18, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 19, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 20; or

[0015] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO:25, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO:26, the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO:27, the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:28, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:29, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:30; or

[0016] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO:35, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO:36, the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO:37, the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:38, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:39, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:40.

[0017] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of an anti-NKG2A antibody, wherein:

[0018] The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 12, SEQ ID NO: 22 or SEQ ID NO: 32, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 12, SEQ ID NO: 22 or SEQ ID NO: 32, and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 14, SEQ ID NO: 24 or SEQ ID NO: 34, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 14, SEQ ID NO: 24 or SEQ ID NO: 34.

[0019] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of an anti-NKG2A antibody, wherein:

[0020] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 12, SEQ ID NO: 22 or SEQ ID NO: 32, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 14, SEQ ID NO: 24 or SEQ ID NO: 34.

[0021] In some embodiments, the nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 12, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 12, and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 14, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 14.

[0022] In some embodiments, the aforementioned nucleic acid molecule comprises nucleic acid sequences encoding the heavy chain variable region and light chain variable region of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 12, and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO: 14.

[0023] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of an anti-NKG2A antibody, wherein:

[0024] The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO:22, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:22, and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO:24, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:24.

[0025] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 22, and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO: 24. In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of an anti-NKG2A antibody, wherein:

[0026] The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO:32, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:32, and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO:34, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:34.

[0027] In some embodiments, the aforementioned nucleic acid molecule comprises nucleic acid sequences encoding the heavy chain variable region and light chain variable region of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO:32, and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO:34.

[0028] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding a human antibody heavy chain constant region and a light chain constant region. In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding a human antibody heavy chain and a light chain, wherein:

[0029] The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31, and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33.

[0030] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding an anti-NKG2A antibody heavy chain and a light chain, wherein:

[0031] The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 11, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 11, and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 13, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 13.

[0032] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding an anti-NKG2A antibody heavy chain and a light chain, wherein:

[0033] The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO:21, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:21, and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO:23, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:23.

[0034] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding an anti-NKG2A antibody heavy chain and a light chain, wherein:

[0035] The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO:31, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:31, and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO:33, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:33.

[0036] In some embodiments, the aforementioned nucleic acid molecule comprises nucleic acid sequences encoding the heavy and light chains of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO:11, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO:13.

[0037] In some embodiments, the aforementioned nucleic acid molecule comprises nucleic acid sequences encoding the heavy and light chains of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO: 21, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO: 23.

[0038] In some embodiments, the aforementioned nucleic acid molecule comprises nucleic acid sequences encoding the heavy and light chains of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO: 31, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO: 33.

[0039] In some embodiments, the aforementioned nucleic acid molecule comprises a signal peptide sequence encoding the heavy and light chains of an anti-NKG2A antibody. The signal peptide can be any signal peptide known in the art suitable for antibody expression. In some embodiments, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:41. In some embodiments, the nucleic acid sequence encoding the signal peptide is as shown in SEQ ID NO:42.

[0040] In a second aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule of the present invention.

[0041] In some embodiments, the recombinant vector is an expression vector.

[0042] In a third aspect, the present invention provides a vector system for expressing an anti-NKG2A antibody, comprising a first vector and a second vector, wherein:

[0043] The first vector comprises nucleic acid sequences encoding HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of an anti-NKG2A antibody, wherein:

[0044] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 15, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 16, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 17; or

[0045] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 26, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 27; or

[0046] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 35, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 36, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 37; and

[0047] The second vector comprises nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3 of the light chain variable region of an anti-NKG2A antibody, wherein:

[0048] The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 18, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 19, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 20; or

[0049] The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:28, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:29, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:30; or

[0050] The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:38, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:39, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:40.

[0051] In some embodiments, in the aforementioned vector system for expressing an anti-NKG2A antibody, the first vector comprises nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 15, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 16, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 17; and

[0052] The second vector contains nucleic acid sequences encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 18, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 19, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 20.

[0053] In some embodiments, in the aforementioned vector system for expressing an anti-NKG2A antibody, the first vector comprises nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 26, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 27; and

[0054] The second vector contains nucleic acid sequences encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 28, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 29, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 30.

[0055] In some embodiments, in the aforementioned vector system for expressing an anti-NKG2A antibody, the first vector comprises nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 35, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 36, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 37; and

[0056] The second vector contains nucleic acid sequences encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:38, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:39, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:40.

[0057] In some embodiments, the aforementioned vector system for expressing anti-NKG2A, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of the anti-NKG2A antibody, wherein:

[0058] The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 12, SEQ ID NO: 22 or SEQ ID NO: 32, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 12, SEQ ID NO: 22 or SEQ ID NO: 32; and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 14, SEQ ID NO: 24 or SEQ ID NO: 34, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 14, SEQ ID NO: 24 or SEQ ID NO: 34.

[0059] In some embodiments, the aforementioned vector system for expressing anti-NKG2A, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of the anti-NKG2A antibody, wherein:

[0060] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 12, SEQ ID NO: 22 or SEQ ID NO: 32, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 14, SEQ ID NO: 24 or SEQ ID NO: 34.

[0061] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of the anti-NKG2A antibody, wherein:

[0062] The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 12, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 12; and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 14, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 14.

[0063] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of the anti-NKG2A antibody, wherein:

[0064] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 12, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 14.

[0065] In some embodiments, the aforementioned vector system for expressing anti-NKG2A, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of the anti-NKG2A antibody, wherein:

[0066] The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO:22, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:22; and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO:24, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:24.

[0067] In some embodiments, the aforementioned vector system for expressing anti-NKG2A, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of the anti-NKG2A antibody, wherein:

[0068] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 22, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 24.

[0069] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of the anti-NKG2A antibody, wherein:

[0070] The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO:32, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:32; and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO:34, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO:34.

[0071] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of the anti-NKG2A antibody, wherein:

[0072] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 32, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 34.

[0073] In some embodiments, the aforementioned vector system for expressing anti-NKG2A antibodies, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain constant region of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain constant region of the anti-NKG2A antibody.

[0074] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of the anti-NKG2A antibody, wherein:

[0075] The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31; and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33.

[0076] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of the anti-NKG2A antibody, wherein:

[0077] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33.

[0078] In some embodiments, the aforementioned vector system for expressing anti-NKG2A antibodies, wherein the first vector comprises a nucleic acid sequence encoding the constant region of the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the constant region of the light chain of the anti-NKG2A antibody.

[0079] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of the anti-NKG2A antibody, wherein:

[0080] The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 11, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 11; and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 13, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 13.

[0081] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of the anti-NKG2A antibody, wherein:

[0082] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO:11, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO:13.

[0083] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of the anti-NKG2A antibody, wherein:

[0084] The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO:21, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:21; and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO:23, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:23.

[0085] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of the anti-NKG2A antibody, wherein:

[0086] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO: 21, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO: 23.

[0087] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of the anti-NKG2A antibody, wherein:

[0088] The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO:31, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:31; and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO:33, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:33.

[0089] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of the anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of the anti-NKG2A antibody, wherein:

[0090] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO:31, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO:33.

[0091] In some embodiments, the aforementioned vector system for expressing an anti-NKG2A antibody comprises a first vector comprising a signal peptide sequence encoding the heavy chain of the anti-NKG2A antibody, and a second vector comprising a signal peptide sequence encoding the light chain of the anti-NKG2A antibody; the signal peptides can be any signal peptide known in the art suitable for antibody expression. In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO:41. In some embodiments, the nucleic acid sequence encoding the signal peptide is set forth in SEQ ID NO:42.

[0092] In a fourth aspect, the present invention provides a recombinant cell comprising the nucleic acid molecule or recombinant vector or anti-NKG2A antibody-expressing vector system of the present invention.

[0093] In some embodiments, the recombinant cells are obtained by transfecting the anti-NKG2A antibody-expressing vector system of the present invention.

[0094] In some embodiments, the host cell of the recombinant cell is a CHO cell, Expicho-S, CHO-S, CHO-K1 or CHO DG44. In some embodiments, the host cell of the recombinant cell is a CHO-K1 cell.

[0095] In a fifth aspect, provided is the use of the nucleic acid molecule, recombinant vector, anti-NKG2A antibody vector system or recombinant cell of the present invention in preparing anti-NKG2A antibodies.

[0096] In a sixth aspect, a method for preparing an anti-NKG2A antibody is provided, comprising culturing the recombinant cell of the present invention to express the anti-NKG2A antibody. In some embodiments, the method further comprises purifying and recovering the antibody.

[0097] In a seventh aspect, a method for preparing an anti-NKG2A antibody is provided, comprising the following steps:

[0098] a. introducing the aforementioned nucleic acid molecule, recombinant vector or vector system for expressing anti-NKG2A antibody of the present invention into a host cell;

[0099] b. culturing host cells to express anti-NKG2A antibodies; and optionally,

[0100] c. Purification and recovery of anti-NKG2A antibodies.

[0101] Any method known in the art can be used to introduce nucleic acid molecules, recombinant vectors or vector systems into host cells. Such operations are well known to those skilled in the art and can enable the expression of anti-NKG2A antibodies.

[0102] For example, a vector system expressing an anti-NKG2A antibody can be introduced in the following manner:

[0103] In one embodiment, the first vector and the second vector in the anti-NKG2A antibody expression vector system of the present invention are introduced into the host cell simultaneously.

[0104] In one embodiment, the first vector and the second vector in the vector system for expressing an anti-NKG2A antibody of the present invention are sequentially introduced into a host cell:

[0105] First, the first vector in the anti-NKG2A antibody expression vector system of the present invention is introduced into a host cell, and then the second vector in the anti-NKG2A antibody expression vector system is transfected into the same host cell; or

[0106] The second vector in the anti-NKG2A antibody expression vector system of the present invention is first introduced into a host cell, and then the first vector in the anti-NKG2A antibody expression vector system is transfected into the same host cell.

[0107] The present invention targets the expression characteristics of anti-NKG2A antibodies and designs nucleic acid molecules capable of efficiently expressing them. The expression level of the anti-NKG2A antibody is as high as 0.237 g / L, and the expressed product contains a high content of intact antibody and a low level of antibody fragments. Using the codons of the present invention to produce anti-NKG2A antibodies significantly reduces production costs, providing strong support for the industrialized production of these antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 Schematic diagram of the structure of the pcDNA3.4-hu-43VL(N26S-G34A)-HC expression vector;

[0109] Figure 2 Schematic diagram of the structure of the pcDNA3.4-hu-43VL(N26S-G34A)-LC expression vector;

[0110] Figure 3 The results of expression of different codons are shown;

[0111] Figure 4A Figure 2 is the result of binding of hu-43VL (N26S-G34A) antibody to CHOK1 cells overexpressing human NKG2A; Figure 4B Figure 2 is the result of binding of hu-43VL (N26S-G34A) antibody to CHOK1 cells overexpressing monkey NKG2A;

[0112] Figure 5 This figure shows the killing results of LCL721.221 cells by NK92 cells mediated by hu-43VL (N26S-G34A) antibody.

[0113] Figure 6 This figure shows the results of the hu-43VL (N26S-G34A) antibody-mediated killing experiment of LCL721.221 cells by primary NK cells. DETAILED DESCRIPTION

[0114] definition

[0115] In the present invention, the term "antibody" refers to an immunoglobulin that can specifically recognize and bind to an antigen, and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or antibody fragments. In particular, the amino acid sequence of the heavy chain of the anti-NKG2A antibody (hu-43VL(N26S-G34A) of the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; the three CDR sequences of the heavy chain are shown in SEQ ID NOs: 5, 6, and 7, respectively, and the three CDR sequences of the light chain are shown in SEQ ID NOs: 8, 9, and 10, respectively.

[0116] The term "variable region" refers to the domain of an antibody heavy or light chain that recognizes and specifically binds an antigenic epitope.

[0117] CDR regions, or "complementarity determining regions," refer to regions of an antibody variable region that are highly variable in sequence and form structurally defined loops and / or contain antigen-contacting amino acid residues. CDRs are primarily responsible for binding the antibody to the antigen epitope and determine the antibody's specificity. Within a given heavy or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one or a combination of many well-known numbering conventions, including, for example, Kabat, Contact, AbM, and Chothia.

[0118] In the present invention, "identity" refers to the percentage of amino acids (or bases) in the two sequences being compared that are identical when the sequences of two peptides or two nucleic acid molecules are aligned. The alignment and homology percentage or sequence identity can be determined using software programs known in the art, such as the software program described by Ausubel et al. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One such alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP. Exemplarily, "the nucleic acid sequence encoding the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 12" means that the nucleic acid sequence encoding the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 15, HCDR2 set forth in SEQ ID NO: 16, and HCDR3 set forth in SEQ ID NO: 17, and codon mutations are allowed to be introduced in regions outside the CDRs, thereby having at least 90% sequence identity with SEQ ID NO: 12", and so on.

[0119] As used herein, "nucleic acid molecule" refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically linked together by phosphodiester bonds. The nucleic acid molecules of the present invention can be isolated nucleic acid molecules, meaning nucleic acid molecules separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule, such as those that are substantially free of other cellular material or culture medium when prepared by recombinant techniques, or that are substantially free of chemical precursors or other chemical components when chemically synthesized. The nucleic acid molecules of the present invention are not limited to the sequences shown, but also include complementary sequences thereof and modifications to the nucleic acid molecules known in the art.

[0120] In the present invention, an "expression cell" refers to a cell capable of receiving, maintaining, replicating, and amplifying a vector, and may also be referred to as a "host cell." Expression cells can also be used to express polypeptides encoded by the vector. When the expression cells divide, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Expression cells can be eukaryotic or prokaryotic cells. Suitable expression cells include, but are not limited to, CHO cells such as CHO-K1 cells, NSO, COS, SP2 cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0121] In the present invention, as used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is connected. The term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the host cell genome into which they have been incorporated. When the vector is transformed into an appropriate expression cell, one or more heterologous proteins can be expressed from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction enzyme digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into expression cells, for amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are usually kept free, but can be designed so that genes or parts thereof are integrated into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

[0122] "Expression vector" includes a vector capable of expressing DNA, and the DNA is operably linked to a regulatory sequence that can affect the expression of such DNA fragments, such as a promoter region. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate expression cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or that are integrated into the genome of the expression cell. Commercially available expression vectors, particularly those expressing in mammalian cells, include pIRES (from Clontech, Palo Alto, USA), pCI-neo vectors (from Promega, Madison, USA), pCMV-Script (from Stratagene, LaJolla, USA), and pCDNA vectors (from Invitrogen, Paisley, UK).

[0123] As used herein, "codon" has a meaning generally known in the art and refers to a triplet of nucleotide residues in mRNA (or DNA), each triplet encoding a specific amino acid. Codons are degenerate; with the exception of methionine and tryptophan, each amino acid has at least two codons. Different species may have preferences for multiple codons for the same amino acid.

[0124] In this context, "codon optimization" is a technique used to improve protein expression levels in vivo by increasing the translation efficiency of target genes. Protein expression sequences are redesigned by avoiding rare codons, utilizing preferred codons, simplifying mRNA secondary structure, optimizing repetitive sequences, eliminating restriction enzyme sites, and adjusting GC content to improve translation efficiency and, consequently, protein expression levels.

[0125] A "vector system" refers to a combination of two or more vectors. A vector system is generally used to express a specific antibody or fusion protein, such as used to express an anti-NKG2A antibody in the present invention. It is well known to those skilled in the art that a dual-vector expression system can be used to express antibodies. Typically, in a dual-vector expression system, a first vector expressing an antibody light chain and a second vector expressing an antibody heavy chain are included, and the first vector and the second vector are introduced into a host cell simultaneously or sequentially for antibody expression. Sequential introduction into a host cell refers to first introducing the first vector and then the second vector, or first introducing the second vector and then the first vector. Methods for introducing nucleic acids or vectors into cells are well known to technicians, such as electroporation, injection, transfection and / or transformation.

[0126] "Host cell" and "cell line" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and their progeny, regardless of the number of passages through the progeny. The progeny may not be completely identical to the parent cell in terms of nucleic acid and may contain mutations, as long as the progeny with the mutations have the same desired function or activity as the original cell.

[0127] The host cell cannot develop into a complete animal or plant individual.

[0128] Host cells include prokaryotic and eukaryotic host cells. Eukaryotic host cells include, but are not limited to, mammalian cells, insect cells, plant cells, and fungal cells. Mammalian cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Exemplary host cells include, but are not limited to, CHO, NSO, COS, SP2 cells, HeLa cells, BHK cells, human hepatocellular carcinoma cells, A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast, such as, but not limited to, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, and Kluyveromyces.

[0129] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur. As used in the specification and claims, the singular forms "a", "an" include the corresponding plural referents unless the content clearly dictates otherwise.

[0130] The present invention will be described below with reference to specific embodiments, but the present invention is not limited thereto.

[0131] Unless otherwise specified, the reagents and instruments used in the following methods are commonly used reagents and instruments in the field and can be obtained commercially; the methods used are conventional methods in the field, and those skilled in the art can undoubtedly perform the methods and obtain corresponding results based on the contents described in the examples.

[0132] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Compendium, 3rd edition, John Wiley & Sons, Inc., 1995. The use of restriction endonucleases follows the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the present invention.

[0133] The room temperature described in the examples is the conventional room temperature in the art, generally 18-26°C.

[0134] Unless otherwise specified, the mice, proteins, and cells used in the following examples were provided by Shanghai Ruizhi Chemical Research Co., Ltd.

[0135] In the following examples, the positive antibody Z270 used is Monalizumab jointly developed by Innate Pharma and AstraZeneca, and the amino acid sequences of its heavy chain and light chain are shown in SEQ ID NO: 43 and SEQ ID NO: 44, respectively.

[0136] The specific sequence is as follows:

[0137] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGRIDPY

[0138] DSETHYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLYWFF

[0139] DVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT

[0140] SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP

[0141] CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA

[0142] KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ

[0143] VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0144] SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:43);

[0145] DIQMTQSPSS LSASVGDRVT ITCRASENIY SYLAWYQQKP GKAPKLLIYN AKTLAEGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQH HYGTPRTFGG GTKVEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC(SEQ ID NO:44)

[0146] Example 1. Screening and humanization of anti-NKG2A antibodies

[0147] Hybridoma technology was used to generate monoclonal antibodies (mAbs) with specificity and high affinity for human and monkey NKG2A, and which did not bind to human NKG2C.

[0148] Fourteen groups of mice (including four groups of Balb / c mice, nine groups of SJL mice, and one group of NOD mice, all female mice aged 6 to 8 weeks) were immunized with the following immunogens: 293F-hNKG2A / CD94 stable cell line, recombinant hNKG2A / CD94-ECD-Fc protein, 293F-cynoNKG2A / CD94 stable cells + recombinant hNKG2A / CD94-ECD-Fc protein, and hNKG2A / CD94 expression plasmid + recombinant hNKG2A / CD94-ECD-Fc protein.

[0149] After immunization, serum titers were assessed by FACS and ELISA. FACS assays were performed using CHOK1-hNKG2A / CD94, CHOK1-cynoNKG2A / CD94, and CHOK1-hNKG2C / CD94 stably transfected cells, with CHOK1-blank cells serving as a control. ELISA assays were performed using hNKG2A / CD94-ECD-Fc and cynoNKG2A / CD94-ECD-Fc proteins.

[0150] Mice with high immune titers are selected, and spleen cells are fused with myeloma cells. Two rounds of screening are performed using FACS. After the primary screening, positive hybridoma clones are transferred to 24-well plates for culture and then undergo a secondary screening. Positive clones obtained from the screening are subcloned and tested again using the primary and secondary screening methods. Positive monoclones are selected for antibody production, cryopreservation, and sequencing.

[0151] After sequencing, a sequence-specific monoclonal antibody was obtained, which was humanized using CDR grafting. The antibody's CDR region was grafted onto a matching human variable region gene framework sequence, and back mutations were designed. At the same time, potential post-translational modification sites (such as glycosylation sites, deamidation, etc.) in the first CDR region of the light chain were mutated. The antibody was then combined with the IgG4 heavy chain constant region and the kappa light chain constant region to ultimately obtain the humanized antibody hu-43VL (N26S-G34A). The specific sequences of the heavy chain, heavy chain variable region, light chain, and light chain variable region of the hu-43VL (N26S-G34A) antibody are as follows:

[0152] hu-43VL(N26S-G34A) heavy chain:

[0153] QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYIHWVRQAPGQGLEWMGRIDPASGSTEYAPKFQGRVTMTADTSTNTGYMELSSLRSEDTAVYYCARYGNFLYYYSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:1)

[0154] hu-43 VL (N26S-G34A) heavy chain variable region:

[0155] QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYIHWVRQAPGQGLEWMGRIDPAS GSTEYAPKFQGRVTMTADTSTNTGYMELSSLRSEDTAVYYCARYGNFLYYYSMDYWGQ GTLVTVSS(SEQ ID NO:2)

[0156] hu-43 VL (N26S-G34A) light chain:

[0157] DIVMTQSPLSLPVTLGQPASISCRSSKSLLHSNANTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:3)

[0158] hu-43VL(N26S-G34A) light chain variable region:

[0159] DIVMTQSPLSLPVTLGQPASISCRSSKSLLHSNANTYLYWFQQRPGQSPRLLIYRMSN LASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK (SEQ ID NO: 4).

[0160] The CDR sequence of hu-43VL(N26S-G34A) is shown in Table 1 below.

[0161] Table 1: CDR sequences of hu-43VL (N26S-G34A)

[0162]

[0163]

[0164] Example 2. Optimization of anti-NKG2A antibody nucleic acid sequence

[0165] The full-length amino acid sequence of the anti-NKG2A antibody (hu-43VL(N26S-G34A)) (heavy chain and light chain as shown in SEQ ID NOs: 1 and 3, respectively) was reverse transcribed to generate nucleotide sequences encoding the same original amino acid sequence. Multiple nucleotide sequences with the highest theoretical expression levels in a selected host (e.g., CHO cells) were generated using different codons for subsequent screening.

[0166] After multiple rounds of multi-system optimization, we finally obtained three groups of highly expressed nucleic acid sequences with different sequences, namely codon group 1, codon group 2, and codon group 3. The relevant sequences are as follows:

[0167] Codon group 1 heavy chain:

[0168] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGCGCTAGCGTGAAGGTGAGCTGCAA GGCTAGCGGCTTCAACATTCAGAACACCTACATCCACTGGGTGAGACAAGCCCCTGGCCAAGGCCTGGAGTGGATG GGCAGAATCGACCCTGCTAGCGGCAGCACCGAGTACGCCCCTAAGTTCCAAGGCAGAGTGACCATGACCGCCGACA CAAGCACCAACACCGGCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCTAGATA CGGCAACTTCCTGTACTATTACAGCATGGACTACTGGGGCCAAGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCAGCAGAAGCACAAGCGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAAGACTACTTCCCTGAGCCTGTCACCGTCAGCTGGAATAGCGGCGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTCCCTAGCAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTAGCAACACCAAGGTGGACAAGAGAGTGGAGAGCAAGTACGGCCCTCCCTGCCCCCCCTGTCCTGCTCCCGAGTTCGAGGGCGGCCCTAGCGTGTTTCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAGGTGACCTGCGTGGTCGTGGCCGTGAGCCAAGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGACAGCCTAGAGAGCCTCAAGTGTACACCCTGCCTCCTAGCCAAGAGGAGATGACCAAGAACCAAGTGAGCCTGACCTGCCTGGTGAAAGGCTTCTACCCTAGCGACATCGCCGTGGAGTGGGAGAGCAACGGACAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGAGCCTGAGCCTGAGCCTGGGCAAGTGATAA(SEQ IDNO:11)

[0169] Codon group 1 heavy chain variable region:

[0170] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGCGCTAGCGTGAAGGTGAGCTGCAAGGCTAGCGGCTTCAACATTCAGAACACCTACATCCACTGGGTGAGACAAGCCCCTGGCCAAGGCCTGGAGTGGATGGGCAGAATCGACCCTGCTAGCGGCAGCACCGAGTACGCCCCTAAGTTCCAAGGCAGAGTGACCATGACCGCCGACACAAGCACCAACACCGGCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCTAGATACGGCAACTTCCTGTACTATTACAGCATGGACTACTGGGGCCAAGGCACCCTGGTGACCGTGAGCAGC(SEQ IDNO:12)

[0171] Codon group 1 light chain:

[0172] GACATCGTGATGACACAGAGCCCTCTGAGCCTGCCTGTGACCCTGGGACAGCCTGCTAGCATCAGCTG CAGAAGCAGCAAGAGCCTGCTGCACAGCAACGCCAACACCTACCTGTACTGGTTTCAGCAGAGACCTGGACAGAGC CCTAGACTGCTGATCTACAGAATGAGCAACCTGGCTAGCGGCGTGCCTGACAGATTCAGCGGCAGCGGCAGCGGCA CCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGAA CCCTTACACCTTCGGCCAAGGCACCAAGCTGGAGATCAAG AGAACCGTGGCCGCCCCTAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAAGAGAGCGTGACCGAGCAAGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAAGGCCTGAGCAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAGTGCTGATAA(SEQ ID NO:13) Codon group 1 light chain variable region:

[0173] GACATCGTGATGACACAGAGCCCTCTGAGCCTGCCTGTGACCCTGGGACAGCCTGCTAGCATCAGCTGCAGAAGCAGCAAGAGCCTGCTGCACAGCAACGCCAACACCTACCTGTACTGGTTTCAGCAGAGACCTGGACAGAGCCCTAGACTGCTGATCTACAGAATGAGCAACCTGGCTAGCGGCGTGCCTGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAGAACCCTTACACCTTCGGCCAAGGCACCAAGCTGGAGATCAAG(SEQ ID NO:14)

[0174] Table 2: CDR coding sequence of codon group 1

[0175]

[0176]

[0177] Heavy chain of codon group 2:

[0178] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCCGTGAAGGTGTCCTGCAA AGCCTCCGGGTTCAACATCCAGAACACCTATATCCACTGGGTGAGGCAGGCACCCGGCCAGGGACTGGAGTGGATG GGAAGGATCGACCCCGCTTCTGGCTCTACCGAGTACGCTCCCAAGTTCCAGGGGAGGGTGACCATGACAGCCGACA CCTCCACCAACACCGGATACATGGAGCTGTCCAGTCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGTA CGGAAACTTCCTGTATTACTACTCCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCAGC GCCTCCACCAAAGGCCCAAGCGTGTTCCCACTGGCCCCCTGTTCCCGCTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCCGTGACAGTGTCCTGGAACTCCGGCGCCCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCTCTGGCCTGTACTCTCTGTCTTCTGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAAGTGGATAAAAGGGTGGAGTCTAAATACGGCCCCCCCTGCCCCCCCTGCCCTGCTCCTGAGTTCGAAGGCGGCCCCTCCGTGTTCCTGTTCCCCCCTAAGCCCAAAGACACCCTGATGATCTCCCGCACCCCCGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCAGGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAACCCCGCGAGGAGCAGTTTAACAGTACCTACAGGGTGGTGAGTGTGCTGACAGTGCTGCATCAGGACTGGCTGAACGGGAAGGAGTACAAGTGCAAGGTGTCCAACAAAGGCCTGCCCAGCTCTATCGAGAAAACCATCTCCAAGGCCAAGGGACAGCCAAGGGAGCCACAGGTGTACACACTGCCCCCCTCCCAGGAAGAAATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCCGACATCGCTGTGGAATGGGAGTCCAACGGCCAGCCAGAAAACAACTACAAAACCACCCCCCCCGTGCTGGACAGCGACGGATCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGGAAGGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCCCTGCACAACCACTATACCCAGAAGTCCCTGTCCCTGTCCCTGGGCAAGTGATAA(SEQ IDNO: 21) Codon group 2 heavy chain variable region:

[0179] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCCGTGAAGGTGTCCTGCAAAGCCTCCGGGTTCAACATCCAGAACACCTATATCCACTGGGTGAGGCAGGCACCCGGCCAGGGACTGGAGTGGATGGGAAGGATCGACCCCGCTTCTGGCTCTACCGAGTACGCTCCCAAGTTCCAGGGGAGGGTGACCATGACAGCCGACACCTCCACCAACACCGGATACATGGAGCTGTCCAGTCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGTACGGAAACTTCCTGTATTACTACTCCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCAGC(SEQ IDNO:22)

[0180] Codon group 2 light chain:

[0181] GACATCGTGATGACCCAGTCCCCCCTGTCCCTGCCCGTGACCCTGGGACAGCCCGCTTCCATCTCCTG CCGCTCCTCCAAATCCCTGCTGCACTCCAACGCCAACACATACCTGTATTGGTTCCAGCAGAGACCCGGCCAGTCC CCCCGCCTGCTGATCTACCGCATGTCCAATCTGGCCTCCGGCGTGCCCGACCGGTTCTCTGGATCCGGCTCCGGCA CCGACTTCACCCTGAAGATCTCCCGGGTGGAGGCTGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAAAA TCCCTATACCTTCGGCCAGGGCACCAAGCTGGAAATCAAG AGGACCGTGGCAGCCCCCTCCGTGTTCATTTTTCCCCCCTCCGATGAGCAGCTGAAGTCCGGCACCGCCTCTGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAAGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCCGGTAACTCCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGTACCTATAGCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGATTACGAGAAACATAAGGTGTATGCCTGCGAAGTGACACACCAGGGACTGAGCAGCCCCGTGACAAAATCCTTTAACAGGGGGGAGTGCTGATAA(SEQ ID NO:23)

[0182] Codon group 2 light chain variable region:

[0183] GACATCGTGATGACCCAGTCCCCCCTGTCCCTGCCCGTGACCCTGGGACAGCCCGCTTCCATCTCCTGCCGCTCCTCCAAATCCCTGCTGCACTCCAACGCCAACACATACCTGTATTGGTTCCAGCAGAGACCCGGCCAGTCCCCCCGCCTGCTGATCTACCGCATGTCCAATCTGGCCTCCGGCGTGCCCGACCGGTTCTCTGGATCCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGGGTGGAGGCTGAGGACGTGGGCGTGTACTACTGCATGCAGCACCTGGAAAATCCCTATACCTTCGGCCAGGGCACCAAGCTGGAAATCAAG(SEQ ID NO:24)

[0184] Table 3: CDR coding sequence of codon group 2

[0185]

[0186]

[0187] Codon group 3 heavy chain:

[0188] CAGGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAGCCTGGCGCCTCCGTGAAGGTGAGCTGCAA GGCTTCCGGCTTCAACATCCAGAATACCTACATCCACTGGGTGAGGCAGGCTCCCGGCCAGGGACTGGAGTGGATG GGCAGAATCGACCCCGCTTCCGGCTCCACCGAGTATGCCCCCAAGTTTCAGGGCAGGGTGACCATGACCGCCGATA CCTCCACCAATACCGGCTATATGGAGCTGAGCAGCCTGAGGTCCGAGGATACCGCTGTGTACTATTGCGCCCGGTA CGGCAATTTTCTGTATTACTACTCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCC GCCTCCACCAAGGGCCCTTCCGTGTTTCCCCTGGCTCCCTGCAGCCGGAGCACCTCTGAGAGCACCGCCGCCCTGGGCTGCCTGGTTAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAATAGCGGCGCCCTGACCAGCGGCGTGCACACATTCCCCGCCGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTAGCTCCAGCCTGGGCACCAAGACCTACACCTGTAACGTGGATCACAAGCCCAGCAATACCAAGGTGGATAAGCGGGTGGAGTCCAAGTACGGCCCCCCTTGCCCTCCTTGCCCCGCTCCTGAGTTCGAGGGCGGCCCTAGCGTGTTTCTGTTCCCCCCTAAGCCTAAGGATACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGTGTGGTGGTGGCCGTGTCCCAGGAGGACCCCGAGGTGCAGTTTAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGGGAGGAGCAGTTTAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCTCCATCGAGAAGACCATCTCCAAGGCTAAGGGCCAGCCCAGGGAGCCTCAGGTGTACACCCTGCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAATAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGATCCTTCTTTCTGTATAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTGTTTAGCTGTTCCGTGATGCACGAGGCTCTGCACAATCACTATACCCAGAAGTCCCTGTCCCTGTCCCTCGGCAAGTGATAA(SEQ IDNO:31)

[0189] Codon group 3 heavy chain variable region:

[0190] CAGGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAGCCTGGCGCCTCCGTGAAGGTGAGCTGCAAGGCTTCCGGCTTCAACATCCAGAATACCTACATCCACTGGGTGAGGCAGGCTCCCGGCCAGGGACTGGAGTGGATGGGCAGAATCGACCCCGCTTCCGGCTCCACCGAGTATGCCCCCAAGTTTCAGGGCAGGGTGACCATGACCGCCGATACCTCCACCAATACCGGCTATATGGAGCTGAGCAGCCTGAGGTCCGAGGATACCGCTGTGTACTATTGCGCCCGGTACGGCAATTTTCTGTATTACTACTCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCC(SEQ IDNO:32)

[0191] Codon group 3 light chain:

[0192] GATATCGTGATGACCCAGAGCCCTCTGTCCCTGCCCGTGACCCTGGGCCAGCCTGCTAGCATCAGCTG TAGGTCCAGCAAGAGCCTGCTGCACAGCAACGCTAATACCTACCTGTACTGGTTTCAGCAGAGGCCCGGCCAGAGC CCTCGGTTGCTGATCTACCGGATGAGCAACCTGGCTAGCGGCGTGCCCGACCGGTTTAGCGGCAGCGGATCCGGCA CCGACTTTACCCTGAAGATCTCCCGGGTGGAGGCTGAGGACGTGGGCGTGTACTACTGTATGCAGCACCTGGAGAA TCCCTATACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG AGGACCGTGGCTGCTCCTTCCGTGTTTATCTTTCCCCCCTCCGATGAGCAGCTGAAGTCCGGCACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCTAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAGAGCGGCAACAGCCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACTCCACCTATTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCTGATTACGAGAAGCACAAGGTGTATGCCTGCGAGGTGACCCACCAGGGCCTGTCCTCCCCCGTGACCAAGTCCTTCAACAGGGGCGAGTGTTGATAA(SEQ ID NO:33)

[0193] Codon group 3 light chain variable region:

[0194] GATATCGTGATGACCCAGAGCCCTCTGTCCCTGCCCGTGACCCTGGGCCAGCCTGCTAGCATCAGCTGTAGGTCCAGCAAGAGCCTGCTGCACAGCAACGCTAATAACCTACCTGTACTGGTTTCAGCAGAGGCCCGGCCAGAGCCCTCGGTTGCTGATCTACCGGATGAG CAACCTGGCTAGCGGCGTGCCCGACCGGTTTAGCGGCAGCGGATCCGGCACCGACTTTACCCTGAAGATCTCCCGGGTGGAGGCTGAGGACGTGGGCGTGTACTACTGTATGCAGCACCTGGAGAATCCCTATACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG(SEQ ID NO:34)

[0195] Table 4: CDR coding sequences of codon group 3

[0196]

[0197] Sequence alignment revealed the following sequence homologies for different codon sets: for the full-length antibody nucleic acid sequence, the codon set 2 heavy chain shared 84.0% identity with the codon set 1 heavy chain, and the codon set 3 heavy chain shared 88.1% identity with the codon set 1 heavy chain. The codon set 2 light chain shared 83.1% identity with the codon set 1 light chain, and the codon set 3 light chain shared 87.3% identity with the codon set 1 light chain.

[0198] For the variable regions, the heavy chain variable region of codon set 2 was 84.8% identical to that of codon set 1, and the heavy chain variable region of codon set 3 was 86.1% identical to that of codon set 1. The light chain variable region of codon set 2 was 80.1% identical to that of codon set 1, and the light chain variable region of codon set 3 was 87.9% identical to that of codon set 1.

[0199] Example 3. Plasmid construction

[0200] Nucleic acid sequences encoding the amino acids of the light and heavy chains of the anti-NKG2A antibody hu-43VL(N26S-G34A) were synthesized separately. The nucleic acid sequences encoding the light and heavy chains containing signal peptides were recombined into the pcDNA3.4 vector (signal peptide + heavy chain, signal peptide + light chain) using the restriction enzyme sites AflII and EcoRV, respectively. The recombinant plasmids pcDNA3.4-hu-43VL(N26S-G34A)-HC and pcDNA3.4-hu-43VL(N26S-G34A)-LC were constructed. The plasmid maps of pcDNA3.4-hu-43VL(N26S-G34A)-HC and pcDNA3.4-hu-43VL(N26S-G34A)-LC are shown in. Figure 1 and Figure 2 .

[0201] The light and heavy chains of the antibody use the same signal peptide sequence, which is as follows:

[0202] Signal peptide of hu-43VL(N26S-G34A) antibody:

[0203] MHSSALLCCLVLLTGVRA(SEQ ID NO:41)

[0204] Nucleic acid sequence encoding signal peptide:

[0205] ATGCACAGCAGCGCCCTGCTTGTGCTGCCTGGTGCTGCTGACCGGCGTGAGAGCC

[0206] (SEQ ID NO:42).

[0207] The six recombinant plasmids with different nucleic acid sequences were named pcDNA3.4-Codon 1-HC, pcDNA3.4-Codon 1-LC, pcDNA3.4-Codon 2-HC, pcDNA3.4-hu-Codon 2-LC, pcDNA3.4-Codon 3-HC, and pcDNA3.4-Codon 3-LC. The light and heavy chain plasmid maps were consistent. The recombinant plasmids were transformed into Escherichia coli DH5α (Takara, 9057) and amplified. The recombinant plasmids were isolated and sequenced to confirm their identity. The transformed E. coli were then stored at -20°C for subsequent transient transfection experiments.

[0208] Example 4. Plasmid amplification and extraction

[0209] The recombinant plasmid obtained in Example 3 was transformed into Escherichia coli and cultured using LB medium. According to the resistance of the pcDNA3.4 plasmid, ampicillin (Shanghai Sangon, A100339) was added to the LB medium for amplification.

[0210] E. coli cells were harvested by centrifugation after expansion and lysed using an alkaline lysis method using a plasmid extraction kit (Axygen, AP-MX-P-25). Endotoxins were removed during the process (Shanghai Sangon, B641718), and plasmids were extracted. Plasmid concentrations were measured using a microspectrophotometer (Hangzhou Aosheng, Nano-300) and stored at -20°C for subsequent transient transfection experiments.

[0211] Example 5. Plasmid transfection into CHO cells

[0212] Take out 1 CHO-K1 (ATCC, product number CCL-61 TM ) Frozen cells were thawed in a 37°C water bath. Cell count was performed at 0.5×10 6 The cells were passaged at a viable cell density of 0.2 × 10 cells / mL in complete CDCHO medium (Gibco, 10743029) containing 6 mmol / L L-Gln (Sigma, V900419-500G). CHO-K1 cells were cultured in a humidified CO2 shaker incubator (Kuhner, ISF4-XC) at 37°C and 5% CO2. Subculture was performed every 2-4 days at a viable cell density of 0.2 × 10 cells / mL. 6 ~0.5×10 6 cells / mL. After cell viability recovered, cells were transferred to Dynamis complete medium (Dynamis medium + 6mmol / L L-Gln, Gibco, A26175) for passage. 24 hours before transfection, cells were passaged in Dynamis complete medium at a viable cell density of 3×10 6 ~4×10 6 cells / mL.

[0213] On the day of transient transfection, the viable cell density was adjusted to 6 × 10 cells / mL using Dynamis complete medium. 6cells / mL. The transient transfection system consisted of 10.5 μg PEI (Polyscoences, 23966), 1.75 μg recombinant heavy chain plasmid, 1.75 μg recombinant light chain plasmid, and 2.625 μg inert DNA (Sigma, 31149-10G-F) per mL of cell culture medium. Four hours after transient transfection, the total volume was supplemented with 4% Cell Boost 7a solution (Cytiva, SH31026.02), 0.4% Cell Boost 7b solution (Cytiva, SH31027.07CN), and 0.125% DMA. On the first day of transient transfection, supplement the culture medium with D-glucose to 7 g / L and cool to 32°C. On the second day of transient transfection, supplement the culture medium with 2 mM L-Gln. On the fifth day of transient transfection, supplement the culture medium with 6% Cell Boost 7a solution, 0.6% Cell Boost 7b solution, and 4 mM L-Gln. On the seventh day of transient transfection, harvest the culture supernatant of the transiently transfected cells. Harvest the supernatant for antibody expression analysis and one-step affinity purification.

[0214] Example 6. Purification of antibodies

[0215] The antibody was purified using a one-step affinity chromatography method, which included centrifugation (12,000 g for 10 min) to collect the culture supernatant of cells transiently transfected with the recombinant plasmid. The supernatant was then purified using a Protein A affinity column (Cytiva, 17543803) on a protein purification chromatography system (Cytiva, AKTA avant). The target protein was eluted using pH 3.0 acetic acid solution (McLean, A801295). The purified antibody samples were subjected to SEC-HPLC, reduced / non-reduced CE-SDS, and iCIEF quality control (see Example 7 for details).

[0216] Example 7. Detection of Antibody Expression

[0217] Detect the expression level of the antibody using HPLC:

[0218] Based on the Protein A one-step affinity chromatography method, the antibody was eluted using a low pH solution. The areas of the standard and sample elution peaks were calculated to establish a linear relationship between the standard concentration and the chromatographic peak area. The antibody concentration in the sample was calculated based on the standard linear relationship and the sample peak area.

[0219] Instrument: High Performance Liquid Chromatograph (Agilent, 1260 Infinity II Prime). Chromatographic Column: Thermo Fish POROS A / 20, 1502412 chromatographic column, size 2.1*30 mm.

[0220] Detection mobile phase A: 50 mM Na2HPO4 (Sinopharm, 10020318) and 150 mM NaCl (Sinopharm, 10019318), pH 7.0; flow rate: 3.0 mL / min.

[0221] Detection mobile phase B: 150 mM NaCl (Sinopharm, 10019318), pH 1.9; flow rate: 3.0 mL / min.

[0222] Injection volume: 80 μL; Column temperature: 25.0 ± 5.0°C; Sample room temperature: 2-8°C; Detection wavelength: 280 nm, detection time: 3 min.

[0223] During the test, the column was first equilibrated using 100% mobile phase A at a flow rate of 3.0 mL / min until the baseline was stable. The sample was then injected, the sample concentration was determined, and the chromatogram was recorded. The sample was determined using gradient elution, as shown in Table 5:

[0224] Table 5: Chromatographic elution gradient for protein content determination by HPLC

[0225] Time (min) 0 0.50 0.51 1.50 1.51 3.00 A(％) 100 100 0 0 100 100 B(％) 0 0 100 100 0 0

[0226] The protein content was obtained by integrating the chromatogram and analyzing the peak area of ​​the target peak of the standard curve solution and the test solution. The results are shown in Table 6 and Figure 3 :

[0227] Table 6: Antibody expression levels in different codon groups

[0228] codon Codon group 1 Codon group 2 Codon group 3 Expression level (g / L) 0.237 0.104 0.076

[0229] The expression results show that codon group 1 has the highest expression level, reaching 0.237 g / L, which is significantly higher than codon group 2 and codon group 3.

[0230] Example 8. Antibody quality detection

[0231] 8.1SEC-HPLC method

[0232] Instrument: High-performance liquid chromatograph (Agilent, 1260 Infinity II Prime). Chromatographic column: TOSOHG3000SWxL column, specifications: 7.8*300 mm, 5 μm.

[0233] The detection mobile phase was composed of 0.2 M sodium chloride (Sinopharm, 10019318), 0.04 M sodium dihydrogen phosphate dihydrate (Sinopharm, 20040718) and 0.04 M disodium hydrogen phosphate dodecahydrate (Sinopharm, 10020318), pH 7.0.

[0234] Injection volume: 25 μL; Column temperature: 25.0 ± 5.0°C; Sample room temperature: 2-8°C; Detection wavelength: 280 nm; Detection time: 30 min.

[0235] During the detection, the chromatographic column was first balanced, that is, the system was balanced with 100% mobile phase at a flow rate of 0.5 mL / min until the baseline was stable, and then the sample was injected, the sample was measured, and the chromatogram was recorded.

[0236] The chromatogram was integrated and the peak area of ​​the target peak was analyzed and calculated to obtain the aggregate content and monomer peak content.

[0237] 8.2 Reducing / Non-reducing CE-SDS Method

[0238] Reduced CE-SDS samples were diluted to 1.0 mg / mL in diluent (pH 6.5, containing 0.04 M sodium dihydrogen phosphate dihydrate, 0.04 M sodium hydrogen phosphate dodecahydrate, and 1% SDS). 5% mercaptoethanol (Sigma, M3148) was added and mixed thoroughly. The mixture was incubated at 70°C in a water bath for 15 minutes. After cooling, the mixture was centrifuged at 6000 rpm for 1 minute, and 75 μL of the supernatant was used as the loading sample.

[0239] The non-reduced CE-SDS sample was diluted to 1.0 mg / mL in diluent (pH 6.5, containing 0.04 M sodium dihydrogen phosphate dihydrate, 0.04 M sodium hydrogen phosphate dodecahydrate, and 1% SDS). 800 mM iodoacetamide (TCI, I0044) was added at 5% of the total volume and mixed thoroughly. The mixture was incubated at 70°C in a water bath for 5 minutes. After cooling, the sample was centrifuged at 6000 rpm for 1 minute, and 75 μL of the supernatant was used as the loading sample.

[0240] Instrument: Capillary electrophoresis instrument (Agilent, 7100). Chromatographic column: CE-SDS capillary (Agilent, G1600-6411), column length 224 mm / 310 mm.

[0241] Sample buffer: 0.04 M sodium dihydrogen phosphate dihydrate, 0.04 M disodium hydrogen phosphate dodecahydrate and 1% SDS, pH 6.5.

[0242] Running buffer: SCIEX gel.

[0243] Injection: injection - 10 kV, 30 / 40 s (reducing / non-reducing), run - 15 kV, 40 min; column temperature: 20 °C.

[0244] Sample tray temperature: 18-22°C. DAD detection wavelength: 220 nm / 360 nm, detection time: 40 min.

[0245] The chromatogram was integrated and the peak area ratio of the target peak was calculated by analysis to obtain the low molecular weight fragment content and monomer peak content.

[0246] 8.3.iCIEF Method

[0247] Instruments: Fully automatic protein capillary electrophoresis analysis system (Protein Simple, KF-1757), iCIEF cartridge (Protein Simple, PS-MC02-C).

[0248] Reagents: 1% Methyl Cellulose (Protein Simple, 101876), iCE Electrolyte Kit (Protein Simple, 102506), Ampholytes 3-10 (Cytiva, 17045601), Ampholytes 8-10.5 (Cytiva, 17045501), Pi marker pI=6.61 (Protein Simple, 102409), Pi marker pI=9.50 (Protein Simple, 101996), Urea (Sigma, U5128), L-Arg (Pfanstiehl, A-70).

[0249] The sample was replaced with ultrapure water three times and finally diluted with ultrapure water to 1 mg / mL.

[0250] Prepare the sample solution as shown below, mix thoroughly, and centrifuge at 12,000 rpm for 3 minutes. Add 80 μL of the supernatant to a 96-well plate for detection.

[0251] Table 7: iCIEF solution configuration

[0252] Reagents volume 1% Methyl Cellulose 35μL Amphoteric electrolytes 3-10 3.5 μL Ampholytes 8~10.5 1.5 μL Marker: pI = 6.61 0.25 μL Marker: pI = 9.50 0.25 μL 1 mg / mL sample solution / blank control 25 μL 6M Urea-20mM L-Arg solution 34.5μL

[0253] The isoelectric focusing electrophoresis procedure is shown in the following table:

[0254] Table 8: iCIEF instrument procedures

[0255] step Experimental conditions Focus on the first time period 1min, 1500V Focus on the second time period 9min, 3000V Loading 55s

[0256] Autosampler temperature: 10°C.

[0257] The chromatogram was integrated and the contents of the charge variant acidic peak, main peak and basic peak were obtained by analyzing and calculating the peak area of ​​the target peak of the test solution.

[0258] The quality of hu-43VL(N26S-G34A) antibody expressed with different codon sets is shown in the table below:

[0259] Table 9-1: Antibody quality results expressed by different codon groups

[0260]

[0261] Table 9-2: Antibody quality results expressed by different codon groups

[0262]

[0263] The SEC-HPLC data showed that the aggregates of hu-43VL (N26S-G34A) antibody expressed in codon group 3 were relatively high; the non-reducing CE-SDS data showed that the fragments of antibodies expressed in codon groups 2 and 3 were relatively high;

[0264] Reduction of CE-SDS data showed that the expression ratios of antibody light and heavy chains expressed by the three codons were consistent. The proportions of antibody light chains plus glycosylated heavy chains expressed by codon group 1 and codon group 2 were similar, while the proportion of antibody light chains plus glycosylated heavy chains expressed by codon group 3 was slightly lower.

[0265] The iCIEF data showed that the isoelectric point of the main peak of the protein was 7.5. The acidic peak content of the antibody expressed by codon group 1 was significantly lower than that of the antibodies expressed by codon group 2 and codon group 3, and the main peak content was significantly higher than that of the antibodies expressed by codon group 2 and codon group 3.

[0266] In summary, considering the expression level, polymer ratio, low molecular weight fragment ratio and charge variant, codon set 1 is superior to codon set 2 and codon set 3, so codon set 1 is preferred.

[0267] Example 9. Antibody Binding Activity Detection

[0268] The hu-43VL (N26S-G34A) antibody was prepared using the above example. Human NKG2A-overexpressing cells (CHOK1-hNKG2A / CD94) and monkey NKG2A-overexpressing cells (CHOK1-cynoNKG2A / CD94) were digested with TrypLE (purchased from Gibco). Cells were harvested by centrifugation and resuspended to 2E6 / mL in FACS buffer (PBS + 2% FBS). 100 μL of cells were added to each well of a 96-well plate and centrifuged to remove the supernatant. 100 μL of hu-43VL (N26S-G34A) antibody and control antibody Z270 were serially diluted in FACS buffer (starting at 200 nM, with 5-fold dilutions, including 0, for a total of 10 concentrations). The cells were then added to the cells at 100 μL / well, mixed, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. Add secondary antibody: anti-human IgG fluorescent secondary antibody 488 (purchased from Thermofisher A-11013), incubate at 4℃ for one hour, wash twice with FACS buffer, resuspend in PBS and analyze with FACS instrument. Figure 4A and 4B .

[0269] The results showed that hu-43VL(N26S-G34A) antibody could bind to CHOK1-hNKG2A / CD94 and CHOK1-cynoNKG2A / CD94 cells with high activity.

[0270] Example 10. Antibody-mediated killing of target cells by NK92 cells

[0271] Target B lymphocyte blasts (LCL721.221) were harvested by centrifugation and resuspended at 2E6 / mL in 1640 medium supplemented with 10% fetal bovine serum. 0.5 mL of the target cell suspension was added to a 24-well plate, which was then incubated at 37°C overnight. NK92 cells (purchased from Enzyme Biotechnology) were harvested by centrifugation, washed twice with PBS, and counted using trypan blue staining to adjust the cell density to 4E5 / mL. 50 μL of the NK92 cell suspension was added to a 96-well plate. The test antibody was diluted in 1640 medium supplemented with 10% fetal bovine serum, and 50 μL of the diluted antibody was added to the 96-well plate (starting at 200 nM, diluted 5-fold, including the zero concentration, for a total of 10 concentrations). The plate was incubated at 37°C for 0.5 hour. Target cells were harvested by centrifugation, washed twice with PBS, and resuspended at 1E6 / mL in 1640 medium supplemented with 10% fetal bovine serum. Add the marker BATDA (purchased from PerkinElmer) (2 μL / mL) to the target cell suspension, and place the target cells in a 37°C incubator for labeling for 20 minutes. Wash the labeled target cells 4 times by centrifugation, and resuspend the cells to 1E5 / mL in 1640 culture medium containing 10% fetal bovine serum. Add 100 μL of target cell suspension to a 96-well culture plate, and place the culture plate in a 37°C incubator for incubation for 2 hours. Centrifuge the culture plate at 300g for 5 minutes, take 25 μL of culture supernatant to another 96-well flat-bottom plate, and add 200 μL of Eu-solution (purchased from PerkinElmer). Read the data using an Envision instrument and analyze the data using Graphpad software. See the results. Figure 5 .

[0272] The results showed that the hu-43VL (N26S-G34A) antibody had a high killing effect on target cells, and the killing activity was comparable to that of the positive control Z270 antibody.

[0273] Example 11. Antibody-mediated killing of target cells by primary NK cells

[0274] Primary NK cells were isolated from PBMC (purchased from Miaoshun Bio) using an NK cell isolation kit and induced for 5-7 days to a cell density of 2E6 / mL. LCL721.221 cells were collected by centrifugation and resuspended to 2E6 / mL in 1640 medium (containing 1mM inducing peptide) containing 10% fetal bovine serum. 0.5mL of target cell suspension was added to a 24-well plate, and the 24-well plate was placed in a 37°C incubator overnight. Primary NK cells were collected by centrifugation, washed twice with PBS, and counted by trypan blue staining to adjust the cell density to 4E5 / mL. 50μL of NK cell suspension was added to a 96-well plate. Dilute the hu-43VL (N26S-G34A) antibody to be tested in 1640 medium supplemented with 10% fetal bovine serum. Add 50 μL of the diluted hu-43VL (N26S-G34A) antibody to a 96-well plate (starting at 200 nM, using 5-fold dilutions, including the 0-point assay, for a total of 10 concentrations). Incubate the plate at 37°C for 0.5 hours. Collect LCL721.221 cells by centrifugation, wash twice with PBS, and resuspend at 1E6 / mL in 1640 medium supplemented with 10% fetal bovine serum. Add the marker BATDA (purchased from PerkinElmer) (2 μL / mL) to the LCL721.221 cell suspension, and label the target cells in a 37°C incubator for 20 minutes. Wash the LCL721.221 cells four times by centrifugation and resuspend at 1E5 / mL in 1640 medium supplemented with 10% fetal bovine serum. 100 μL of LCL721.221 cell suspension was added to a 96-well culture plate and incubated in a 37°C incubator for 2 hours. The plate was centrifuged at 300 g for 5 minutes, and 25 μL of the culture supernatant was transferred to another 96-well flat-bottom plate. 200 μL of Eu-solution was added. Data were read using an Envision instrument and analyzed using Graphpad software. Results are shown in the table. Figure 6 .

[0275] The results showed that primary NK cells mediated by hu-43VL (N26S-G34A) antibody had a killing effect on LCL721.221 cells, and the killing activity was significantly better than the control molecule Z270, indicating that it has good drug development prospects.

Claims

1. A nucleic acid molecule comprising nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of an anti-NKG2A antibody, and nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3 of the light chain variable region of an anti-NKG2A antibody, wherein: The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 15, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 16, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 17; or The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 26, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 27; or The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 35, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 36, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 37; and The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 18, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 19, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 20; or The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:28, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:29, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:30; or The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:38, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:39, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:40; Preferably, The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 15, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 16, the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 17, and The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 18, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 19, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 20; or The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 26, the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 27, and The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:28, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:29, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:30; or The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 35, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 36, the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 37, and The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:38, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:39, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:

40.

2. The nucleic acid molecule according to claim 1, wherein The nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of an anti-NKG2A antibody, wherein: the nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 32, or has at least 90% sequence identity to SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 32, and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 14, SEQ ID NO: 24, or SEQ ID NO: 34, or has at least 90% sequence identity to SEQ ID NO: 14, SEQ ID NO: 24, or SEQ ID NO: 34; Preferably, The nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 12, or has at least 90% sequence identity thereto, and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO: 14, or has at least 90% sequence identity thereto; or The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 22, or has at least 90% sequence identity thereto, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 24, or has at least 90% sequence identity thereto; or The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 32, or has at least 90% sequence identity thereto, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 34, or has at least 90% sequence identity thereto.

3. The nucleic acid molecule according to claim 1 or 2, wherein The nucleic acid molecule comprises a nucleic acid sequence encoding a human antibody heavy chain constant region and a light chain constant region; preferably, The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31, or has at least 85% sequence identity to SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31, and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33, or has at least 85% sequence identity to SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33; More preferably, the nucleic acid molecule comprises nucleic acid sequences encoding the heavy and light chains of an anti-NKG2A antibody, wherein: The nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO: 11, or has at least 85% sequence identity thereto, and the nucleic acid sequence encoding the light chain is as shown in SEQ ID NO: 13, or has at least 85% sequence identity thereto; or, The nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO: 21, or has at least 85% sequence identity thereto, and the nucleic acid sequence encoding the light chain is as shown in SEQ ID NO: 23, or has at least 85% sequence identity thereto; or, The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO: 31, or has at least 85% sequence identity thereto, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO: 33, or has at least 85% sequence identity thereto. A recombinant vector comprising the nucleic acid molecule according to any one of claims 1 to 3.

5. A vector system for expressing an anti-NKG2A antibody, comprising a first vector and a second vector, wherein: The first vector comprises nucleic acid sequences encoding HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of an anti-NKG2A antibody, wherein: The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 15, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 16, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 17; or The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 26, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 27; or The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 35, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 36, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 37; and The second vector comprises nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3 of the light chain variable region of an anti-NKG2A antibody, wherein: The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 18, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 19, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 20; or The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:28, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:29, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:30; or The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:38, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:39, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:40; Preferably, The first vector comprises nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 15, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 16, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 17; and The second vector comprises nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3 of the light chain variable region of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 18, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 19, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 20; or, The first vector comprises nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 26, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 27; and The second vector comprises nucleic acid sequences encoding LCDR1, LCDR2, and LCDR3 of the light chain variable region of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO: 28, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO: 29, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO: 30; or, The first vector comprises nucleic acid sequences encoding HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of an anti-NKG2A antibody, wherein the nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 35, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 36, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 37; and The second vector contains nucleic acid sequences encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of the anti-NKG2A antibody, wherein the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:38, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:39, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:

40.

6. The carrier system according to claim 5, wherein The first vector comprises a nucleic acid sequence encoding the heavy chain variable region of an anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of an anti-NKG2A antibody, wherein: The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 32, or has at least 90% sequence identity to SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 32; and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 14, SEQ ID NO: 24, or SEQ ID NO: 34, or has at least 90% sequence identity to SEQ ID NO: 14, SEQ ID NO: 24, or SEQ ID NO: 34; Preferably, The nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 12, or has at least 90% sequence identity to SEQ ID NO: 12; and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO: 14, or has at least 90% sequence identity to SEQ ID NO: 14; or The nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 22, or has at least 90% sequence identity to SEQ ID NO: 22; and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO: 24, or has at least 90% sequence identity to SEQ ID NO: 24; or The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 32, or has at least 90% sequence identity to SEQ ID NO: 32; and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 34, or has at least 90% sequence identity to SEQ ID NO:

34.

7. The vector system according to claim 5 or 6, wherein The first vector comprises a nucleic acid sequence encoding the heavy chain of an anti-NKG2A antibody, and the second vector comprises a nucleic acid sequence encoding the light chain of an anti-NKG2A antibody, wherein: The nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31, or has at least 85% sequence identity to SEQ ID NO: 11, SEQ ID NO: 21 or SEQ ID NO: 31; and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33, or has at least 85% sequence identity to SEQ ID NO: 13, SEQ ID NO: 23 or SEQ ID NO: 33; Preferably, The nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO: 11, or has at least 85% sequence identity to SEQ ID NO: 11; and the nucleic acid sequence encoding the light chain is as shown in SEQ ID NO: 13, or has at least 85% sequence identity to SEQ ID NO: 13; or The nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO: 21, or has at least 85% sequence identity to SEQ ID NO: 21; and the nucleic acid sequence encoding the light chain is as shown in SEQ ID NO: 23, or has at least 85% sequence identity to SEQ ID NO: 23; or The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO: 31, or has at least 85% sequence identity to SEQ ID NO: 31; and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO: 33, or has at least 85% sequence identity to SEQ ID NO:

33.

8. A recombinant cell comprising the nucleic acid molecule of any one of claims 1 to 3, or the recombinant vector of claim 4, or the vector system of any one of claims 5 to 7; preferably, the host cell of the recombinant cell is a CHO, Expicho-S, CHO-S, CHO-K1 or CHO DG44 cell, more preferably a CHO-K1 cell.

9. Use of the nucleic acid molecule according to any one of claims 1 to 3, or the recombinant vector according to claim 4, or the vector system according to any one of claims 5 to 7, or the recombinant cell according to claim 8 in the preparation of an anti-NKG2A antibody.

10. A method for preparing an anti-NKG2A antibody, comprising culturing the recombinant cell according to claim 8 to express the anti-NKG2A antibody; preferably, the method further comprises purifying and recovering the antibody; or The method comprises the following steps: a. introducing the nucleic acid molecule according to any one of claims 1 to 3, the recombinant vector according to claim 4, or the vector system according to any one of claims 5 to 7 into a host cell; b. culturing host cells to express anti-NKG2A antibodies; and optionally, c. Purification and recovery of anti-NKG2A antibodies.

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