Anti-nkg2a antibodies and uses thereof

Abstract

Provided are anti-NKG2A antibodies or antigen-binding fragments thereof, and applications thereof, the antibodies or antigen-binding fragments thereof comprising a VH-CDR1, a VH-CDR2, a VH-CDR3, a VL-CDR1, a VL-CDR2, and a VL-CDR3. The antibodies or antigen-binding fragments thereof have potential for treating tumors, and provide a selection for clinically tumor immunotherapy drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to anti-NKG2A antibodies. Background Technology

[0002] NKG2A, also known as the natural killer cell lectin-like receptor, is an inhibitory receptor in the NKG2 receptor family, primarily expressed on NK cells and CD8 cells. + On the surface of T cells, Th2 cells, and NKT cells, the heterodimeric complex NKG2A-CD94, formed by NKG2A and CD94 (NK cell surface membrane protein) linked by disulfide bonds, is recognized by HLA-E, a non-classical major histocompatibility complex class I (MHC I) molecule on target cells. 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 conditions, but HLA-E is highly expressed in various tumor cells, and NK and CD8+ cells are also highly expressed at tumor infiltration sites. + T cells highly express NKG2A, enabling tumor cells to evade NK and CD8. + T-cell immune recognition. Additionally, viral infection can induce host cells to express NK and CD8 receptors. + T cells bind to the ligand of the NKG2A inhibitory receptor, evading clearance by 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 cells by blocking this inhibitory signal, thus playing an anti-tumor role. Summary of the Invention

[0003] This invention provides an antibody that specifically binds to NKG2A on the surface of human NK92 cells and blocks the NKG2A and HLA-E signaling pathways, significantly increasing the killing effect of NK92 cells or primary NK cells on HLA-E-positive tumor cells. It exhibits good anti-tumor activity and superior functional activity compared to known anti-NKG2A antibodies. Therefore, the antibody of this invention has the potential to treat tumors, providing an option for clinical tumor immunotherapy.

[0004] In a first aspect, the present invention provides an anti-NKG2A antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDRs: VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises three CDRs: VL-CDR1, VL-CDR2, and VL-CDR3; wherein... The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 2; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 3; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 4; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 6; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 7; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 8; or, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 10; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 11; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 12; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 14; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 15; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 18; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 19; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 20; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 22; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 23; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 24; or, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 26; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 27; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 28; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 30; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 31; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 32; or, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 34; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 35; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 36; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 38; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 39; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 40; or, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 42; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 43; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 44; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 46; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 47; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 48; or, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 50; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 51; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 52; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 54; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 55; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 56; or, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 58; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 59; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 60; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 62; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 63; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 64; or The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 50; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 51; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 52; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 68; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 55; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 56; When the amino acid sequence of VL-CDR1 is as shown in SEQ ID NO: 68, X1 is T, S, Q, A, D, G, E, F, I, K, L, P, V, W, Y, R or H; X2 is N, Q, S or T; X3 is G, A, L, E, K, R, S, N, T or Q; wherein, one and only one of the two conditions, X2 being N and X3 being G, must be present.

[0005] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three CDRs: VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region includes three CDRs: VL-CDR1, VL-CDR2, and VL-CDR3; wherein, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 50; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 51; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 52; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 or 160; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 55; and The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 56.

[0006] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three CDRs: VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region includes three CDRs: VL-CDR1, VL-CDR2, and VL-CDR3; wherein, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 50; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 51; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 52; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 104; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 55; and The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 56.

[0007] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three CDRs: VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region includes three CDRs: VL-CDR1, VL-CDR2, and VL-CDR3; wherein, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 58; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 59; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 60; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 62; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 63; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO: 64.

[0008] In some embodiments, the present invention provides an anti-NKG2A antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a variable heavy chain region and a variable light chain region, wherein: The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 1, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 5; or The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 9, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 13; or The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 17, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 21; or The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 25, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 29; or The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 33, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 37; or The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 41, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 45; or The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 49, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 53; or The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 57, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 61; or The heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 65, 85 or 86, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 66, 87, 88, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159; or The heavy chain variable region includes three complementary determinant regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 89, 90 or 91, and the light chain variable region includes three complementary determinant regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 92, 93 or 94.

[0009] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 65, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 103.

[0010] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 91, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 92.

[0011] Those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondences between different numbering systems are well known to those skilled in the art. In some embodiments, the CDRs are defined according to Kabat, IMGT, Chothia, AbM, or Contact numbering rules.

[0012] In a preferred embodiment, the heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:1; the light chain variable region sequence is as shown in SEQ ID NO:5, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:5; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 9, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 9; the light chain variable region sequence is as shown in SEQ ID NO: 13, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 13; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 17, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 17; the light chain variable region sequence is as shown in SEQ ID NO: 21, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 21; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 25, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 25; the light chain variable region sequence is as shown in SEQ ID NO: 29, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 29; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 33, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 33; the light chain variable region sequence is as shown in SEQ ID NO: 37, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 37; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 41, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 41; the light chain variable region sequence is as shown in SEQ ID NO: 45, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 45; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 57, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 57; the light chain variable region sequence is as shown in SEQ ID NO: 61, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 61; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 65; the light chain variable region sequence is as shown in SEQ ID NO: 66, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 66; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 65; the light chain variable region sequence is as shown in SEQ ID NO: 67, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 67. When the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 67, X1 is T, S, Q, A, D, G, E, F, I, K, L, P, V, W, Y, R, or H; X2 is N, Q, S, or T; X3 is G, A, L, E, K, R, S, N, T, or Q; wherein, one and only one of the two conditions, X2 being N and X3 being G, must be present, or... The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 85, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 85; the light chain variable region sequence is as shown in SEQ ID NO: 87, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 87; or The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 86, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 86; the light chain variable region sequence is as shown in SEQ ID NO: 87, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 87; or The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 65; the light chain variable region sequence is as shown in SEQ ID NO: 88, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 88; or The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 89, 90 or 91, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 89, 90 or 91, and the light chain variable region sequence is as shown in SEQ ID NO: 92, 93 or 94, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 92, 93 or 94.

[0013] In some embodiments, the heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, and the light chain variable region sequence is as shown in SEQ ID NO: 66, 87, 88, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159.

[0014] In some embodiments, the heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 85, and the light chain variable region sequence is as shown in SEQ ID NO: 66, 87, 88, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159.

[0015] In some embodiments, the heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:86, and the light chain variable region sequence is as shown in SEQ ID NO:66,88,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157 or 159.

[0016] In some embodiments, the heavy chain variable region sequence of the antibody or its antigen-binding fragment is shown as SEQ ID NO: 89, 90 or 91, and the light chain variable region sequence is shown as SEQ ID NO: 92, 93 or 94.

[0017] In some embodiments, the heavy chain variable region sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO:65, and the light chain variable region sequence is shown in SEQ ID NO:103.

[0018] In some embodiments, the heavy chain variable region sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO:91, and the light chain variable region sequence is shown in SEQ ID NO:92.

[0019] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment further includes a human or mouse frame region, and / or the light chain variable region of the antibody or its antigen-binding fragment further includes a human or mouse frame region.

[0020] In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; the heavy chain constant region and / or light chain constant region is human or mouse-derived.

[0021] In some embodiments, the heavy chain constant region of the antibody is the heavy chain constant region of human IgG4. In some embodiments, the heavy chain constant region of the antibody contains S228P, L235E, D265A, and / or R409K mutations. In some embodiments, the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO: 95 or 161. In some embodiments, the light chain constant region contains the amino acid sequence shown in SEQ ID NO: 96.

[0022] In a preferred embodiment, the antibody or its antigen-binding fragment is an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.

[0023] In some embodiments, the anti-NKG2A antibody or its antigen-binding fragment, wherein the antibody comprises a heavy chain and a light chain, wherein, The heavy chain has an amino acid sequence as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 162 or 166, and the light chain has an amino acid sequence as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 163.

[0024] In some embodiments, the anti-NKG2A antibody or its antigen-binding fragment, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 164 or 167, or contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 164 or 167, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 165, or contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 165.

[0025] In some embodiments, the anti-NKG2A antibody or its antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163.

[0026] In some embodiments, the anti-NKG2A antibody or its antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 164, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 165.

[0027] In some embodiments, the anti-NKG2A antibody or its antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163.

[0028] In some embodiments, the anti-NKG2A antibody or its antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 167, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 165.

[0029] In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody, a double-chain antibody, a single-chain antibody, a bispecific antibody, a single-domain antibody, or a multispecific antibody.

[0030] In some embodiments, the antibody or its antigen-binding fragment is a full-length antibody protein or an antigen-binding fragment.

[0031] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', and (Fab'). 2 Fv, Fd, scFv and dAb are connected by disulfide bonds.

[0032] In the second aspect, a biomaterial is provided, which is: 1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention; preferably, the nucleic acid molecule encodes the heavy chain variable region and / or light chain variable region of the antigen or antigen-binding fragment thereof; more preferably, the nucleic acid molecule encodes the heavy chain variable region, the nucleotide sequence of which is shown in SEQ ID NO: 69, 71, 73, 75, 77, 79, 81 or 83; and / or, the nucleic acid molecule encodes the light chain variable region, the nucleotide sequence of which is shown in SEQ ID NO: 70, 72, 74, 76, 78, 80, 82 or 84; 2) A vector comprising the nucleic acid molecule of 1) above; preferably, the vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector; 3) A cell comprising the nucleic acid molecule of 1) above or the carrier of 2) above; preferably, the cell expresses the antibody of the present invention or its antigen-binding fragment. This cell cannot develop into an individual.

[0033] Thirdly, a method for preparing the antibody or its antigen-binding fragment of the present invention is provided, comprising: 1) Chemical synthesis method, synthesized based on the amino acid sequence of the antibody or its antigen-binding fragment; or, 2) Bioprocessing, including culturing the cells described in the second aspect above and harvesting the antibody or its antigen-binding fragment.

[0034] In a fourth aspect, a composition is provided comprising the anti-NKG2A antibody of the present invention or an antigen-binding fragment thereof, or biological material.

[0035] In some embodiments, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

[0036] In some embodiments, the pharmaceutical composition is a liquid formulation; in some embodiments, the pharmaceutical composition is an injectable formulation; in some embodiments, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody or antigen-binding fragment of the present invention, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0037] Fifthly, a kit is provided comprising the antibody or antigen-binding fragment thereof of the present invention, biological material, or antibody composition; said kit is used for diagnosing or detecting NKG2A protein in an in vitro sample or cells expressing NKG2A protein.

[0038] In a sixth aspect, the invention provides applications of the antibody or its antigen-binding fragment, biological material, or antibody composition, wherein the applications are: 1) To prepare drugs for the prevention and / or treatment of diseases associated with abnormal NKG2A expression or function; or 2) Prepare drugs that enhance the cytotoxic activity of NK92 cells or primary NK cells; or 3) Prepare drugs to stimulate the human immune response.

[0039] In some implementations, the diseases associated with abnormal NKG2A expression or function are selected from: cancer, viral diseases, autoimmune diseases, or inflammatory diseases; more preferably, the cancers are selected from: non-small cell lung cancer, pharyngeal tumors, melanoma, colon cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, bladder cancer, glioma, glioblastoma, thyroid cancer, esophageal cancer, prostate cancer, breast cancer, lymphocytic leukemia, small cell lung cancer, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, gastric cancer, peritoneal tumors, non-leukemic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, pilocytic lymphoma, Burketts lymphoma, acute and chronic myeloid leukemia, and promyelocytic leukemia.

[0040] In some implementations, the cancer is a solid tumor, such as non-small cell lung cancer, small cell lung cancer, fallopian tube cancer, peritoneal tumor, ovarian tumor, breast cancer, head and neck squamous cell carcinoma, etc. In some implementations, the cancer is an HLA-E-expressing cancer.

[0041] In some embodiments, the cancer is a hematologic malignancy, such as chronic lymphocytic leukemia. In some embodiments, the hematologic malignancy is a lymphoma that expresses CD94 / NKG2A.

[0042] In some embodiments, the autoimmune or inflammatory disease is selected from: polyarteritis nodosa, systemic lupus erythematosus, Wegener's granulomatosis, autoimmune hepatitis, Behcet's disease, Crohn's disease, primary biliary cirrhosis, scleroderma, ulcerative colitis, Sjögren's syndrome, type 1 diabetes mellitus, uveitis, Graves' disease, thyroiditis, type 1 diabetes mellitus, myocarditis, rheumatic fever, scleroderma, ankylosing spondylitis, rheumatoid arthritis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, Guillain-Barré syndrome, multiple sclerosis, alopecia areata, pemphigus / pemphigoid, psoriasis, and vitiligo.

[0043] In a seventh aspect, the present invention provides a method for preventing and / or treating diseases associated with abnormal NKG2A expression or function, the method comprising administering to the individual an effective amount of an anti-NKG2A antibody or antigen-binding fragment thereof, as described above, or a biological material, or a composition as described above.

[0044] In an eighth aspect, the present invention provides a method for enhancing the cytotoxic activity of NK92 cells or primary NK cells, the method comprising administering to the human individual an effective amount of an anti-NKG2A antibody or its antigen-binding fragment, as described above, or the biological material, or the composition described above.

[0045] In a ninth aspect, the present invention provides a method for stimulating an immune response in a human individual, the method comprising administering to the human individual an effective amount of an anti-NKG2A antibody or an antigen-binding fragment thereof, as described in any of the preceding claims, or a biological material, or a composition, as described in the preceding claims. In some embodiments, the human individual has a tumor and an antitumor immune response is stimulated. In other embodiments, the human individual has a chronic viral infection and an antiviral immune response is stimulated.

[0046] The anti-NKG2A antibody or its antigen-binding fragment described in this invention, or the biological material described herein, or the composition as described above, can be used as a pharmaceutical. In some embodiments, it is used as a pharmaceutical for the prevention and / or treatment of diseases associated with abnormal NKG2A expression or function. In some embodiments, it is used as a pharmaceutical for enhancing the cytotoxic activity of NK92 cells or primary NK cells. In some embodiments, it is used as a pharmaceutical for stimulating an immune response in a human individual.

[0047] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0048] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields.

[0049] NKG2A Natural killer (NK) cells are a crucial type of lymphocyte in the body, playing a vital role in both innate and adaptive immunity. NK cells possess two types of surface receptors, classified into inhibitory and activating receptors based on their function. These receptors mediate different recognition patterns of NK cells, transmitting different activation and inhibition signals. The CD94 / NKG2 family is a well-studied receptor family, mainly including members such as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H. Among them, NKG2A is an inhibitory receptor, and its ligand is the non-classical major histocompatibility complex class I molecule—HLA-E. HLA-E molecules expressed on target cells bind to NKG2A, inhibiting the killing function of NK cells. Therefore, antibodies inhibiting CD94 / NKG2A may enhance the killing activity of tumor-specific lymphocytes against tumor cells. Furthermore, certain lymphomas, such as NK-lymphoma, are characterized by CD94 / NKG2A expression. In such patients, therapeutic antibodies that target and kill CD94 / NKG2A-expressing cells may be able to eradicate tumor cells.

[0050] Antibody As used in this article, a natural "antibody" refers to a protective immune protein produced by the body in response to antigen stimulation, capable of binding to the antigen, and secreted by plasma cells (effector B cells); an "immunoglobulin" refers to a globulin with antibody (Ab) activity or chemical structure, similar to an antibody molecule. Antibodies typically consist of two pairs of polypeptide chains (each pair containing one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (k) and lambda (lambda) light chains. Heavy chains are classified into five classes based on the antigenicity of their constant region: μ, γ, α, δ, and ε, corresponding to the immunoglobulins IgM, IgG, IgA, IgD, and IgE, respectively. Antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a variable region (VH) and a constant region (CH). The constant region of the heavy chain consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Within both the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. The VH and VL regions can be further subdivided into highly denatured regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0051] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev.Comparat. Immunol. 27:55-77, 2003). The numbering rules of this invention are shown in Table 1 below.

[0052] Table 1. Relationship between CDR numbering systems

[0053] Unless otherwise stated, the variable region and CDR sequence in the embodiments of the present invention are governed by the "Kabat" numbering rule. Although a numbering system (such as Kabat) is used to define amino acid residues in a specific implementation, the corresponding technical solutions of other numbering systems are considered equivalent.

[0054] As used herein, the term "framework region" or "FR" residue refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above. The term "antibody" is not limited to any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0055] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', and F(ab'). 2 Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabody, single-domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechno1, 23: 1126-1136.

[0056] “Fab” consists of a light chain, a heavy chain CH1, and a variable region.

[0057] “Fab” is a Fab fragment with one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0058] "F(ab')2" is a divalent segment formed by connecting two Fab' segments through a disulfide bridge in the hinge region.

[0059] "Fab'-SH" refers to a Fab' that has a free thiol group on the cysteine ​​residue in the constant region.

[0060] "Single-chain Fab" is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; wherein the linker is a peptide linker. In some embodiments, the peptide linker is a polypeptide of at least 30 amino acids, preferably a polypeptide of 32-50 amino acids.

[0061] "Diabodies" have two antigen-binding sites, including a heavy chain variable domain (VH) and a light chain variable domain (VL) that are linked together in the same polypeptide chain.

[0062] A “linear antibody” contains a pair of tandem Fd fragments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding regions.

[0063] "Fv" has the VL and VH domains of one arm of the antibody.

[0064] A "single-domain antibody" is an antibody fragment that contains all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain.

[0065] A single-chain antibody fragment (scFv) is an antibody composed of a heavy chain variable region and a light chain variable region linked together by a linker.

[0066] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native and modified Fc regions. In some embodiments, the Fc region comprises two subunits with the same or different sequences. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise stated, Fc regions are numbered using the EU index.

[0067] The term "IgG antibody" refers to an antibody with a "heavy" chain and a "light" chain cross-linked via intra- and inter-chain disulfide bonds, exhibiting a typical "Y" structure. Each heavy chain consists of an N-terminal HCVR and a heavy chain constant region ("HCCR"). Each light chain consists of an LCVR and a light chain constant region ("LCCR").

[0068] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0069] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or epitope. In some embodiments, the KD of an antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, e.g., cynomolgus macaques (Macaca fascicularis) or chimpanzees (Callithrix jacchus) or common marmosets (Callithrix jacchus).

[0070] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0071] The monoclonal antibodies of this invention can be prepared using a variety of techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al., Nature 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0072] Antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to achieve immunospecific antibody purification. For purification of immunoglobulins, see, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0073] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP4, 816, 567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). In some embodiments, the term "chimeric antibody" may include antibodies (e.g., human-mouse co-antibody) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a murine antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0074] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three (heavy and / or light immunoglobulin chains) of the receptor CDR of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDR is called the "donor," and the immunoglobulin providing the framework is called the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the receptor framework can be a naturally occurring human framework or a sequence having approximately 85%, 90%, 95%, 99%, or higher identity compared to it. Humanized antibodies typically retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, and reactivity. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0075] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned. During alignment, gaps may be introduced where necessary to achieve the maximum percentage of sequence identity, but any conserved substitutions are not considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0076] The term “nucleic acid” is used interchangeably with the term “polynucleotide” herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or links, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). “Isolated” nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. An isolated nucleic acid encoding the antigen-binding molecule refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitution can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a degenerate base and / or a deoxyinosine residue.

[0077] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding molecules or antibodies described herein, along with other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0078] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0079] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids, phage particles, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0080] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0081] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless explicitly stated otherwise, the terms “patient” or “subject” are used interchangeably herein. As used herein, the term “cynomolgus monkey” refers to the cynomolgus monkey (Macacafascicularis). In some embodiments, the individual or subject is a human.

[0082] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0083] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within the subject's body. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0084] "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention intended to be applied to the individual being treated, and may be implemented for preventative purposes or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, the molecules disclosed herein are used to delay the onset of disease or slow its progression.

[0085] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state. In some embodiments, an effective dose is a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when administered to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete therapeutic or preventative effect may not occur after a single dose is administered, but may occur after a series of doses. Therefore, a therapeutically or preventatively effective dose may be administered in a single or multiple-dose manner. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved health status in the patient.

[0086] HLA-E is a non-classical MHC molecule that is expressed on the cell surface and regulated by binding to peptides derived from signaling sequences of other MHC class 1 molecules. HLA-E binds to natural killer (NK) cells and some T cells, specifically binding to CD94 / NKG2A, CD94 / NKG2B, and CD94 / NKG2C (see, for example, Braud et al. (1998) Nature 391:795-799, the full text of which is incorporated herein by reference). Surface expression of HLA-E is sufficient to protect target cells from lysis by CD94 / NKG2A+NK, T, or NKT cell clones. As used herein, “HLA-E” refers to any variant, derivative, or isotype of the HLA-E gene or the protein it encodes. It also includes any nucleic acid or protein sequence that shares one or more biological properties or functions with wild-type, full-length HLA-E and shares at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher nucleotide or amino acid identity.

[0087] NK cells refer to a subset of lymphocytes involved in non-traditional immunity. NK cells can be identified by certain characteristics and biological properties, such as the expression of specific surface antigens including CD16, CD56, and / or CD57; the absence of α / β or γ / δ TCR complexes on the cell surface; the ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activating specific cell-lysing enzymes; the ability to kill tumor cells or other disease cells expressing ligands for NK activation receptors; and the ability to release protein molecules called cytokines that stimulate or inhibit immune responses. Any of these characteristics or activities can be used to identify NK cells using methods well known in the art.

[0088] Beneficial effects of the invention The antibody of this invention can specifically bind to the NKG2A protein and the soluble extracellular domain of NKG2A on the surface of NK cells, and significantly increase the killing effect of NK92 cells or primary NK cells on HLA-E positive tumor cells, exhibiting superior functional activity compared to known anti-NKG2A antibodies. The NKG2A antibody of this invention has high specificity and no cross-reactivity with human NKG2C family protein antigens. Therefore, the antibody of this invention (especially the humanized antibody) has significant clinical value. Attached Figure Description

[0089] Figure 1 The results of the ELISA detection of the binding reaction between the NKG2A chimeric antibody and the extracellular region of human NKG2A protein (hNKG2A / CD94-ECD-Fc) were obtained.

[0090] Figure 2The results of the ELISA detection of the binding reaction between the NKG2A chimeric antibody and the extracellular region of monkey NKG2A protein (cynoNKG2A / CD94-ECD-Fc) were obtained.

[0091] Figure 3 The results of FACS detection of the binding reaction between the NKG2A chimeric antibody and the human NKG2A overexpressing cell line (CHOK1-hNKG2A / CD94).

[0092] Figure 4 The binding reaction between the NKG2A chimeric antibody and the monkey NKG2A overexpressing cell line (CHOK1-cynoNKG2A / CD94) was detected by FACS.

[0093] Figure 5 The results of FACS detection of the binding reaction between the NKG2A chimeric antibody and the human NKG2C overexpressing cell line (CHOK1-hNKG2C / CD94).

[0094] Figure 6 The results show the activity of the NKG2A chimeric antibody in the assay of NK92 cells killing LCL721.221 tumor cells (the antibody enhances the killing activity of NK92 cells).

[0095] Figure 7 The results show the activity of the NKG2A chimeric antibody in the assay of NK92-mediated killing of MOLM-13 tumor cells (the antibody enhances the killing activity of NK92 cells).

[0096] Figure 8 The results show the activity of the NKG2A chimeric antibody in the assay of primary NK cells killing LCL721.221 tumor cells (PBMC donor-1). The antibody enhances the killing activity of primary NK cells.

[0097] Figure 9 A~9E represent the results of FACS detection of the binding reaction between humanized NKG2A antibody and CHOK1-hNKG2A / CD94.

[0098] Figures 10A-10C show the results of FACS detection of the binding reaction between humanized NKG2A antibody and CHOK1-cynoNKG2A / CD94.

[0099] Figure 11 shows the activity detection results of the humanized NKG2A antibody in the experiment mediating the killing of LCL721.221 tumor cells by NK92 (the antibody enhances the killing activity of NK92 cells).

[0100] Figures 12A-12D show the activity of humanized NKG2A antibody in the assay of primary NK cells killing LCL721.221 tumor cells (PBMC donor-2, donor-3, donor-4). The antibody enhances the killing activity of primary NK cells. Detailed Implementation

[0101] The present invention will be described below with reference to specific embodiments, but the scope of the present invention is not limited thereto.

[0102] Unless otherwise specified, the reagents and instruments used in the following methods are commonly used in the art and can be obtained commercially; the methods used are conventional methods in the art, and those skilled in the art can perform the methods and obtain the corresponding results without any doubt based on the description of the embodiments.

[0103] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0104] The room temperature described in the examples is the conventional room temperature in the art, generally 10 to 30°C.

[0105] Unless otherwise specified, the proteins, cells, and mice used in the following examples were provided by Shanghai Ruizhi Chemical Research Co., Ltd.

[0106] In the following examples, the constructed antibodies have a constant region of human IgG4 antibody. Those skilled in the art know and can obtain the sequence of the human IgG4 antibody constant region.

[0107] Example 1: Generation of anti-NKG2A antibodies This invention uses hybridoma technology to produce monoclonal antibodies (mAbs) with specificity and high affinity for human and monkey NKG2A, and these antibodies do not bind to human NKG2C overexpressing cells.

[0108] Fourteen groups of mice (including four Balb / c mice, nine SJL mice, and one NOD mouse, all 6-8 week old females) were immunized with the following immunogens: 293F-hNKG2A / CD94 stable cell line, recombinant hNKG2A / CD94-ECD-Fc protein, 293F-cynoNKG2A / CD94 stable cell line + recombinant hNKG2A / CD94-ECD-Fc protein, and hNKG2A / CD94 expression plasmid + recombinant hNKG2A / CD94-ECD-Fc protein. Serum immunotiter levels were measured by FACS and ELISA after immunization. FACS was performed using CHOK1-hNKG2A / CD94, CHOK1-cynoNKG2A / CD94, and CHOK1-hNKG2C / CD94 stable cells, with CHOK1-blank cells serving as a control. ELISA was performed using hNKG2A / CD94-ECD-Fc and cynoNKG2A / CD94-ECD-Fc proteins.

[0109] Mice with high immunogenicity were selected, and spleen cells were fused with myeloma cells. Two rounds of screening were performed using the FACS method. After the initial screening, positive hybridoma clones were transferred to 24-well plates for culture, followed by a second screening. The resulting positive clones were then subcloned, and the subclones were further tested using the same initial and second screening methods. Positive monoclonal clones were selected for antibody production, cryopreservation for library preparation, and sequencing. This project yielded a total of 52 positive monoclonal clones through 12 hybridoma fusions.

[0110] Example 2: Sequencing, production, and purification of chimeric antibodies The subclonal cells collected in Example 1 were cryopreserved and mailed to Bio-Tech for antibody light and heavy chain variable region gene sequencing and analysis. Sequencing yielded eight sequence-specific monoclonal antibodies. The monoclonal numbers, names of the produced chimeric antibodies, and their light and heavy chain variable regions and CDR sequences are shown in Table 2 below.

[0111] Table 2: Antibody IDs and their amino acid sequence numbers

[0112] Using recombinant DNA technology, the light and heavy chain variable region genes of the sequenced monoclonal antibody were linked to the human IgG4 antibody constant region gene. The gene was then incorporated into the expression vector pcDNA3.4 using total gene synthesis technology. The chimeric antibody was transfected into CHOK1 mammalian cells (purchased from ATCC) to express the chimeric antibody. Purification was performed using protein A affinity chromatography to separate the anti-human NKG2A human-mouse chimeric antibody from serum-free culture supernatant containing the target antibody. The amino acid sequence of the heavy chain constant region of the chimeric antibody is shown in SEQ ID NO: 95, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 96.

[0113] Simultaneously, based on the humZ270 sequence disclosed in IMGT / 2Dstructure-DB card for INN 10113, a positive control antibody Z270 (Monalizumab jointly developed by Innate Pharma and AstraZeneca) for NKG2A was generated. The sequence link is https: / / www.imgt.org / 3Dstructure-DB / cgi / details.cgi?pdbcode=10113.

[0114] The nucleotide sequences of the variable regions of the antibody heavy and light chains are shown in Table 2.

[0115] Table 3: Nucleotide sequence numbers of the variable region of the heavy and light chains of NKG2A chimeric antibody:

[0116] Example 3: ELISA detection of the binding activity between chimeric antibody and protein Dilute two antigen proteins, human NKG2A extracellular domain protein (hNKG2A / CD94-ECD-Fc) and monkey NKG2A extracellular domain protein (cynoNKG2A / CD94-ECD-Fc), to 1 μg / mL with PBS. Add 100 μL / well to an ELISA microplate and incubate overnight at 4°C. Block with ELISA blocking buffer (PBS phosphate buffer containing 1% BSA, pH 7.4, percentages are by weight) at 37°C for two hours. Add serially diluted antibody to eight wells (starting at 100 nM, 10-fold serial dilution, including well 0). Add 100 μL / well and incubate at 37°C for 1 hour. Wash the plate three times and add secondary antibody (Thermofisher): anti-human IgG (Fab specific)-HRP (1:5000 dilution), 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate three times, add 100 μL of TMB chromogenic buffer per well, incubate at 37°C for 10 minutes, and then add 50 μL of 1N hydrochloric acid to terminate the chromogenic reaction. Read the OD at 450 nm using a microplate reader.

[0117] The results are as follows Figure 1 As shown, the chimeric antibody tested bound to the hNKG2A / CD94-ECD Fc protein. Figure 2 As shown, the chimeric antibody tested bound to the cynoNKG2A / CD94-ECD Fc protein.

[0118] Example 4: Flow cytometry (FACS) assay to detect the binding of chimeric antibody to NKG2A / CD94 expressing cells Human NKG2A overexpressing cells (CHOK1-hNKG2A / CD94), monkey NKG2A overexpressing cells (CHOK1-cynoNKG2A / CD94), and human NKG2C overexpressing cells (CHOK1-hNKG2C / CD94) were digested using TrypLE (purchased from Gibco). After centrifugation to collect cells, they were resuspended in FACS buffer (PBS + 2% FBS) to a concentration of 2E6 / mL. Cells were added to 96-well plates at 100 μL per well, centrifuged, and the supernatant was discarded. Antibodies were serially diluted with FACS buffer (starting at 200 nM, 5-fold dilution, including 0 point, for a total of 10 concentration points), and 100 μL was added to each well of the cells. The cells were incubated at 4°C for 1 hour. After incubation, the cells were centrifuged at 300g for 5 min and washed twice with FACS buffer. Secondary antibody was added: anti-human IgG fluorescent secondary antibody 488 (purchased from Thermofisher A-11013). After incubation at 4°C for one hour, the sample was washed twice with FACS buffer, resuspended in PBS, and analyzed using an FACS instrument.

[0119] The experimental results are shown in Figure 3 , Figure 4 The chimeric antibodies detected all bound to CHOK1-hNKG2A / CD94 cells and CHOK1-cynoNKG2A / CD94 cells. Figure 5 As shown, none of the chimeric antibodies tested bound to CHOK1-hNKG2C / CD94 cells, among which mAb011 was a positive control antibody against NKG2C (purchased from Wise Chemical).

[0120] Example 5: Anti-NKG2A antibody-mediated killing effect of NK92 cells on target cells Target cells, LCL721.221 B lymphocyte blast cells or MOLM-13 human acute myeloid leukemia cells (provided by Shanghai Ruizhi Chemical), were collected by centrifugation and resuspended in 1640 medium containing 10% fetal bovine serum to a density of 2E6 / mL. 0.5 mL of the cell suspension was added to a 24-well plate, and the plate was incubated overnight at 27°C. NK92 cells (purchased from Enzyme Research Biotechnology) were collected by centrifugation, washed twice with PBS, and then stained with trypan blue for counting. The cell density was adjusted to 4E5 / mL. 50 μL of NK92 cell suspension was added to a 96-well plate. The antibody to be tested was diluted with 1640 medium containing 10% fetal bovine serum, and 50 μL of the diluted antibody was added to a 96-well plate (starting concentration 200 nM, 5-fold dilution, including 0 point, for a total of 10 concentration points). The plate was incubated at 37°C for 0.5 hours. Target cells were collected by centrifugation, washed twice with PBS, and resuspended in 1640 medium containing 10% fetal bovine serum to a density of 1E6 / mL. BATDA (PerkinElmer) (2 μL / mL) was added to the target cell suspension, and the cells were incubated at 37°C for 20 minutes. The labeled target cells were washed four times by centrifugation, and resuspended in 1640 medium containing 10% fetal bovine serum to a density of 1E5 / mL. 100 μL of the target cell suspension was added to a 96-well plate, and the plate was incubated at 37°C for 2 hours. The plate was centrifuged at 300g for 5 minutes, and 25 μL of the culture supernatant was transferred to another 96-well plate. 200 μL of Eu- solution (PerkinElmer) was added. Data were read using an Envision instrument and analyzed using Graphpad software.

[0121] The experimental results are shown in Figure 6 In the experiment of NK92 cells killing LCL721.221 cells, the chimeric antibodies tested all showed good activity and could enhance the killing effect of NK92 on tumor cells. Among them, cAb-9, cAb-43, and cAb-52 were all superior to the control antibody Z270. Figure 7 As shown, in the experiment of NK92 cells killing MOLM-13 cells, cAb-9, cAb-14, cAb-43, and cAb-52 all showed strong activity and were superior to the control antibody Z270.

[0122] Example 6: Detection of the killing effect of chimeric antibody-mediated primary NK cells on target cells Primary NK cells were isolated from PBMCs (purchased from Miaoshun Biotechnology) 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 in 1640 medium (containing 1 mM inducing peptide) with 10% fetal bovine serum to a density of 2E6 / mL. 0.5 mL of the cell suspension was added to a 24-well plate and incubated overnight at 27°C. Primary NK cells were collected by centrifugation, washed twice with PBS, and then stained with trypan blue for cell counting. The cell density was adjusted to 4E5 / mL. 50 μL of cell suspension was added to a 96-well plate. The antibody was diluted with 1640 medium containing 10% fetal bovine serum, and 50 μL of the diluted antibody (starting concentration 200 nM, 5-fold dilution, including 0 point, for a total of 10 concentration points) was added to the 96-well plate. The plate was incubated at 37°C for 0.5 hours. LCL721.221 cells were collected by centrifugation, washed twice with PBS, and resuspended in 1640 medium containing 10% fetal bovine serum to a density of 1E6 / mL. BATDA (PerkinElmer) (2 μL / mL) was added to the LCL721.221 cell suspension, and the cells were incubated at 37°C for 20 minutes. The LCL721.221 cells were washed four times by centrifugation and resuspended in 1640 medium containing 10% fetal bovine serum to a density of 1E5 / mL. 100 μL of LCL721.221 cell suspension was added to a 96-well plate, and the plate was incubated at 37°C for 2 hours. The plate was centrifuged at 300g for 5 minutes, and 25 μL of the culture supernatant was transferred to another 96-well plate with 200 μL of Eu- solution added. Data were read using an Envision instrument and analyzed using Graphpad software.

[0123] Experimental results are as follows Figure 8 As shown, among the antibodies tested, cAb-17 did not mediate the killing activity of primary NK cells against LCL721.221 cells. Other antibodies showed good activity in this experiment. cAb-9 and cAb-14 were significantly better than the control antibody Z270. Compared with cAb-9, cAb-43 and cAb-52 were better than cAb-9.

[0124] Example 7: Preparation of humanized antibodies Monoclonal antibodies 43 and 52 were selected for humanization. The humanization design was completed by Baiying Biotechnology, employing a CDR transplantation approach. The CDR region of molecule 43 or antibody 52 was transplanted into a matching human variable region gene framework sequence, and a reversion mutation was designed to obtain the heavy and light chain variable regions of the hu-43 and hu-52 humanized antibodies. Specifically, monoclonal antibody 43 was humanized using two heavy chain variable region templates and three light chain variable region templates from the human antibodies. The sequences of the heavy chain variable regions of the hu-43 humanized antibody are shown in SEQ ID NO: 65, 85, and 86, and the sequences of the light chain variable regions are shown in SEQ ID NO: 66, 87, and 88, respectively. The monoclonal antibody 52 was humanized using three heavy chain variable region templates and three light chain variable region templates of human antibodies. The sequences of the heavy chain variable regions of the obtained hu-52 humanized antibody are shown in SEQ ID NO: 89, 90 and 91, and the sequences of the light chain variable regions are shown in SEQ ID NO: 92, 93 and 94, respectively.

[0125] The variable regions of the obtained humanized antibodies are combined and linked with the heavy chain constant regions (such as the heavy chain constant region of IgG4 as shown in SEQ ID NO: 95) and light chain constant regions (such as the light chain constant region of kappa as shown in SEQ ID NO: 96) of human antibodies to obtain full-length humanized antibodies. Examples of hu-43 humanized antibodies include hu-43VH-11 / VL-21, hu-43VH-11 / VL-11, hu-43VH-13 / VL-11, hu-43VH-13 / VL-21, hu-43VH-13 / VL-31, hu-43VH-21 / VL-11, hu-43VH-21 / VL-21, hu-43VH-21 / VL-31, and hu-43VH-11 / VL-31. Humanized antibodies against hu-52 include hu-52VH-13 / VL-11, hu-52VH-23 / VL-11, hu-52VH-33 / VL-11, hu-52VH-13 / VL-21, hu-52VH-23 / VL-21, hu-52VH-33 / VL-21, hu-52VH-13 / VL-31, hu-52VH-23 / VL-31, and hu-52VH-33 / VL-31.

[0126] Analysis of the variable region sequence of the hu-43VH-11 / VL-21 humanized antibody revealed potential post-translational modification sites (hereinafter referred to as hotspots) in the first CDR region of the light chain, including glycosylation NKS sites and deamidation NG sites. Therefore, mutations were made in the light chain N26 to T, S, Q, A, D, G, E, F, I, K, L, P, V, W, Y, R, or H, and G34 to A, L, E, K, R, S, N, T, or Q, or N33. The mutation is Q, S, or T (either G34 or N33 is chosen here), the heavy chain remains unchanged, and the designed mutation sequence is named according to the hotspot mutation site of the light chain, i.e., hu-43VL(N26X1-N33X2 (or G34X3)), where amino acid X1 is T, S, Q, A, D, G, E, F, I, K, L, P, V, W, Y, R or H; X2 is Q, S or T; and X3 is A, L, E, K, R, S, N, T or Q.

[0127] The amino acid sequence of the heavy chain variable region of the humanized hotspot mutant molecule is shown in SEQ ID NO: 65, and the sequences of CDR1, CDR2, and CDR3 of the heavy chain are shown in SEQ ID NO: 50, 51, and 52, respectively; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 67, and the sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID NO: 68, 55, and 56, respectively. In SEQ ID NO: 67 and 68, X1 is T, S, Q, A, D, G, E, F, I, K, L, P, V, W, Y, R, or H; X2 is N, Q, S, or T; and X3 is G, A, L, E, K, R, S, N, T, or Q. One and only one of the two conditions, X2 being N and X3 being G, must be present. For example, the sequences of antibodies obtained after mutation are shown in Table 4.

[0128] Table 4: Variable region sequence numbers of humanized antibodies and their hotspot mutant molecules

[0129] Multiple humanized hotspot mutant molecules were reverse-translated into base sequences and cloned into the mammalian expression vector pCDNA3.4 to obtain recombinant expression vectors. These recombinant expression vectors were then transfected into HEK293 or CHO-S cells using a transient transfection method. After 5 days of serum-free culture, the cell culture supernatant was harvested, purified by affinity chromatography, and the antibody protein was obtained. The obtained hu-43VH-11 / VL-21 mutant antibody contains the heavy chain constant region of IgG4 as shown in SEQ ID NO: 95 and the light chain constant region of kappa as shown in SEQ ID NO: 96. The in vitro activity and function of multiple humanized hotspot mutant molecules of hu-43VH-11 / VL-21 and Z270 were evaluated.

[0130] To further reduce Fc-mediated ADCP activity and enhance antibody stability, the inventors mutated the heavy chain constant region of the hu43-VL(N26S-G34A) and hu-52VH33 / VL11 antibodies at the L235E, D265A, and R409K sites, obtaining new antibody molecules hu43(N26S-G34A)-IgG4 mutant antibody and hu-52VH33 / VL11-IgG4 mutant antibody.

[0131] Example 8: In vitro activity and function detection of humanized antibodies FACS detection of the binding activity of humanized antibodies to human NKG2A overexpressing cell lines (FACS) refers to Example 4: binding of hu-43VH-11 / VL-21 hotspot mutant molecule, cAb-43, and control molecule Z270 to cells as follows. Figure 9 As shown in (AE), the maximum molecular weight fraction (MFI) of cAb-43 and its humanized mutant antibodies binding to human NKG2A-overexpressing cell lines was greater than that of the control Z270 antibody. The binding activity of the hu-43VH-11 / VL-21 hotspot mutant molecules was slightly lower than that of cAb-43, but the activities of most molecules were comparable. The binding activity of the hu-52 humanized antibody is shown in Table 5. Table 5 shows that the maximum MFI of the hu-52 humanized antibody was higher than that of the control Z270 antibody, indicating that the humanized hu-52 had good binding activity with human NKG2A-overexpressing cell lines.

[0132] Table 5. Binding activity of hu-52 humanized antibody to human NKG2A overexpressing cell lines

[0133] FACS detection of the binding activity of humanized antibodies to monkey NKG2A overexpressing cells (FACS) Referring to Example 4: The binding of hu-43VH-11 / VL-21 site-directed mutant molecule, cAb-43, and control molecule Z270 to cells is shown in Figure 10 (AC). The binding activities of cAb-43 and control molecule Z270 are comparable, while the binding activity of the hu-43VH11 / VL21 hotspot mutant molecule is slightly decreased, but the activities of most molecules are comparable. This indicates that multiple mutations at this amino acid site are effective mutations.

[0134] The killing effect of humanized antibodies on LCL721.221 cells mediated by NK92 cells was investigated as described in Example 5. The results are shown in Figure 11 and Tables 6-1 and 6-2. Tables 6-1 and 6-2 show that the humanized antibodies retained a high killing effect on target cells, and their killing activity was superior to Z270. Figure 11 shows that the two humanized hotspot mutant molecules hu-43VH-11 / VL-21, hu-43VL (N26S-G34A) and hu-43VL (N26T-G34A), mediated the killing activity of NK92 cells on target cells with comparable activity to Z270, but slightly weaker than the chimeric antibody cAb-43.

[0135] Table 6-1. Killing effect of hu-43 humanized antibody on target cells

[0136] Table 6-2. Killing effect of hu-52 humanized antibody on target cells

[0137] Humanized antibodies mediated the killing of LCL721.221 cells by primary NK cells, as described in Example 6. The results are shown in Figure 12 (AD). Multiple experiments examined the killing effects of hu-43 and hotspot mutant molecules hu-43VL (N26S-G34A) and Z270 on LCL721.221 cells mediated by primary NK cells from different donors. All results showed that the killing activity of hu-43VL (N26S-G34A) was significantly superior to that of the control molecule Z270.

[0138] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

[0139] Sequence List: 1: cAb-9 VH EVQLQQSVAELVRPGASVKLSCTASGFNIQNTYMHWVKERPEQGLEWIGRIDPANGDTEFAPKFQGKATLTTDTSSNTAYLQLSSLTSEDTAIYYCGRERYFYGSRGFAYWGQGTLVTVSA 2:cAb-9 VH-CDR1 NTYMH 3:cAb-9 VH-CDR2 RIDPANGDTEFAPKFQG 4:cAb-9 VH-CDR3 ERYFYGSRGFAY 5:cAb-9 VL DILMTQSPPSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK 6:cAb-9 VL-CDR1 HASQGISSNIG 7:cAb-9 VL-CDR2 HGTNLED 8:cAb-9 VL-CDR3 VQYAQFPWT 9:cAb-14 VH EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYIHWVKQRPEQGLEWIGRIDPANGNTKYAPKFQGKASIIVDTSSNTAYLQLSSLTSEDTAIYYCARYYASSPYYYNMDYWGQGTSVTVSS 10:cAb-14 VH-CDR1 NTYIH 11:cAb-14 VH-CDR2 RIDPANGNTKYAPKFQG 12:cAb-9 VH-CDR3 YYASSPYYYNMDY 13:cAb-9 VL DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTFTFGSGTKLEIK 14:cAb-14 VL-CDR1 RASENIYSNLA 15:cAb-14 VL-CDR2 AATNLAD 16:cAb-14 VL-CDR3 QHFWGTFT 17:cAb-17 VH EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYIDWVKQRPEQGLEWIGNIDPANGNTKYAPKFQGKATITADTSSNTAYVQLSSLTSEDTAIYYCARWGGWDVRFAYWGQGTLVTVSA 18:cAb-17 VH-CDR1 NTYID 19:cAb-17 VH-CDR2 NIDPANGNTKYAPKFQG 20:cAb-17 VH-CDR3 WGGWDVRFAY 21:cAb-17 VL DIKMTQSPSSMYASLGERVTITCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQNYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK 22:cAb-17 VL-CDR1 KASQDINTYLS 23:cAb-17 VL-CDR2 RANRLVD 24:cAb-17 VL-CDR3 LQYDEFPLT 25:cAb-24 VH EVQLQQSVAELVRPGASVKLSCTASGFNIQNTYMHWVKQRPEQGLEWIGRIDPANGNTKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCARYYDSRYGYWGQGTTLTVSS 26:cAb-24 VH-CDR1 NTYMH 27:cAb-24 VH-CDR2 RIDPANGNTKYAPKFQG 28:cAb-24 VH-CDR3 YYDSRYGY 29:cAb-24 VL DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPTTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDLGIYFCLQYDEFPWTFGGGTKLEIK 30:cAb-24 VL-CDR1 KASQDINSYLS 31:cAb-24 VL-CDR2 RANRLVD 32:cAb-24 VL-CDR3 LQYDEFPWT 33:cAb-33 VH EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMDWVKQRPEQGLEWIGRIDPANGNTKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCAREGNWDDWFVYWGQGTLVTVSA 34:cAb-33 VH-CDR1 NTYMD 35:cAb-33 VH-CDR2 RIDPANGNTKYAPKFQG 36:cAb-33 VH-CDR3 EGNWDDWFVY 37:cAb-33 VL DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPPTFGGGTKLEIK 38:cAb-33 VL-CDR1 RSSQSIVHSNGNTYLE 39:cAb-33 VL-CDR2 KVSNRFS 40:cAb-33 VL-CDR3 FQGSHVPPT 41:cAb-35 VH EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMDWVKQRPEQGLEWIGRIDPANGNTKHAPKFQGKATITTDTSSNTAYLQLSSLTSEDTAIYYCARWGGWDVRFAYWGQGTLVTVSA 42:cAb-35 VH-CDR1 NTYMD 43:cAb-35 VH-CDR2 RIDPANGNTKHAPKFQG 44:cAb-35 VH-CDR3 WGGWDVRFAY 45:cAb-35 VL DIKMTQSPSSMYASLGERVTINCQASQDINSYLSWFQQKPGKSPKTLIHRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQFDEFPLTFGAGTKLELK 46:cAb-35 VL-CDR1 QASQDINSYLS 47:cAb-35 VL-CDR2 RANRLVD 48:cAb-35 VL-CDR3 LQFDEFPLT 49:cAb-43 VH EVQLQQSVAELVRPGASVKLSCTASGFNIQNTYIHWVKQRPEQGLEWIGRIDPASGSTEYAPKFQGKATITADTSSNSGYLQLSSLTSEDTAIYYCARYGNFLYYYSMDYWGQGTSVTVSA 50: cAb-43 VH-CDR1 NTYIH 51: cAb-43 VH-CDR2 RIDPASGSTEYAPKFQG 52: cAb-43 VH-CDR3 YYNFLYYYSMDY 53: cAb-43 VL DIVMTQAAPSVPVTPGESVSISCRSNKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLENPYTFGGGTKLEIK 54: cAb-43 VL-CDR1 RSNKSLLHSNGNTYLY 55: cAb-43 VL-CDR2 RMSNLAS 56: cAb-43 VL-CDR3 MQHLENPYT 57: cAb-52 VH EVQLQQSVAELVRPGASVKLSCTASGFNIQNTYLHWVKQRPEQGLEWIGRIDPANDNIKYVPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCTRHGLFYEGWFAYWGQGTLVTVST 58: cAb-52 VH-CDR1 NTYLH 59: cAb-52 VH-CDR2 RIDPANDNIKYVPKFQG 60: cAb-52 VH-CDR3 HGLFYEGWFAY 61: cAb-52 VL EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSGTSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPYTFGSGTKLEIK 62: cAb-52 VL-CDR1 SVSSSISSSNLH 63: cAb-52 VL-CDR2 GTSNLAS 64: cAb-52 VL-CDR3 QQWSSYPYT 65: hu-43 VH-11 QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYIHWVRQAPGQGLEWMGRIDPASGSTEYAPKFQGRVTMTADTSTNTGYMELSSLRSEDTAVYYCARYGNFLYYYSMDYWGQGTLVTVSS 66: hu-43 VL-21 DIVMTQSPLSLPVTLGQPASISCRSNKSLLHSNGNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 67: Humanized hot-spot mutant molecule light chain variable region DIVMTQSPLSLPVTLGQPASISCRSX1KSLLHSX2X3NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 68: Humanized hot-spot mutant molecule VL-CDR1 RSX1KSLLHSX2X3NTYLY 69: cAb-9 VH GAGGTACAGTTGCAACAGTCTGTTGCAGAGCTTGTGAGGCCAGGTGCCTCTGTCAAACTGTCTTGTACTGCCAGCGGGTTCAATATCCAGAACACATATATGCACTGGGTAAAGGAGAGGCCAGAGCAGGGCTTGGAGTGGATAGGAAGAATCGACCCAGCCAATGGCGACACTGAGTTTGCTCCTAAGTTCCAGGGGAAAGCTACCCTCACAACAGATACCTCTAGCAACACTGCCTATCTCCAGCTGTCCTCTCTGACCTCCGAGGATACTGCCATCTACTATTGCGGCAGAGAGAGATACTTCTACGGAAGCAGAGGCTTCGCCTATTGGGGGCAAGGAACACTGGTTACAGTGTCTGCA 70:cAb-9 VL GACATATTGATGACACAGTCTCCTCCCTCTATGTCAGTCTCACTGGGCGATACTGTTTCAATTACATGCCACGCCAGCCAGGGCATTTCATCAAACATCGGCTGGTTGCAGCAGAAACCAGGCAAGTCTTTCAAGGGACTCATCTATCACGGCACCAACTTGGAGGATGGTGTGCCTTCCCGATTCTCCGGATCAGGCTCCGGCGCCGATTACTCCCTGACCATTTCTAGCCTGGAATCCGAGGACTTCGCCGACTACTATTGTGTACAGTATGCTCAGTTCCCATGGACATTCGGAGGGGGAACCAAGCTCGAAATAAAA 71:cAb-14 VH GAAGTACAGCTTCAACAGTCAGTAGCCGAGTTGGTGAGACCAGGTGCATCAGTGAAGTTGAGCTGTACTGCTAGTGGTTTCAACATTAAGAACACATATATCCACTGGGTCAAACAGAGGCCCGAGCAAGGACTGGAGTGGATAGGCAGGATCGATCCAGCAAACGGAAATACCAAATACGCTCCTAAGTTTCAGGGGAAGGCTTCCATTATTGTGGATACCAGTTCTAACACGGCCTACCTGCAGCTGTCCAGCCTCACCTCCGAGGATACCGCCATCTACTACTGCGCCCGATATTATGCCTCCAGCCCCTATTATTATAATATGGATTACTGGGGCCAGGGCACTTCCGTTACAGTGAGCTCC 72:cAb-14 VL GATATTCAGATGACCCAGTCTCCGGCAAGCCTGTCAGTGAGTGTGGGCGAGACAGTGACAATCACCTGTAGGGCCAGCGAAAACATCTATAGCAATCTCGCATGGTATCAACAGAAACAGGGTAAGTCACCACAGCTGCTTGTGTATGCTGCCACCAACCTGGCCGATGGAGTCCCATCCAGATTTTCTGGCTCTGGGAGCGGGACACAATACTCCCTCAAGATTAACAGCCTCCAAAGCGAGGACTTTGGGTCCTACTACTGCCAGCACTTTTGGGGAACATTTACGTTCGGCAGCGGGACAAAACTCGAAATCAAG 73:cAb-17 VH GAAGTTCAGCTTCAACAGTCTGTGGCTGAACTTGTACGCCCAGGGGCATCCGTGAAGTTGAGTTGTACAGCCAGCGGCTTCAATATCAAAAACACCTACATCGATTGGGTGAAACAGCGACCTGAGCAGGGACTGGAATGGATCGGCAACATTGACCCGGCTAACGGCAACACCAAATACGCTCCGAAATTCCAGGGTAAAGCTACTATTACAGCTGATACCTCCTCCAACACCGCCTACGTCCAGCTGAGCAGCCTGACCTCTGAAGATACAGCTATCTACTACTGTGCCAGGTGGGGAGGTTGGGACGTGAGGTTCGCCTATTGGGGACAGGGAACCCTCGTCACTGTCAGCGCT 74:cAb-17 VL GACATCAAGATGACGCAGTCACCAAGCTCTATGTACGCCTCACTGGGGGAGCGAGTAACCATCACTTGTAAGGCATCCCAGGATATCAACACTTACCTGAGCTGGTTTCAGCAAAAGCCTGGCAAGTCCCCCAAGACCCTCATCTATAGAGCTAATCGCCTCGTGGACGGAGTGCCCAGCCGGTTTAGTGGATCCGGATCCGGCCAGAACTATTCCCTGACAATCAGCAGCCTGGAGTATGAGGACATGGGGATTTATTACTGTCTGCAATATGATGAGTTCCCGCTCACTTTCGGAGCCGGAACCAAGCTTGAGTTGAAG 75:cAb-24 VH GAGGTACAGCTGCAGCAATCTGTTGCCGAATTGGTGAGACCAGGTGCCTCAGTGAAACTGTCTTGCACTGCCTCTGGATTCAATATCCAGAATACTTACATGCATTGGGTCAAGCAGAGACCCGAACAGGGGCTCGAATGGATAGGAAGGATTGATCCCGCTAACGGCAACACAAAGTACGCTCCAAAATTCCAAGGAAAAGCCACTATAACCGCCGATACTAGCAGCAATACCGCTTATCTCCAGCTGTCTTCCCTGACAAGTGAGGACACGGCCATTTATTATTGTGCCAGATATTACGATAGTAGGTACGGATATTGGGGACAGGGAACAACACTGACAGTGTCCTCC 76:cAb-24 VL GACATCAAGATGACACAGTCCCCATCTAGTATGTACGCTAGCCTTGGTGAGCGGGTGACAATCACTTGCAAAGCCTCCCAGGACATCAACTCATACCTGTCCTGGTTCCAGCAGAAGCCCGGGAAGAGTCCAACCACACTGATATACCGAGCCAACCGACTGGTAGATGGCGTGCCTTCTCGGTTCTCCGGCTCAGGTAGCGGGCAGGACTACTCCCTGACTATTTCCTCTCTCGAATACGAAGATCTGGGCATCTACTTCTGTCTCCAGTACGACGAATTTCCCTGGACGTTCGGAGGAGGAACCAAGCTTGAGATTAAG 77:cAb-33 VH GAGGTGCAGTTGCAGCAAAGCGTTGCAGAACTTGTTAGACCTGGTGCTAGTGTGAAACTGAGCTGCACCGCCAGTGGATTTAACATCAAGAACACCTACATGGACTGGGTCAAACAGAGGCCCGAGCAGGGGTTGGAGTGGATTGGACGAATTGATCCCGCAAACGGGAACACAAAATACGCTCCTAAGTTTCAGGGAAAAGCCACCATTACAGCAGACACCAGTAGCAATACCGCTTATCTGCAGCTGTCTAGCCTGACCAGCGAGGACACCGCAATATACTACTGCGCCAGAGAAGGGAATTGGGATGATTGGTTCGTATACTGGGGACAGGGCACACTGGTGACAGTCTCTGCT 78:cAb-33 VL GATGTCCTGATGACCCAGACCCCACTGAGTCTCCCAGTAAGTTTGGGCGACCAGGCATCAATCTCATGTAGAAGCAGCCAGTCTATCGTGCACAGCAACGGCAATACATACCTCGAGTGGTTCCTCCAGAAACCAGGTCAGTCCCCTAAGCTGCTCATCTACAAGGTCAGCAACCGGTTTAGCGGTGTACCAGACAGATTCTCTGGGTCCGGCTCCGGAACTGACTTTACCCTTAAGATCTCTAGGGTGGAAGCCGAAGATCTCGGAGTGTACTACTGTTTCCAGGGGTCACATGTGCCACCTACTTTTGGAGGCGGGACTAAGTTGGAGATAAAG 79:cAb-35 VH GAAGTGCAACTGCAGCAATCTGTCGCTGAACTCGTGAGACCTGGTGCATCTGTCAAATTGTCATGTACGGCATCAGGCTTTAACATTAAAAATACATACATGGATTGGGTCAAACAGCGGCCAGAACAGGGTCTGGAATGGATCGGAAGGATTGATCCAGCTAATGGTAACACCAAGCATGCTCCCAAGTTTCAGGGAAAGGCCACCATTACCACTGATACAAGCAGCAATACTGCTTACCTGCAGCTGAGCTCCTTGACCAGCGAGGACACCGCAATTTATTATTGTGCCAGGTGGGGAGGATGGGATGTGCGCTTTGCCTACTGGGGCCAGGGTACCCTGGTAACTGTCTCCGCC 80:cAb-35 VL GACATCAAAATGACTCAGAGTCCTAGCAGTATGTACGCATCTCTCGGGGAGCGAGTTACCATCAACTGCCAGGCCAGCCAAGATATCAACTCTTACCTGTCATGGTTCCAGCAGAAGCCCGGAAAGAGTCCGAAGACCTTGATTCACCGAGCTAATAGGCTGGTGGATGGCGTGCCAAGCCGCTTCAGTGGTAGTGGCTCTGGACAGGACTATTCACTGACCATTAGCAGCTTGGAATACGAGGATATGGGCATCTACTACTGTCTGCAATTTGACGAGTTCCCACTGACATTCGGCGCCGGCACAAAACTTGAGTTGAAA 81:cAb-43 VH GAAGTCCAGCTTCAGCAGTCTGTTGCAGAACTGGTAAGACCTGGGGCCTCAGTAAAGTTGAGCTGCACTGCTAGCGGCTTCAACATCCAGAACACCTACATCCACTGGGTGAAACAGAGACCCGAACAAGGATTGGAGTGGATTGGCCGAATAGATCCCGCCTCCGGGAGTACTGAGTACGCTCCTAAGTTCCAGGGCAAGGCCACAATCACAGCCGATACCTCCTCTAACAGCGGTTACCTGCAGCTGAGCAGCTTGACCTCCGAGGATACTGCAATTTACTATTGTGCAAGATACGGCAACTTTCTGTATTATTACAGTATGGACTACTGGGGACAGGGAACCTCCGTCACTGTGTCTGCC 82:cAb-43 VL GACATCGTTATGACACAGGCTGCCCCTTCTGTTCCCGTAACACCAGGGGAGAGCGTGAGCATTAGCTGTCGGTCAAACAAGTCTCTTCTGCACAGCAACGGAAATACCTATCTGTACTGGTTTCTGCAGCGCCCAGGACAGAGTCCCCAGCTTTTGATCTACAGAATGAGTAACTTGGCCAGCGGGGTGCCAGATCGCTTTTCCGGATCAGGAAGCGGCACCGCCTTTACCCTGCGGATTTCTCGGGTCGAGGCAGAGGATGTGGGGGTGTACTACTGCATGCAGCACCTTGAAAATCCATACACTTTCGGGGGAGGAACCAAGCTCGAGATCAAG 83:cAb-52 VH GAAGTACAGCTTCAACAGAGTGTTGCAGAGTTGGTGCGCCCTGGAGCCTCTGTGAAACTGAGCTGCACAGCATCCGGTTTCAACATCCAGAATACTTACCTGCATTGGGTGAAGCAGCGGCCTGAGCAGGGTCTGGAGTGGATTGGAAGAATCGACCCTGCAAATGATAATATCAAGTATGTGCCAAAGTTCCAGGGCAAGGCAACCATTACAGCCGATACTAGCAGCAATACCGCCTACCTGCAGCTTTCAAGCCTTACATCTGAGGACACTGCTATCTACTACTGCACAAGACACGGTCTGTTCTACGAGGGGTGGTTTGCTTACTGGGGGCAGGGGACCCTTGTTACCGTGTCTACA 84:cAb-52 VL GAAATTGTACTGACACAGTCTCCAGCCTTGATGGCTGCCTCCCCTGGTGAGAAGGTGACTATTACCTGTAGCGTGAGCAGTTCTATTTCCAGCTCCAACCTGCACTGGTATCAGCAGAAGAGCGGCACCAGTCCAAAGCCCTGGATATATGGCACCTCCAACCTTGCATCCGGCGTGCCCGTGCGATTCAGTGGCTCAGGTTCCGGTACTTCCTACTCTCTCACCATCTCCTCTATGGAAGCCGAAGACGCAGCTACATACTACTGCCAGCAGTGGTCCAGTTACCCCTACACTTTCGGCTCTGGAACCAAGCTGGAAATCAAG 85:hu-43VH-13: QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYIHWVRQAPGQGLEWIGRIDPASGSTEYAPKFQGRATITADTSTNTGYMELSSLRSEDTAVYYCARYGNFLYYYSMDYWGQGTLVTVSS 86:hu-43VH-21: QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYIHWVRQAPGQGLEWMGRIDPASGSTEYAPKFQGRVTMTADTSINTGYMELSRLRSDDTAVYYCARYGNFLYYYSMDYWGQGTLVTVSS 87:hu-43VL-11: DIVMTQSPLSLPVTPGEPASISCRSNKSLLHSNGNTYLYWFLQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 88:hu-43VL-31 DIVMTQTPLSLSVTPGQPASISCRSNKSLLHSNGNTYLYWFLQKPGQPPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEI 89:hu-52VH-13 QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYLHWVRQAPGQGLEWMGRIDPANDNIKYVPKFQGRVTMTADTSTNTAYMELSSLRSEDTAVYYCTRHGLFYEGWFAYWGQGTLVTVSS 90:hu-52VH-23 QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYLHWVRQAPGQGLEWMGRIDPANDNIKYVPKFQGRVTMTADTSINTAYMELSRLRSDDTAVYYCTRHGLFYEGWFAYWGQGTLVTVSS 91: hu-52VH-33 QVQLVQSGAEVKKPGSSVKVSCKASGFNIQNTYLHWVRQAPGQGLEWMGRIDPANDNIKYVPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCTRHGLFYEGWFAYWGQGTLVTVSS 92:hu-52VL-11 DIQLTQSPSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPLIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSYPYTFGQGTKLEIK 93: hu-52VL-21 DIQLTQSPSTLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPLIYGTSNLASGVPS RFSGSGSGTEYTLTISSLQPDDAATYYCQQWSSYPYTFGQGTKLEIK 94: hu-52VL-31 EIVLTQSPATLSLSPGERATLSCSVSSSISSSNLHWYQQKPGQAPRPLIYGTSNLASGIPA RFSGSGSGTDYTLTISSLEPEDAAVYYCQQWSSYPYTFGQGTKLEIK 95: IgG4 heavy chain constant region: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 96: Light chain constant region (Kappa): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 97: hu43-VL(N26S-N33S) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHS S GNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 98: hu43-VL(N26S-N33S) LCDR1: RS S KSLLHS S GNTYLY 99: hu43-VL(N26S-N33Q) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHS Q GNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 100: hu43-VL(N26S-N33Q) LCDR1: RS S KSLLHS Q GNTYLY 101: hu43-VL(N26S-N33T) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHS T GNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 102: hu43-VL(N26S-N33T) LCDR1: RS S KSLLHS T GNTYLY 103: hu43-VL(N26S-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 104: hu43-VL(N26S-G34A) LCDR1: RS S KSLLHSN A NTYLY 105: hu43-VL(N26T-N33S) VL: DIVMTQSPLSLPVTLGQPASISCRS T KSLLHS S GNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 106: hu43-VL(N26T-N33S) LCDR1: RS T KSLLHS S GNTYLY 107: hu43-VL(N26T-N33Q) VL: DIVMTQSPLSLPVTLGQPASISCRS T KSLLHS Q GNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 108: hu43-VL(N26T-N33Q) LCDR1: RS T KSLLHS Q GNTYLY 109: hu43-VL(N26T-N33T) VL: DIVMTQSPLSLPVTLGQPASISCRS T KSLLHS T GNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 110: hu43-VL(N26T-N33T) LCDR1: RS T KSLLHS T GNTYLY 111: hu43-VL(N26T-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS T KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 112: hu43-VL(N26T- G34A) LCDR1: RS T KSLLHSN A NTYLY 113: hu43-VL(N26A-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS A KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 114: hu43-VL(N26A- G34A) LCDR1: RS A KSLLHSN A NTYLY 115: hu43-VL(N26D-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS D KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 116: hu43-VL(N26D- G34A) LCDR1: RS D KSLLHSN A NTYLY 117: hu43-VL(N26E-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS E KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 118: hu43-VL(N26E- G34A) LCDR1: RS E KSLLHSN A NTYLY 119: hu43-VL(N26F-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS F KSLLHSNANTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 120: hu43-VL(N26F- G34A) LCDR1: RS F KSLLHSN A NTYLY 121: hu43-VL(N26G-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS G KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 122: hu43-VL(N26G- G34A) LCDR1: RS G KSLLHSN A NTYLY 123: hu43-VL(N26H-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS H KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 124: hu43-VL(N26H- G34A) LCDR1: RS H KSLLHSN A NTYLY 125: hu43-VL(N26I-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS I KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 126: hu43-VL(N26I- G34A) LCDR1: RS I KSLLHSN A NTYLY 127: hu43-VL(N26K-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS K KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 128: hu43-VL(N26K- G34A) LCDR1: RS K KSLLHSN A NTYLY 129: hu43-VL(N26L-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS L KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 130: hu43-VL(N26L- G34A) LCDR1: RS L KSLLHSN A NTYLY 131: hu43-VL(N26P-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS P KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 132: hu43-VL(N26P- G34A) LCDR1: RS P KSLLHSN A NTYLY 133: hu43-VL(N26Q-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS Q KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 134: hu43-VL(N26Q- G34A) LCDR1: RS Q KSLLHSN A NTYLY 135: hu43-VL(N26R-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS R KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 136: hu43-VL(N26R- G34A) LCDR1: RS R KSLLHSN A NTYLY 137: hu43-VL(N26V-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS V KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 138: hu43-VL(N26V- G34A) LCDR1: RS V KSLLHSN A NTYLY 139: hu43-VL(N26W-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS W KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 140: hu43-VL(N26W- G34A) LCDR1: RS W KSLLHSN A NTYLY 141: hu43-VL(N26Y-G34A) VL: DIVMTQSPLSLPVTLGQPASISCRS Y KSLLHSN A NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 142: hu43-VL(N26Y- G34A) LCDR1: RS Y KSLLHSN A NTYLY 143: hu43-VL(N26S-G34E) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN E NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 144: hu43-VL(N26S-G34E) LCDR1: RS S KSLLHSN E NTYLY 145: hu43-VL(N26S-G34G) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN GNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 146: hu43-VL(N26S-G34G) LCDR1: RS S KSLLHSN G NTYLY 147: hu43-VL(N26S-G34K) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN K NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 148: hu43-VL(N26S-G34K) LCDR1: RS S KSLLHSN K NTYLY 149: hu43-VL(N26S-G34L) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN L NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 150: hu43-VL(N26S-G34L) LCDR1: RS S KSLLHSN L NTYLY 151: hu43-VL(N26S-G34N) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN N NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 152: hu43-VL(N26S-G34N) LCDR1: RSS KSLLHSN N NTYLY 153: hu43-VL(N26S-G34Q) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN Q NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 154: hu43-VL(N26S-G34Q) LCDR1: RS S KSLLHSN Q NTYLY 155: hu43-VL(N26S-G34R) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN R NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 156: hu43-VL(N26S-G34R) LCDR1: RS S KSLLHSN R NTYLY 157: hu43-VL(N26S-G34S) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN S NTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 158: hu43-VL(N26S-G34S) LCDR1: RS S KSLLHSN S NTYLY 159: hu43-VL(N26S-G34T) VL: DIVMTQSPLSLPVTLGQPASISCRS S KSLLHSN TNTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIK 160: hu43-VL(N26S-G34T) LCDR1: RS S KSLLHSN T NTYLY 161: IgG4 constant region mutation sequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 162: Heavy chain sequence of hu43-VL(N26S-G34A)-IgG4 mutant antibody: QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYIHWVRQAPGQGLEWMGRIDPASGSTEYAPKFQGRVTMTADTSTNTGYMELSSLRSEDTAVYYCARYGNFLYYYSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 163: hu43-VL(N26S-G34A)-IgG4 mutant antibody light chain sequence: DIVMTQSPLSLPVTLGQPASISCRSSKSLLHSNANTYLYWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 164: hu-52VH-33 / VL11-IgG4 mutant antibody heavy chain: QVQLVQSGAEVKKPGSSVKVSCKASGFNIQNTYLHWVRQAPGQGLEWMGRIDPANDNIKYVPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCTRHGLFYEGWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 165: hu-52VH-33 / VL11-IgG4 mutant antibody light chain: DIQLTQSPSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPLIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 166: hu43(N26S-G34A) antibody heavy chain: QVQLVQSGAEVKKPGASVKVSCKASGFNIQNTYYIHWVRQAPGQGLEWMGRIDPASGSTEYAPKFQGRVTMTADTSTNTGYMELSSLRSEDTAVYYCARYGNFLYYYSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 167:hu-52VH33 / VL11 antibody heavy chain: QVQLVQSGAEVKKPGSSVKVSCKASGFNIQNTYLHWVRQAPGQGLEWMGRIDPANDNIKYVPKFQGRVTITADTSTNTAYMELSSLRSEDATAVYYCTRHGLFYEGWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPPAPEFLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Claims

1. An anti-NKG2A antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDRs: VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises three CDRs: VL-CDR1, VL-CDR2, and VL-CDR3; wherein, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 50; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 51; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 52; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 54, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 or 160; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 55; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO:

56.

2. The anti-NKG2A antibody or its antigen-binding fragment according to claim 1, wherein, The amino acid sequence of VH-CDR1 is shown in SEQ ID NO: 50; The amino acid sequence of VH-CDR2 is shown in SEQ ID NO: 51; The amino acid sequence of VH-CDR3 is shown in SEQ ID NO: 52; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO: 104; The amino acid sequence of VL-CDR2 is shown in SEQ ID NO: 55; The amino acid sequence of VL-CDR3 is shown in SEQ ID NO:

56.

3. An anti-NKG2A antibody or its antigen-binding fragment thereof, said antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein: According to the Kabat numbering rules, the heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 49, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 53; or According to the Kabat numbering rules, the heavy chain variable region includes three complementarity-determining regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 65, and the light chain variable region includes three complementarity-determining regions (CDRs) of the light chain variable region as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159.

4. The anti-NKG2A antibody or its antigen-binding fragment according to claim 3, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein: According to the Kabat numbering rules, the heavy chain variable region includes three complementary determinant regions (CDRs) of the heavy chain variable region as shown in SEQ ID NO: 65, and the light chain variable region includes three complementary determinant regions (CDRs) of the light chain variable region as shown in SEQ ID NO:

103.

5. The anti-NKG2A antibody or its antigen-binding fragment according to claim 1, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 80% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 80% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 80% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 80% sequence identity.

6. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 85% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 85% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 85% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 85% sequence identity.

7. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 90% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 90% sequence identity.

8. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 91% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 91% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 91% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 91% sequence identity.

9. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 92% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 92% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 92% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 92% sequence identity.

10. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 93% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 93% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 93% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 93% sequence identity.

11. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 94% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 94% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 94% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 94% sequence identity.

12. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 95% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 95% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 95% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 95% sequence identity.

13. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 96% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 96% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 96% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 96% sequence identity.

14. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 97% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 97% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 97% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 97% sequence identity.

15. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 98% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 98% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 98% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 98% sequence identity.

16. The anti-NKG2A antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 49, or has at least 99% sequence identity with the sequence shown in SEQ ID NO: 49; the light chain variable region sequence is as shown in SEQ ID NO: 53, or has at least 99% sequence identity with the sequence shown in SEQ ID NO: 53; or, The heavy chain variable region sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 65, or has at least 99% sequence identity with the sequence shown in SEQ ID NO:

65. The sequence of the light chain variable region is as shown in SEQ ID NO: 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 or 159, or is identical to that of SEQ ID NO:

65. The sequences shown as 66, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 159 have at least 99% sequence identity.

17. The anti-NKG2A antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region and / or light chain constant region are of human or mouse origin.

18. The anti-NKG2A antibody or its antigen-binding fragment according to claim 17, wherein the heavy chain constant region is the heavy chain constant region of human IgG4.

19. The anti-NKG2A antibody or its antigen-binding fragment according to claim 17, wherein the heavy chain constant region comprises S228P, L235E, D265A and / or R409K mutations.

20. The anti-NKG2A antibody or its antigen-binding fragment according to any one of claims 1 to 19, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

163.

21. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

163.

22. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

163.

23. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 91% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 91% sequence identity with SEQ ID NO:

163.

24. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 92% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 92% sequence identity with SEQ ID NO:

163.

25. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 93% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 93% sequence identity with SEQ ID NO:

163.

26. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 94% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 94% sequence identity with SEQ ID NO:

163.

27. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

163.

28. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 96% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 96% sequence identity with SEQ ID NO:

163.

29. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 97% sequence identity with SEQ ID NO:

163.

30. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 98% sequence identity with SEQ ID NO:

163.

31. The anti-NKG2A antibody or its antigen-binding fragment according to claim 20, wherein the antibody comprises a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 162 or 166, or contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 162 or 166, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 163, or contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

163.

32. The anti-NKG2A antibody or its antigen-binding fragment according to any one of claims 1 to 19, wherein the antibody is an IgG type antibody; and the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv and scFv.

33. A biomaterial, comprising: 1) A nucleic acid molecule encoding the anti-NKG2A antibody or its antigen-binding fragment as described in any one of claims 1 to 32; 2) A vector containing the nucleic acid molecule described in 1) above; 3) Cells containing nucleic acid molecules from 1) above or carriers from 2) above.

34. The biomaterial according to claim 33, wherein: The nucleic acid molecule encodes the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment.

35. The biomaterial according to claim 33 or 34, wherein: The nucleic acid molecule encodes the heavy chain variable region, the nucleotide sequence of which is shown in SEQ ID NO: 81; and the nucleic acid molecule encodes the light chain variable region, the nucleotide sequence of which is shown in SEQ ID NO:

82.

36. The biomaterial according to claim 33, wherein: The vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or retrovirus.

37. The biomaterial according to claim 33, wherein: The cells express the anti-NKG2A antibody or its antigen-binding fragment as described in any one of claims 1 to 32.

38. The method for preparing the anti-NKG2A antibody or its antigen-binding fragment according to any one of claims 1 to 32 is as follows: 1) Chemical synthesis method, synthesized based on the amino acid sequence of the antibody or its antigen-binding fragment; or, 2) Bio-preparation, comprising culturing the cells of claim 33 or 37 and harvesting the anti-NKG2A antibody or its antigen-binding fragment.

39. A composition comprising the anti-NKG2A antibody or its antigen-binding fragment as described in any one of claims 1 to 32, or the biological material as described in any one of claims 33 to 37.

40. The composition according to claim 39, wherein, The composition is a pharmaceutical composition.

41. The composition according to claim 39 or 40, wherein the composition comprises a pharmaceutically acceptable carrier.

42. A kit comprising an anti-NKG2A antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 32, or a biological material as claimed in any one of claims 33 to 37, or a composition as claimed in any one of claims 39 to 41; said kit being used for diagnosing or detecting NKG2A protein or cells expressing NKG2A protein in ex vivo samples.

43. The use of the anti-NKG2A antibody or its antigen-binding fragment as described in any one of claims 1 to 32, or the biological material as described in any one of claims 33 to 37, or the composition as described in any one of claims 39 to 41, wherein the use is for the preparation of a medicament for the prevention and / or treatment of cancer. The cancers mentioned are selected from: non-small cell lung cancer, colon cancer, ovarian cancer, endometrial cancer, head and neck squamous cell carcinoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and acute myeloid leukemia.

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