CN121358774A

CN121358774A - Antibodies or antigen-binding fragments that bind NKG2A and uses thereof

Info

Publication number: CN121358774A
Application number: CN202480040596.XA
Authority: CN
Inventors: 高婧; 高瑞; 孙大为; 刘泽宇; 仇全; 于景丰; 周红萍; 施梅; 孙俊; 王春年; 卢宏韬
Original assignee: Kewang Shanghai Biomedical Technology Co ltd; Kewang Suzhou Biomedical Technology Co ltd
Current assignee: Kewang Shanghai Biomedical Technology Co ltd; Kewang Suzhou Biomedical Technology Co ltd
Priority date: 2023-06-21
Filing date: 2024-06-12
Publication date: 2026-01-16
Also published as: WO2024260280A1; TW202515914A

Abstract

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

Description

Antibodies or antigen-binding fragments that bind NKG2A and uses thereof

Technical Field

The present disclosure relates to antibodies or antigen-binding fragments, polynucleotides, vectors, host cells, and pharmaceutical compositions thereof that bind to NKG 2A. The disclosure also relates to the use of the antibodies or antigen binding fragments.

Background

The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Immunooncology has become a revolution in cancer treatment. Tumor control has achieved unprecedented improvements by releasing therapeutic blocking antibodies to immunosuppressive 'checkpoints' (immune checkpoint inhibitors, ICI). However, only a few patients respond to these immunotherapies. Currently, the identification of predictive biomarkers for therapeutic response is an important topic of research and a number of factors have been identified. These factors include the number of T cells in the tumor and the total mutational load of the tumor cells, suggesting that ICI depends on natural immunity targeting neoantigens presented by HLA molecules (van Hall T, andr en P et al, J Immunother Cancer [ J.Care. Immunol., 10 months, 17, 7 (1): 263.)

NKG2A, also known as KLRC1 (killer cell lectin-like receptor C1) in humans, belongs to the NKG2 family and is a group of transmembrane proteins expressed on both T cells and NK cells. The protein family is characterized by type II membrane orientation and the presence of a type C lectin domain. NKG2A is expressed as a heterodimer with CD94 (killer cell lectin-like receptor D1, also known as KLRD 1) on the cell surface of humans and mice, and recognizes the non-classical class I major histocompatibility complex (MHC-I) molecule Human Leukocyte Antigen (HLA) -E in humans and Qa-1 ^b in mice. Binding of NKG2A/CD94 to its cognate ligand inhibits T-cell effector function and NK-cell effector function (Le Drean et al, 1998, rapaport et al, 2015).

Disclosure of Invention

For the above purpose, the present disclosure relates to antibodies or antigen-binding fragments thereof that bind NKG 2A. Antibodies or antigen-binding fragments thereof that bind NKG2A may improve immune responses (e.g., innate immune responses). In addition, antibodies or antigen-binding fragments thereof that bind to NKG2A may be used in the diagnosis, prevention or treatment of oncologic related diseases (e.g., cancer treatment), inflammatory or autoimmune diseases and infectious diseases.

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

In one embodiment, the VH and VL are selected from a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 11, b) a VL comprising an amino acid sequence having at least 80%, 98%, 99%, 100% sequence identity to SEQ ID NO: 12, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 13, a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO. 13, c) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% sequence identity to SEQ ID NO. 14, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 15, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 16, d) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17, and a VL comprising an amino acid sequence having at least 80%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the amino acid sequence of SEQ ID NO: 18, e) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% sequence identity to SEQ ID NO: 19, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 20, f) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 22, g) a VL comprising an amino acid sequence having at least 80%, 99%, 100% sequence identity to SEQ ID NO: 22, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 23, a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO. 23, h) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% sequence identity to SEQ ID NO. 24, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 25, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO. 25, i) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 26, and a VH comprising an amino acid sequence having at least 80%, 98%, 99% or 100% sequence identity to SEQ ID NO. 27, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the amino acid sequence of SEQ ID NO. 28, j) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 28, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% sequence identity to SEQ ID NO. 29, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 30, k) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 31, l) a VL comprising an amino acid sequence having at least 80%, 99% and 100% sequence identity to SEQ ID NO: 32, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 33, a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO. 33, m) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% sequence identity to SEQ ID NO. 34, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 35, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO: 42, n) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13, and a VL comprising an amino acid sequence having at least 80%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the amino acid sequence of SEQ ID NO. 43, o) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 43, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% sequence identity to SEQ ID NO. 29, VL of amino acid sequences of 95%, 96%, 97%, 98%, 99%, 100% sequence identity.

In some embodiments, the antibody or antigen-binding fragment thereof that binds NKG2A does not bind NKG2E (killer lectin-like receptor C3, KLRC 3) or binds little to NKG 2E.

In some embodiments, an antibody or antigen-binding fragment thereof that binds NKG2A improves an immune response (e.g., an innate immune response).

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

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

In some embodiments, an antibody or antigen-binding fragment thereof that binds NKG2A increases the response of Natural Killer (NK) cells (e.g., NK cells expressing NKG 2A).

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

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to NKG2A, which antibody or antigen-binding fragment thereof binds to a peptide as set forth in SEQ ID No. 8.

In another aspect, the present disclosure provides a binding site comprising an amino acid sequence as set forth in SEQ ID NO. 8.

In another aspect, the disclosure relates to an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided above that binds NKG 2A.

In another aspect, the disclosure relates to an isolated vector comprising a polynucleotide as described above.

In another aspect, the present disclosure relates to a host cell comprising an isolated polynucleotide or an isolated vector as described above.

In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds NKG2A, an isolated polynucleotide, an isolated vector, or a host cell, and a pharmaceutically acceptable carrier.

In another aspect, provided herein is the use of an antibody or antigen-binding fragment thereof, an isolated polynucleotide, an isolated vector, a host cell, or a pharmaceutical composition that binds NKG2A in the manufacture of a therapeutic agent for diagnosing, preventing, or treating a disease.

In another aspect, provided herein is a method of diagnosing, preventing or treating a subject suffering from a oncologically-related disease, inflammatory or autoimmune disease, or infectious disease, comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment thereof, isolated polynucleotide, isolated vector, host cell, or pharmaceutical composition provided above that binds NKG 2A.

In some embodiments, the method comprises enhancing an immune response or reducing adverse effects. In some embodiments, the disease is ameliorated by stimulation of immune cells.

Drawings

The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

FIG. 1 shows the binding activity of cAb017, tab1, tab2, tab3 and isotype hIgG4 to CHOK1-hNKG2A/CD94 cells detected by FACS.

FIG. 2 shows the binding activity of cAb017, tab1, tab2, tab3 and isotype hIgG4 to CHOK1-cynoNKG A/CD94 cells detected by FACS.

FIG. 3 shows the binding activity of cAb017, tab1, tab2, tab3 and isotype hIgG4 to NK92 cells detected by FACS.

FIG. 4A shows the binding specificity of cAb017, tab1, tab2, tab3 and isotype hIgG4 to CHOK1-hNKG2A/CD94 cells detected by FACS.

Figure 4B shows binding of the cAb017, tab1, tab2, tab3, positive control (antibodies recognized by NKG 2E) and human IgG4 isotype to NKG2E detected by ELISA assay, each test antibody at a concentration of 200 nM, 20 nM, 2 nM, 0 nM, respectively, from left to right.

FIG. 5 shows blocking activity of cAb017, tab1, tab2, tab3 and isotype hIgG4 binding to CHOK1-NKG2A/CD94 cells.

FIG. 6 shows the effect of cAb017, tab1, tab2, tab3 and isotype hIgG4 on NK92 mediated cytotoxicity detected by FACS.

FIGS. 7A and 7B show the effect of cAb017, tab1, tab2, tab3 and isotype hIgG4 on CD107A degranulation in NKG2A ⁺ NK cells or NKG2A ^- NK cells, respectively.

FIG. 8 shows the effect of cAb017, tab1, tab2, tab3 and isotype hIgG4 on primary NK mediated cytotoxicity.

FIG. 9 shows the functional activity of cAb017, tab1, tab2, tab3 and isotype hIgG4 in CHO/OKT3/HLA-E stimulated Jurkat cell lines expressing NKG2A/CD94 to reverse the inhibition of NFAT signaling.

FIG. 10 shows the functional activity of cAb017 and Tab1 to activate NFAT signaling in Jurkat cell lines expressing NKG2C/CD94/DAP 12. hIgG4 is a human IgG4 isotype.

FIG. 11 shows the binding activity of cAb017, H17.H28, H17.H29, H17.H230 and isotype hIgG4 to 293T-hNKG2A/CD94 cells detected by FACS.

FIG. 12 shows the activity of cAb017 and cAb 017-derived humanized variants H17.H28, H17.H29, H17.H230 to block the interaction between NKG2A/CD94 and HLA-E as assessed by competitive FACS.

FIG. 13 shows binding activity of cAb017 and cAb017 derived humanized variants H17.H68, H17.H69, H17.H70, H17.H71, H17.H72, H17.H73, H17.H74, H17.H75, H17.H76 and isotype hIgG4 to 293T/NKG2A/CD94 cells detected by FACS.

FIGS. 14A and 14B show the functional activity of cAb017 and cAb017 derived humanized variants H17.H28, H17.H68, H17.H69, H17.H70, H17.H71, H17.H72, H17.H73, H17.H74, H17.H75, H17.H76 and isotype hIgG4 in CHO/OKT3/HLA-E stimulated Jurkat cell lines expressing NKG2A/CD94 to reverse inhibition of NFAT signaling.

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

FIG. 16 shows the functional activity of cAb017, cAb 017-derived humanized variants (H17.H28, H17.H68, H17.H69, H17.H70, H17.H71, H17.H72), hIgG4 and cells only (from left to right) to activate NFAT signaling in Jurkat cell lines expressing NKG2C/CD94/DAP 12.

Figure 17 shows the functional activity of cAb017, a humanized variant derived from cAb017 (h17.h28, h17.h73, h17.h74, h17.h75, h17.h76), hIgG4 and cells only (from left to right) to activate NFAT signaling in Jurkat cell lines expressing NKG2C/CD94/DAP 12.

Fig. 18 shows the functional activity of cAb017, h17.h73, hotspot removal variants h17.h73.dr02, h17.h28, hotspot removal variants h17.h28.dr02, higg4 and Jurkat alone (left to right) to activate NFAT signaling in Jurkat cell lines expressing NKG2C/CD94/DAP 12.

FIGS. 19A and 19B show the functional activity of cAb017 and Tab1 to activate NK cell activity in the NKG2C+ population. hIgG4 is a human IgG4 isotype.

Figure 20 shows the effect of cAb017, h73.Dr02 and h28.dr02 in NK92 mediated cytotoxicity assays.

Detailed Description

The present disclosure will be explained in more detail below. This description is not intended to be an inventory of all the different ways in which the invention may be implemented or all the features that may be added to the invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, many variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in view of this disclosure without departing from the invention. Thus, the following description is intended to illustrate some specific embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the test practice of the present disclosure, the preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.

Antibodies or antigen-binding fragments thereof that bind NKG2A

Provided herein are novel antibodies or antigen-binding fragments thereof that bind to NKG2A, wherein the antibodies or antigen-binding fragments comprise a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 36, RIDPANXNTKYAPNFQG (SEQ ID NO: 37), and 38, respectively, and LCDR1, LCDR2, and LCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 39, 40, and 41, respectively.

In some embodiments, X is G or a. In some embodiments, X is G. In some embodiments, X is a.

As used herein, "antibody" refers to a polypeptide of the immunoglobulin (Ig) family that binds to an antigen. For example, a naturally occurring IgG-type "antibody" is a tetramer comprising at least two heavy (H) chains and two light (L) chains, which are interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region is composed of one domain. VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. See generally Fundamental Immunology [ basic immunology ], chapter 7 (Paul, W.edition, 2 nd edition, raven Press (RAVEN PRESS), new York (1989)).

The term "antigen-binding fragment" refers to antibody fragments, including Fab, F (ab) ₂、Fab'、F(ab')₂, fv, domain antibodies (dabs), other monovalent and bivalent fragments, complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv, scFab, and scFabAC), chimeric antibodies, diabodies, triabodies, minibodies, nanobodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding to the polypeptide, as well as fusions and derivatives of the foregoing. Antigen binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. See, for example, holliger and Hudson, nature Biotechnology [ Nature Biotechnology ] 23:1126-1136 (2005) and Hust et al, BMC Biotech [ BMC Biotechnology ] 7:14 (2007).

In some embodiments, to generate humanized antibodies using CDR grafting, the Kabat definition is used to define CDRs in addition to heavy chain CDR1, which is defined using a combination of Kabat and Chothia systems.

Kabat and Chothia systems are known to those skilled in the art, see, for example, kabat E A, wu T, perry H M et al, sequence of Proteins of Immunological Interest [ sequences of immunologically interesting proteins ] [ J ], 1991, chothia, C.et al, (1987) J.mol. Biol. [ journal of molecular biology ], 196:901917. AlLazikani et al, (1997) J.molecular.biol. [ journal of molecular biology ] 273:927948。

In some embodiments, HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOS: 36, RIDPANXNTKYAPNFQG (SEQ ID NOS: 37) and 38, respectively, X is G, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOS: 39, 40 and 41, respectively.

In some embodiments, HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOS: 36, RIDPANXNTKYAPNFQG (SEQ ID NOS: 37) and 38, respectively, X is A, and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOS: 39, 40 and 41, respectively.

Amino acids in the present disclosure are shown in standard single letter codes, as is well known to those skilled in the art.

In some embodiments, VH and VL are selected from the following:

a) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 10, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 11;

b) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 12, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 13;

c) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 14, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 15;

d) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 16, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 17;

e) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 18, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 19;

f) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 20, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 21;

g) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 22, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 23;

h) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 24, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 25;

i) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 26, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 27;

j) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 28, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 29;

k) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 30, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 31;

l) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 32, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO. 33;

m) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 34, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO. 35;

n) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 42, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO. 13;

o) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 43, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID NO. 29.

The percent identity of two amino acid sequences is determined by dividing the number of identical residues by the total number of amino acid residues and multiplying the quotient by 100 to obtain the percent. The estimated identity space-time bits are not counted. Thus, two copies of the identical sequence have 100% identity, but sequences with deletions, additions or substitutions may have a lower degree of identity. Those skilled in the art will recognize that there are several computer programs that can be used to determine sequence identity, such as those that use algorithms such as BLAST. BLAST nucleotide searches were performed using the NBLAST program and BLAST protein searches were performed using the BLASTP program, with default parameters for each program.

In the present disclosure, antibodies or antigen-binding fragments thereof that bind NKG2A include conservative substitutions within their sequences, preferably without significantly affecting the desired activity of the polypeptide, antibody, or antigen-binding fragment. Substitutions may be naturally occurring or may be introduced, for example, using mutagenesis (e.g., hutchinson et al, 1978, J. Biol. Chem. J. Biochemistry. 253:6551). For example, the amino acids glycine, alanine, valine, leucine and isoleucine can generally be substituted for each other (amino acids having aliphatic side chains). Among these possible substitutions, glycine and alanine are preferred for substitution with each other (because they have relatively short side chains), and valine, leucine and isoleucine for substitution with each other (because they have larger aliphatic side chains that are hydrophobic). Other amino acids that may be substituted with one another in general include, but are not limited to, phenylalanine, tyrosine, and tryptophan (amino acids having aromatic side chains), lysine, arginine, and histidine (amino acids having basic side chains), aspartic acid and glutamic acid (amino acids having acidic side chains), and asparagine and glutamine (amino acids having amide side chains).

In some embodiments, the VH comprises or has the amino acid sequence shown as SEQ ID NO.10, the VL comprises or has the amino acid sequence shown as SEQ ID NO.11, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 12, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 13, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 14, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 15, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 16, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 17, the VH comprises or has the amino acid sequence shown as SEQ ID NO. 18, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 19, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 20, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 22, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 23, the VL comprises or has the amino acid sequence shown as SEQ ID NO. 24, the VH comprises or has the amino acid sequence shown as SEQ ID NO. 26, the VH comprises or has the amino acid sequence shown as SEQ ID NO. 26, and has the amino acid sequence shown or has the amino acid sequence shown as SEQ ID NO. 28, VL comprises or has the amino acid sequence shown in SEQ ID NO. 31, VH comprises or has the amino acid sequence shown in SEQ ID NO. 32, VL comprises or has the amino acid sequence shown in SEQ ID NO. 33, VH comprises or has the amino acid sequence shown in SEQ ID NO. 34, VL comprises or has the amino acid sequence shown in SEQ ID NO. 35, VH comprises or has the amino acid sequence shown in SEQ ID NO. 42, VL comprises or has the amino acid sequence shown in SEQ ID NO. 13, or VH comprises or has the amino acid sequence shown in SEQ ID NO. 43, VL comprises or has the amino acid sequence shown in SEQ ID NO. 29, or conservative substitutions thereof.

In some embodiments, the antibody or antigen binding fragment encompasses (unless otherwise indicated or otherwise suggested by context) a monoclonal antibody, polyclonal antibody, murine antibody, hamster antibody, goat antibody, rabbit antibody, chimeric antibody, primate-made antibody, humanized antibody, (fully) human antibody, multimeric antibody, heterodimeric antibody, semi-dimeric antibody, bivalent antibody, trivalent antibody or tetravalent antibody, bispecific antibody, single chain antibody (e.g., scFv, scFab, and scFabAC), diav, diabody, triabody or tetrabody, single domain antibody, and modified Fab fragment. In certain embodiments, the antibody or antigen binding fragment is monovalent.

In some embodiments, the antibody or antigen binding fragment comprises an Fc fragment that is to be in communication with the immune system when the antibody binds to its target, the Fc fragment can be any class (e.g., igG, igE, igM, igD or IgA) or subclass of immunoglobulin molecule, preferably the Fc fragment is an IgG molecule. In some embodiments, the Fc fragment is a human wild-type IgG Fc fragment. In certain embodiments, the Fc fragment is a human IgG, e.g., igG1, igG2, igG3, or IgG4, optionally having one or more mutations compared to the wild-type human IgG molecule. An exemplary Fc fragment is human IgG4 with an S228P mutation, which means that amino acid S (standard single letter code) at position 228 (defined by Kabat) is mutated to amino acid P.

The antibody or antigen binding fragment is chimeric or humanized.

Typically, chimeric antibodies comprise the heavy chain variable region and/or the light chain variable region (including CDRs and framework residues) of one species (typically a mouse) fused to the constant region of another species (typically a human). Humanized antibodies typically comprise the heavy and/or light chain CDRs from a murine antibody grafted into a non-human primate antibody variable region framework or a human antibody variable region framework, typically further comprising human constant regions. See, e.g., riechmann et al (1988) Nature, 332:323-327.

Methods of making all of the antibodies or antigen binding fragments described above are well known to those of skill in the art. See, for example, U.S. Pat. No. 5,807,715; morrison et al (1984) Proc. Natl Acad. Sci. USA [ Proc. Natl Acad. Sci. USA (21): 6851-5; sharon et al (1984) Nature [ Nature 309 (5966): 364-7; takeda et al (1985) Nature [ Nature 314 (6010): 452-4).

In certain embodiments, antibodies or antigen binding fragments are produced by the Selective Lymphocyte Antibody Method (SLAM) (Babcook et al, 1996, proc. Natl. Acad. Sci [ Proc. Natl. Acad. Sci., U.S. Sci., 93, 7843-7848; de Wildt et al, 1997, J. Immunol. Methods, 207:61-67 and LAGERKVIST et al, 1995, bioTechniques, biotechnology, 18:862-869) which enable isolation from any type of cells that produce high affinity antibodies during an in vivo immune response. The above method relies on the isolation of individual cells that produce antibodies, followed by clonal expansion of the cells, followed by screening of those clones that produce anti-NKG 2A antibodies, followed by the identification of the sequences of their Variable Heavy (VH) and light (VL) genes. Thus, B cells positive for antibodies against NKG2A were isolated. The B cells may be from human, mouse, rat, hamster, rabbit, goat, or other mammalian species. Antibody genes in these B cells can be cloned and expressed in host cells (e.g., e.coli), for example, by conventional recombinant DNA techniques. Cells expressing the antibody may be purified by conventional methods. If the antibodies are from a non-human source, they may be humanized by conventional methods (e.g., by mutagenesis of their genes). The humanized antibody may then be expressed in the host cell and may be purified.

Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler and Milstein, nature [ Nature ], 1975, 256:495-497), three-source hybridoma technology, human B-cell hybridoma technology (Kozbor et al, immunology Today's Immunology, 1983, 4, 72), and EBV-hybridoma technology (Cole et al, "Monoclonal Antibodies AND CANCER THERAPY [ monoclonal antibody and cancer treatment ]", pages 77-96, alan R.List, inc., 1985). Methods for producing and manufacturing recombinant antibodies are well known in the art (see, e.g., simmons et al, 2002, journal of Immunological Methods [ journal of immunological methods ], 263, 133-147).

Antibodies or antigen binding fragments of the present disclosure may also be generated using a variety of phage display Methods known in the art, including Methods disclosed by Brinkman et al, 1995, J. Immunol Methods [ journal of immunology ], 182:41-50; ames et al, 1995, J. Immunol Methods [ journal of immunology ], 184, 177-186; kettleborough et al, 1994, eur. J. Immunol. [ European journal of immunology ], 24, 952-958.

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

In some embodiments, the antibodies or antigen binding fragments provided herein bind to a peptide as set forth in SEQ ID NO. 8. In some embodiments, the antibodies or antigen binding fragments provided herein bind to a peptide as set forth in SEQ ID NO. 9.

The present disclosure provides a binding site comprising an amino acid sequence as set forth in SEQ ID NO. 8 or 9. In certain embodiments, the binding site comprises the amino acid sequence set forth in SEQ ID NO. 8.

NKG2A is an inhibitory receptor, selectively expressed on cytotoxic lymphocytes, including Natural Killer (NK) and CD8 positive (CD 8 ⁺) T cells. It will be appreciated that blocking of NKG2A and one or more ligand MHC-1 molecules thereof, such as HLA-E (human), qa-1 (mouse), advantageously improves the immune response mediated by natural killer cells (NK) and CD8 ⁺ T cells in a subject (e.g., human, mouse) in need thereof. The NKG2A and CD94 molecules (e.g., co-expressed on NK cells or CD8 ⁺ T cells) form the inhibitory isoform NKG2A/CD94 heterodimer. The present disclosure provides examples of antibodies or antigen binding fragments that bind to NKG2A, thereby blocking the interaction between heterodimeric NKG2A/CD94 and MHC-1 molecules (preferably human MHC-1 protein HLA-E).

The present disclosure provides antibodies or antigen binding fragments thereof that bind NKG2A that improve immune responses. In some embodiments, an antibody or antigen-binding fragment thereof provided herein that binds NKG2A improves an innate immune response.

In some embodiments, the antibody or antigen binding fragment blocks the interaction between NKG2A and HLA-E, wherein IC ₅₀ does not exceed 1 nM, 0.8 nM, or 0.6 nM.

In some embodiments, the antibody or antigen binding fragment reverses inhibition of a T cell response, optionally NKG2A/HLA-E mediated inhibition of a T cell response.

In some embodiments, the antibody or antigen binding fragment activates T cells or increases the response of T cells, such as by activating NFAT signaling.

In some embodiments, the antibody or antigen binding fragment activates NFAT signaling in T cells expressing NKG2C (killer lectin-like receptor C2, KLRC 2). In some embodiments, the antibody or antigen binding fragment activates NFAT signaling in a T cell expressing NKG 2A.

In some embodiments, the antibody or antigen binding fragment activates T cells expressing NKG 2C. In some embodiments, the antibody or antigen binding fragment activates T cells expressing NKG 2A.

In some embodiments, the antibody or antigen binding fragment increases the response of NK cells, optionally, NK cells express NKG2A. In some embodiments, the antibody or antigen binding fragment upregulates CD107a (lysosomal associated membrane protein 1). In some embodiments, the antibody or antigen binding fragment increases NK cell response by upregulating CD107 a.

In some embodiments, the antibody or antigen binding fragment improves lysis of cells expressing the MHC-1 molecule. In some embodiments, the antibody or antigen binding fragment improves lysis of cells expressing HLA-E (e.g., tumor cells expressing HLA-E).

The antibody or antigen binding fragment binds to NKG2A with EC ₅₀ no more than 1nM, 0.8 nM, or 0.6 nM, or KD (affinity constant, kd=koff/kon, or kd=kd/Ka) no more than 9.9E-8 nM, 9.9E-9 nM, or 9.9E-10 nM.

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

The antibody or antigen binding fragment exerts an NK-mediated cytotoxic effect on HLA-E expressing tumor cells, wherein EC ₅₀ does not exceed 1 nM, 0.8 nM, 0.5 nM, or 0.3 nM.

EC ₅₀ or IC ₅₀ may be measured by methods well known in the art, such as FACS assays, competitive FACS (fluorescence activated cell sorting). KD can be measured by methods well known in the art, such as biological layer interferometry (Octet).

In some embodiments, the antibody or antigen binding fragment does not bind to NKG2E or binds to NKG2E barely, and thus the antibody or antigen binding fragment does not reduce or decrease the activation activity mediated by NKG2E or NKG2E/CD94 heterodimers.

In some embodiments, the antibody or antigen binding fragment binds weakly to NKG 2C.

In some embodiments, the antibody or antigen-binding fragment is cross-reactive, optionally, the antibody or antigen-binding fragment binds to cynomolgus NKG2A and human NKG 2A.

Polynucleotides, vectors and host cells

The present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof that binds NKG2A, the antibody or antigen-binding fragment thereof comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of the amino acid sequences set forth in SEQ ID NOs 10-35, 42 and 43.

In some embodiments, the isolated polynucleotide encodes an antibody or antigen-binding fragment thereof that binds NKG2A as shown in SEQ ID NOs 10-35, 42 and 43.

The polynucleotide is DNA, RNA, DNA/RNA hybrid, or a modified nucleic acid sequence. In some embodiments, the polynucleotide is a nucleic acid sequence of DNA. The coding polynucleotide (DNA or RNA) may be a recombinant molecule or a synthetic molecule.

The present disclosure also relates to sequence variants of the polynucleotides described above. For example, the disclosure includes nucleic acid sequences, including fragments thereof and complements thereof, that are about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, 99.5%, 99.9%, or 100% identical to any one of the polynucleotide sequences provided herein. The disclosure also includes polynucleotides that differ from the polynucleotide sequences specifically provided herein by virtue of the degeneracy of the genetic code.

The polynucleotide may further include regulatory sequences (e.g., promoter sequences, untranslated 5 'regions, and untranslated 3' regions) and/or vector sequences. For example, polynucleotides constitute vectors.

As used herein, the term "vector" refers to a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. The vector may include one or more of an origin of replication, one or more regulatory sequences that regulate expression of the polypeptide of interest (e.g., such as a promoter and/or enhancer), and/or one or more selectable marker genes (e.g., such as an antibiotic resistance gene and a gene useful in colorimetric assays, such as β -galactosidase).

In some implementations, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using viral expression systems (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses.

In one aspect, the present disclosure also provides an isolated vector comprising a polynucleotide as described herein. The provided isolated polynucleotides can be inserted into vectors for further cloning (amplification of DNA) or for expression using recombinant techniques known in the art.

The vector component typically includes, but is not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1. Alpha.) and a transcription termination sequence.

In some embodiments, provided herein are vectors, at least one promoter operably linked to a nucleic acid sequence (e.g., SV40, CMV, EF-1 a), and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV 40), lambda phage, and M13 phage, plasmids pcDNA3.3、pMD18-T、pOptivec、pCMV、pEGFP、pIRES、pQD-Hyg-GSeu、pALTER、pBAD、pcDNA、pCal、pL、pET、pGEMEX、pGEX、pCI、pEGFT、pSV2、pFUSE、pVITRO、pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS10、pLexA、pACT2.2、pCMV-SCRIPT.RTM.、pCDM8、pCDNA1.1/amp、pcDNA3.1、pRc/RSV、PCR 2.1、pEF-1、pFB、pSG5、pXT1、pCDEF3、pSVSPORT、pEF-Bos, and the like.

The present disclosure provides host cells containing an isolated polynucleotide as described herein or an isolated vector as described herein.

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

Vectors containing polynucleotide sequences encoding antibodies or antigen binding fragments can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the above polynucleotides (nucleic acid sequences of DNA, RNA or DNA/RNA hybrids) in the vectors herein are, for example, prokaryotic cells, such as e.g. e.coli, or other microbial cells, or eukaryotic cells (including but not limited to mammalian cells, such as e.g. human cells, mouse cells, monkey cells, rabbit cells, goat cells, hamster cells or rat cells, insect cells, avian cells, plant cells and eukaryotic cells).

In some embodiments, the host cell may be, for example, (1) a bacterial cell such as E.coli, (2) a fungal cell and Aspergillus (Aspergillus) cell, a yeast cell such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Kluyveromyces lactis (K.lactis) and the like, (3) an insect cell line such as EXPRESSF (a cell line from Spodoptera frugiperda (Spodoptera frugiperda) cell (Protein science Corp.), meriden, conn., USA [ Mei Lideng, conn.); (4) a mammalian cell, or (5) a plant cell.

Typical mammalian cells include COS1 and COS7 cells, chinese Hamster Ovary (CHO) cells, NS0 myeloma cells, NIH 3T3 cells, 293 cells, HEPG2 cells, heLa cells, C127, 3T3, BHK, bowes melanoma cells, L cells, MDCK, HEK293, WI38, murine ES cell lines (e.g., from lines 129/SV, C57/BL6, DBA-1, 129/SVJ), K562, jurkat cells, and BW5147. The invention thus provides cells expressing the antibodies of the invention, including but not limited to hybridoma cells, B cells, plasma cells, and mammalian and human host cells (e.g., adult embryonic stem cells) recombinantly modified to express the antibodies of the invention. Other useful mammalian cell lines are well known and readily available from the American type culture Collection ("ATCC") (Manassas, va., USA [ Manassas, va.) and the national institute of medical science Human genetic cell Bank (National Institute of GENERAL MEDICAL SCIENCES (NIGMS) Human GENETIC CELL receptor) at the Coriell cell Bank (Camden, N.J., USA [ Carmden, N.J.). These cell types are merely representative and this list is not meant to be an exhaustive list.

In some embodiments, the host cell is a mammalian culture cell line, such as CHO, BHK, NS, 293, and derivatives thereof.

The vector may be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., using recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which may be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

Kit for detecting a substance in a sample

The present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof that binds NKG2A, an isolated polynucleotide, an isolated vector, or a host cell as described above. Such kits may further include, if desired, one or more of a variety of conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to those of skill in the art. Instructions for inserting or labeling as an instruction for the amount of the component to be administered, instructions for administration, and/or instructions for mixing the components may also be included in the kit.

Pharmaceutical composition

The present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds NKG2A as described above, an isolated polynucleotide, an isolated vector, or host cell, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated in any manner known in the art.

The pharmaceutical compositions may be formulated for parenteral (e.g., oral, intranasal, or by inhalation, ocular artery, rectal, intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form (i.e., a physically discrete unit containing a predetermined amount of the active compound for ease of administration and dose uniformity). The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, sterile diluents (e.g., sterile water or saline), fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methyl paraben, phenol, ascorbic acid, thimerosal, and the like), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediamine tetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers.

Formulations of the compositions may be formulated and enclosed in ampules, disposable syringes or multiple dose vials. Where desired (as in, for example, an injectable formulation), proper fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant. Absorption of an antibody or antigen-binding fragment thereof may be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release may be achieved by implant and microcapsule delivery systems, which may include biodegradable biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

In some embodiments, the pharmaceutical composition is formulated as an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as, for example, a liquid solution, suspension, emulsion, or solid form suitable for producing a liquid solution, suspension or emulsion. Formulations for injection may include sterile and/or non-exothermic solutions ready for injection, sterile dried soluble products (e.g., lyophilized powders, including subcutaneous injection tablets) ready for combination with a solvent prior to use, sterile suspensions ready for injection, sterile dried insoluble products ready for combination with a vehicle prior to use, and sterile and/or non-exothermic emulsions. The solution may be aqueous or non-aqueous.

In some embodiments, sterile lyophilized powders are prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain other pharmacological components that improve the stability of the powder or excipients that make the reconstituted solution from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other suitable agents.

The antibody or antigen binding fragment, or pharmaceutical composition containing the same, may be included in a container, package, or dispenser alone, or as part of a kit, along with the label and instructions for administration.

Therapeutic method

The present disclosure provides the use of an antibody or antigen binding fragment, isolated polynucleotide, isolated vector, host cell or pharmaceutical composition as described above in the manufacture of a therapeutic agent for the diagnosis, prevention or treatment of a disease. In some embodiments, the therapeutic agent may reverse NGK 2A-mediated immunosuppression or improve an immune response.

The present disclosure provides the use of an antibody or antigen binding fragment, isolated polynucleotide, isolated vector, host cell, or pharmaceutical composition described above in the manufacture of a therapeutic agent for improving an immune response to a disease.

The present disclosure provides a method for diagnosing, preventing or treating a subject suffering from a oncologically related disease, inflammatory or autoimmune disease, or infectious disease, comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment, isolated polynucleotide, isolated vector, host cell, or pharmaceutical composition described above.

The term "therapeutically effective amount" refers to an amount of a composition or active agent disclosed herein that is effective to "treat" a disease in a subject.

As used herein, the term "treating (treat, treating or treatment)" refers to alleviating or ameliorating a disease (i.e., slowing or arresting the progression of the disease or at least one clinical symptom thereof), or alleviating or ameliorating at least one physical parameter or biomarker associated with a disease, including those physical parameters or biomarkers that may not be discernable by a patient. For example, for cancer, "treating (treat, treating or treatment)" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, removing all or part of a tumor, inhibiting or slowing the growth and metastasis of a tumor, slowing the progression of a tumor, or some combination thereof.

As used herein, the term "prevent" (prevent, preventing or prevention) refers to the prophylactic treatment of a disease, or to the delay of the onset or progression of a disease.

In some embodiments, a therapeutic agent (i.e., an antibody or antigen-binding fragment thereof that binds NKG2A, an isolated polynucleotide, an isolated vector, a host cell, or a pharmaceutical composition) or an active agent (e.g., an antibody or antigen-binding fragment thereof that binds NKG 2A) described above can enhance an immune response to a disease, or can reduce an adverse effect in diagnosing, preventing, or treating a disease.

In some embodiments, the methods may optionally enhance immune responses (e.g., innate immune responses) by blocking NKG2A signaling pathways, which may be helpful in treating a number of diseases.

In some embodiments, the method may increase the response of NK cells and/or T cells. In some embodiments, the disease is ameliorated by stimulation of immune cells (e.g., NK cells, T cells). In some embodiments, the method may activate T cells. In some embodiments, the method may activate NK cells, thereby up-regulating CD107a.

In some embodiments, the method can activate NFAT signaling, thereby up-regulating the response of T cells.

In some embodiments, the methods improve lysis of cells expressing HLA-E (e.g., tumor cells expressing HLA-E), where the tumor can be a malignant tumor or a benign tumor.

In some embodiments, the disease comprises a oncology-related disease, an inflammatory or autoimmune disease, or an infectious disease.

In some embodiments, the oncologically related disease includes a solid tumor or a liquid tumor. In some embodiments, oncologically related diseases include malignant tumors and benign tumors. In some embodiments, the oncologically related disease includes breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urinary tract cancer, or hematological malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small Cell Lung Cancer (SCLC), bladder cancer, or metastatic hormone refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is head and neck Squamous Cell Carcinoma (SCCHN), renal Cell Carcinoma (RCC), triple Negative Breast Cancer (TNBC), and colorectal cancer.

In some embodiments, the infectious disease includes viral infectious disease and bacterial infectious disease.

In some embodiments, the inflammatory or autoimmune disease is characterized by redness, swelling, fever, and pain as a result of capillary dilation and edema and migration of phagocytic leukocytes. Some examples of inflammatory responses include arthritis, contact dermatitis, hyper IgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory diseases. Idiopathic inflammatory diseases include, for example, psoriasis and lupus (e.g., systemic Lupus Erythematosus (SLE), drug-induced lupus erythematosus, and lupus nephritis).

In some embodiments, the disease comprises a tumor. In some embodiments, the disease comprises a cancer such as B-cell lymphoma. Without limitation, the method of treatment reduces the rate at which the volume of a tumor increases over time in a subject, reduces the risk of metastasis, or reduces the risk of additional metastasis in a subject. In some embodiments, the treatment may arrest, slow, delay or inhibit the progression of cancer. In some embodiments, the treatment may result in a reduction in the number, severity, and/or duration of one or more symptoms of cancer in the subject.

The subject contains mammals including primates, rodents, canines, and pigs, such as mice, rats, rabbits, cats, dogs, pigs, monkeys, chimpanzees, gorillas, and the like. In some embodiments, the subject contains mice, cynomolgus monkeys, and humans. Unless noted otherwise, the terms "patient" or "subject" are used interchangeably herein.

Daily doses of therapeutic agents may be obtained from cell culture assays, animal studies or clinical studies. A therapeutically effective amount of a therapeutic agent or active agent (e.g., an antibody or antigen binding fragment) will be an amount that treats a disease in a subject, reduces the severity, frequency, and/or duration of one or more symptoms of the disease in the subject. Effectiveness and administration can be determined by a health care professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in the subject. In addition, it will be understood that the particular dosage level of any particular subject will depend on a variety of factors including the activity of the particular compound employed, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antigen binding fragment in vivo.

Examples

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

Standard references that set forth the general principles of recombinant DNA technology known to those skilled in the art include Ausubel et al Current Protocols In Molecular Biology [ Current protocols in molecular biology ], john Wiley & Sons, new York [ New York John Willi father Press ], (1998 and the journal of 2001); sambrook et al Molecular Cloning: A Laboratory Manual [ molecular cloning: laboratory Manual ], 2 nd edition, cold Spring Harbor Laboratory Press, planview, N.Y. (Cold spring harbor laboratory Press, prain, N.Y. (1989); kaufman et al, editions Handbook Of Molecular And Cellular Methods In Biology AND MEDICINE [ handbook of molecular and cellular methods in biology and medicine ], CRC Press, boca Raton [ CRC Press, bokaraton ] (1995); mcPherson, editions Directed Mutagenesis: A PRACTICAL Aproch [ directed mutagenesis: a practical method ], IRL Press, oxford [ IRL, oxford ] (1991).

Standard references that set forth the general principles of Immunology known to those skilled in the art include Harlow and Lane, antibodies: A Laboratory Manual [ Antibodies: laboratory Manual ], 2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. (Cold spring harbor laboratory Press, new York Cold spring harbor ] (1999), and Roitt et al, immunology [ Immunology ], 3 rd edition, mosby European limited (Mosby-Year Book Europe Limited), london (1993). Standard references illustrating general principles of medical physiology and pharmacology known to those skilled in the art include Fauci et al, editors, harrison 'S PRINCIPLES of INTERNAL MEDICINE [ halisen's medical principles ], 14 th edition, mcGraw-Hill Companies, inc.) (1998).

Materials used in the embodiments of the present disclosure are known and commercially available.

Example 1 Generation of reagents

1.1 Reference antibodies

Reference antibodies Tab1, tab2 and Tab3 have been produced according to patents WO 2020102501 A1 (SEQ ID NOS: 107 and 118), WO 2019126514 A2 (SEQ ID NOS: 13 and 14) and CN 111153995A (SEQ ID NOS: 1 and 5), respectively. The heavy chain variable region (VH) and light chain variable region (VL) sequences of Tab1, tab2 and Tab3 are shown in table 1 using standard single letter codes.

TABLE 1 variable region sequences of Tab1, tab2 and Tab3

1.2 Stable cell lines

Cell lines CHOK1/hNKG2A, 293T/hNKG2A, CHOK1/cynoNKG a and CHOK1/mNKG a stably expressing human, cynomolgus and mouse NKG2A/CD94 have been generated for hybridoma screening and in vitro assays. CHOK1 cells or 293T cells were transfected with human (or "h"), cynomolgus monkey (or "cyno") or mouse (or "m") NKG2A/CD94 expression plasmids and selectively cultured in medium containing 0.2 μg/mL puromycin for 2 weeks. The monoclonal cells were then isolated by limiting dilution and screened by FACS to obtain monoclonal cell lines stably expressing human, cynomolgus monkey or mouse NKG2A/CD 94. To generate a stable pool of Jurkat-NFAT-human NKG2A/CD94, jurkat-NFAT cells were transfected with human NKG2A and CD94 expression plasmids and selectively cultured in medium containing 400. Mu.g/mL hygromycin and 0.2. Mu.g/mL puromycin. To generate a stable pool of Jurkat-NFAT-human NKG2C/CD94, jurkat-NFAT cells were transfected with human NKG2C/CD94 and DAP12 (transmembrane immune signaling linker, TYROBP) expression plasmids and selectively cultured in medium containing 800. Mu.g/mL hygromycin and 0.2. Mu.g/mL puromycin.

1.3 Recombinant proteins

Human NKG2A/CD94 extracellular domain with human Fc tag (ECD, UNIProt_P26715, pro94-Leu233, accession number #Q13241-1, lys32-Ile 179) recombinant proteins were purchased from the intelligent pharmaceutical technologies Co., ltd (CHEMPARTNER) for immunization and hybridoma screening.

Human CD94 extracellular domain (ECD, accession # Q13241-1, lys32-Ile 179) recombinant protein with a 6 x his tag was purchased from Baiposis Biotechnology Co., ltd (Acro Biosystem).

Human NKG2E extracellular domain with human Fc tag (ECD, accession # Q07444-1, glu98-Ser 240) recombinant protein (rhNKG E-ECD-Fc protein) was purchased from R & D System for specific characterization.

HLA-E.times.01.03 HLA-A precursor recombinant protein of 3-11 tetramer-VMAPRTLVL-PE (HLA-E-PE) was purchased from MBL company for cell-based blocking assays.

EXAMPLE 2 hybridoma development and screening

2.1 Immunization and fusion

40 Mice from both strains (SJL, NOD) were immunized with Fc tagged human NKG2A/CD94 ECD-Fc recombinant protein or a human NKG2A/CD94 overexpressing cell line using a rapid immunization strategy. Serum titers of immunized mice were detected by FACS assay using human and cynomolgus NKG2A/CD94 overexpressing cell lines as antigens. When the serum titer reaches high levels, a final boost is performed. Three days after the final boost, pooled spleen cells were harvested and fused with SP2/0 mouse myeloma cells. The fused cells were then seeded into 384-well plates for selection.

2.2 Primary and secondary sieves

10-12 Days after fusion, the supernatants harvested from each well of hybridoma cells were assayed by ACUMEN for primary screening using CHOK1/hNKG2A/CD94 and CHOK1/hNKG2C/CD94 as antigens. Hybridoma cells in positive wells were expanded and rescreened for confirmation using the same ELISA assay as the primary screen. FACS binding with CHOK1/hNKG2A/CD94, CHOK1/cynoNKG A/CD94 and CHOK1 is also included in the rescreen. Hybridoma cells that split antibodies with the highest binding to human NKG2A and cross-reactively bound to cynomolgus monkey activity were then subcloned.

2.3 Subcloning and screening of hybridomas

Selected hybridoma cells were limited diluted into 96-well plates at a density of 1 cell/well to obtain monoclonal hybridoma cells. Supernatants harvested from these monoclonal cells were screened by the same FACS assay as example 2.2. Antibodies secreted by positive clones were quantified by biolayer interferometry, and then the binding affinity of human NKG2A/CD94 and the activity of blocking the interaction between NKG2A/CD94 and its ligand HLA-E were determined. As shown in Table 2, the monoclonal antibody secreted from clone 147B6-2E10 exhibited nanomolar human NKG2A/CD94 binding affinity and good blocking activity.

TABLE 2 characterization summary of mouse antibodies

EXAMPLE 3 production and characterization of chimeric antibodies

3.1 Production of chimeric antibodies

The heavy and light chain variable regions of the monoclonal antibodies secreted by clone 147B6-2E10 were sequenced. Based on the sequencing results, a human IgG4 chimeric antibody with an S228P mutation in the Fc region was generated and designated cAb017, where the suffix "c" represents the chimeric. The variable region of cAb017 is shown below with standard single letter codes, with the CDRs underlined. In addition to the heavy chain CDR1 being defined using a combination of Kabat and Chothia systems, CDRs are defined using the Kabat definition.

VH：EVQLQQSVAELVRPGASVKLSCTGSGFNIQNTYMHWVKQRPEQGLEWIGRI DPANGNTKYAPNFQGKATILAETSSNTAYLQLSSLTSEDTAIYYCAREGGDYYGSSYALAYWGQGASVTVSS（SEQ ID NO: 10）

VL：DVQIIQSPSYLAASPGETITINCRASKSISKFLAWYQEKPGKTNKLLIYSGSTLQ SGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEFPWTFGGGTKVEIK（SEQ ID NO: 11）

3.2 Characterization of chimeric antibodies

3.2.1 Binding Activity

The binding activity of human NKG2A/CD94 of the three reference antibodies Tab1, 2, 3 and human IgG4 isotype (hIgG 4) was detected by FACS assay with cAb017 using CHOK1-hNKG2A/CD94 or 293T-hNKG2A/CD94, CHOK1-cynoNKG2A/CD94, NK92 cells as antigen. All test monoclonal antibodies (mAbs) bound strongly to CHOK1-hNKG2A/CD94 (FIG. 1), CHOK1-cynoNKG A/CD94 (FIG. 2) and NK92 (FIG. 3) with similar affinity. Table 3 summarizes EC ₅₀ and the highest geometric Mean Fluorescence Intensity (MFI) values analyzed using a four parameter nonlinear fit of GRAPHPAD PRISM 9.0.0.

TABLE 3 binding of cAb017 and Tab1, 2, 3 to Membrane NKG2A/CD94

3.2.2 Affinity detection

The binding affinities of cAb017 and Tab1, 2,3 to human NKG2A/CD94 ECD recombinant proteins were determined using biological layer interferometry (Octet). Association and dissociation curves were fitted with a 1:1 binding model and the Ka/Kd values for each antibody were calculated and summarized in table 4. The cAb017 (KD, 0.028 nM) showed higher antigen binding affinity than all reference antibodies in the test.

TABLE 4 human NKG2A/CD94 binding affinities of cAb017, tab1, tab2 and Tab3

3.2.3 Binding specificity

Binding of cAb017, tab1, tab2 and Tab3 to NKG2C was detected by FACS assay using CHOK1-hNKG2C/CD94 cells (fig. 4A) as antigen (see methods described in example 3.2.1). As shown in fig. 4A, cAb017 and Tab1 may weakly bind to NKG2C, whereas Tab2 and Tab3 do not bind to NKG 2C.

Binding of cAb017, tab1, tab2, tab3, NKG2E recognized antibodies (positive control) and human IgG4 isotype to NKG2E was detected by ELISA assay using rhNKG E-ECD-Fc protein (FIG. 4B) as antigen. As shown in fig. 4B, none of the test antibodies recognized the NKG2E protein.

3.2.4 Blocking Activity

The activity of cAb017 and Tabs 1, 2,3 blocking the interaction between NKG2A/CD94 and HLA-E was evaluated by competitive FACS. Briefly, CHOK1-NKG2A/CD94 cells were pre-incubated for 30min with 10. Mu.g/mL of test antibody or IgG4 isotype. Recombinant proteins of HLA-E-PE complex tetramers were then added and incubated for an additional 1 hour. Blocking activity was determined by quantifying the blocking of ligand binding to CHOK1-NKG2A/CD94 cells. As shown in FIG. 5, cAb017 and Tab1, 2 may block completely the interaction between NKG2A/CD94 and HLA-E, while Tab3 may block only partially the interaction between NKG2A/CD94 and HLA-E. Table 5 summarizes IC ₅₀ values and highest blocking rate analyzed using a four parameter nonlinear fit of GRAPHPAD PRISM 9.0.0.

TABLE 5 blocking Activity of cAb017, tab1, tab2 and Tab3

3.2.5 NK92 mediated cytotoxicity assays

The effect of cAb017, tab1, tab2 and Tab3 in NK92 mediated cytotoxicity was studied. Briefly, serial dilutions of antibodies and NK-92 cells (three replicates) were added to U-bottom 96-well microtiter plates for pre-incubation. Target cells (tumor cell line LCL 721.221) were loaded with specific peptides to induce HLA-E expression. Target cells were labeled with fluorescent enhancing ligand (DELFIA bat da reagent) and 100 μl (1×10 ⁴) of target cells were added to each well. After a short centrifugation, the CO-cultures were incubated 2h at 37 ℃ in a 95% humidification chamber containing 5% CO ₂. They were then centrifuged 5 min and 20 μl of supernatant was extracted from each well and added to 100 μl of europium solution (Eu). The percentage of specific release (lysis) was calculated using the following formula:

% specific release = × 100

As shown in FIG. 6, all of the test antibodies improved specific lysis of HLA-E expressing tumor cells. And cAb017 and Tab3 showed the strongest efficacy. EC ₅₀ values were analyzed using a GRAPHPAD PRISM 9.0.0 four parameter nonlinear fit and are shown below.

Span = maximum specific release-minimum specific release

3.3.6 CD107a threshing assay

The effect of cAb017, tab1, tab2 and Tab3 on NK cell degranulation was studied using this assay. Briefly, NK cells were isolated from human PBMC using Ficoll gradient and human NK cell isolation kit (Miltenyi Biotech). NK cells were incubated overnight with recombinant human (rh) IL-2 (400 IU/ml). After activation, cell viability was greater than 90%, as indicated by the cytometer measurements. NK cells were then co-cultured with 5×l0 ⁴ HLA-E expressing human B lymphoblastic target cell lines 721.221 for four hours in the presence of anti-human NKG2A antibodies or hIgG4 isotype control. Cells were then harvested and stained for anti-human CD3, anti-human CD56, live/dead (live/dead), and anti-human CD107a, and anti-NKG 2A antibodies. Fresh cells were obtained on BD Canto II. The percent degranulation (% CD 107) in NKG2a ⁺ or NKG2a ^- NK cells was then analyzed on FlowJo (flow cytometry software). NK cell degranulation was increased in the presence of cAb017, tab1, tab2 and Tab3 as measured by CD107A expression by flow cytometry (fig. 7A). No increase in NK cell response was observed on NKG2A negative (-) NK cells, indicating that the antibody effect was specific for NK cells expressing NKG2A (fig. 7B).

3.2.7 Primary NK cytotoxicity assay

The effect of cAb017, tab1, tab2 and Tab3 on primary NK mediated cytotoxicity was studied using this assay. Briefly, NK cells were isolated from human PBMC using Ficoll gradient and human NK cell isolation kit (meitian gentle biotechnology). NK cells were cultured with recombinant human IL-2 (Peprotech, catalog No. 200-02) and recombinant human IL-12 (Peprotech, catalog No. 200-12) for 4-5 days. After NK activation, serial dilutions of antibodies and NK cells were added to the U-bottom 96-well microtiter plate for pre-incubation. Target cells (tumor cell line LCL 721.221) were loaded with specific peptides to induce HLA-E expression. Target cells were labeled with a fluorescence enhancing ligand (DELFIA bat da reagent) and 100 ul (1×10 ⁴) of target cells were added to each well. After a short centrifugation, the CO-cultures were incubated 2h at 37 ℃ in a 95% humidification chamber containing 5% CO ₂. They were then centrifuged 5 min and 20 μl of supernatant was extracted from each well and added to 100 μl of europium solution (Eu). The percentage of specific release (specific cytotoxicity) was calculated using the following formula:

% specific release = × 100

As shown in fig. 8, cAb017 and Tab3 significantly improved primary NK-mediated specific lysis against HLA-E expressing tumor cells.

3.2.8 Jurkat-NFAT NKG2A/CD94 reporter assay

The functional activity of cAb017, tab1, tab2 and Tab3 to reverse inhibition of NFAT signaling was tested in a CHO/OKT3/HLA-E (Chinese hamster ovary cells engineered to express single chain OKT3 and HLA-E) stimulated Jurkat cell line expressing NKG2A/CD 94. Jurkat effector cells expressing NKG2A were co-cultured with CHO/scOKT3/HLA-E target cells at an effector cell to target cell ratio (E: T) of 6:1. anti-NKG 2A antibodies or hIgG4 isotype antibodies were added to the co-culture at titrated concentrations. After five hours incubation at 37 ℃, one-Glo reagent was used to quantify luciferase activity. Relative Luciferase Unit (RLU) data was plotted using GraphPad software. The NKG2A antibody reversed the NKG2A/HLA-E mediated inhibition of the T-cell response. Specifically, as shown in fig. 9, cAb017 reversed inhibition of NFAT signaling in CHO/OKT3/HLA-E stimulated NKG2A expressing Jurkat T cell line, with EC ₅₀ value of 0.31 nM.

3.2.9 Jurkat-NFAT NKG2C/CD94 reporter assay

The functional activity of cAb017 and Tab1 to activate NFAT signaling was tested in Jurkat cell lines expressing NKG2C/CD94/DAP 12. Test antibodies or isotype antibodies were added to the co-culture at titrated concentrations. After five hours incubation at 37 ℃, one-Glo reagent was used to quantify luciferase activity. Relative Luciferase Unit (RLU) data was plotted using GraphPad software. As shown in fig. 10, in Jurkat T cell line expressing NKG2C, cAb017 can activate NFAT signaling, however Tab1 did not induce NFAT activation.

3.2.10 Epitope analysis

Epitope identification (epi binding) was performed on cAb017, tab1, tab2 and Tab3 by competitive ELISA. Briefly, ab1 was immobilized on ELISA plates overnight at 4 ℃. After blocking with PBS-2% BSA to reduce non-specific binding, the plates were washed with PBS-0.05% Tween 20 and incubated with a mixture of 50. Mu.l/well Ab2 and 50. Mu.l/well biotinylated NKG2A/CD94 ECD recombinant protein for 1 hour at 37 ℃. After washing, horseradish peroxidase (HRP) -labeled detection antibody was added and incubated at 37 ℃ for 1 hour. Color development was performed by adding 100. Mu.l/well TMB solution. After incubation for 10-15 min at Room Temperature (RT), the reaction was stopped by adding 100. Mu.l 1N HCl. The optical density of these plates at 450 nm was then read immediately using a microplate reader.

Finally, the relative binding signal of tandem Ab2 was calculated by normalization. A high relative binding signal indicates low competition between Ab2 and Ab1 for binding antigen. Also, a low relative signal indicates a high competition between Ab2 and Ab 1. As shown in Table 6, all antibodies were in the same bin (bin), although Tab2 was not fully competitive with other antibodies, probably due to the relatively weak affinity for NKG2A/CD 94.

TABLE 6 epitope identification of cAb017, tab1, tab2 and Tab3

The binding epitope for cAb017, tab1, tab2 and Tab3 on NKG2A/CD94 was further mapped using hydrogen deuterium exchange mass spectrometry (HDX-MS). Key peptides "IDNEEEMKF" (SEQ ID NO: 7), "FKHEIKDSDNAEL" (SEQ ID NO: 8) and "LQVNRL" (SEQ ID NO: 9) were identified and designated as peptide-1, peptide-2 and peptide-3, respectively.

TABLE 7 epitope mapping of cAb017, tab1, tab2 and Tab3

Binding of Tab1 and cAb017 resulted in reduced exchange of hydrogen deuterium in peptide-3, indicating that antibody coverage of this region on NKG2A/CD94 may be critical for binding to NKG 2C.

3.2.11 NK activation assay

The effect of cAb017 and Tab1 on NKG 2C-positive NK cells was investigated with this assay. Briefly, NK cells were isolated from human PBMC using Ficoll gradient and human NK cell isolation kit (meitian gentle biotechnology). NK cells were incubated overnight with recombinant human (rh) IL-2 (400 IU/ml). After activation, cell viability was greater than 90%, as indicated by the cytometer measurements. Test antibodies and isotype controls were blotted onto 96-well plates and NK cells were incubated for 6 hours. The percentage of% CD107a and% ifnγ in NKG2C ⁺ NK cells was then analyzed on FlowJo (flow cytometry software). NK cell activity increased in the presence of cAb017 and Tab1 as measured by flow cytometry derived CD107a (FIG. 19A) and% IFN gamma (FIG. 19B) expression. The data shows that the antibody can specifically activate NKG2C positive NK cells and show a stronger potency than the reference antibody (Tab 1).

Example 4 antibody humanization

4.1 Humanized design

The cAb017 was humanized using Complementarity Determining Region (CDR) grafting.

Briefly, IGH1-2 x 06 and IGKV1-33 x 01 were first selected as humanized templates for the heavy and light chains, respectively, based on their homology to the original mouse antibody sequence. In addition to the heavy chain CDR1 being defined using a combination of Kabat and Chothia systems, CDRs are defined using the Kabat definition. For grafting, different combinations of CDRs and classical residues from cAb017 were grafted onto the template. The resulting variants were produced and designated h17.h28 to h17.h30 and h17.h68 to h17.h76, with the prefix "hu" representing "humanized" and the numbers in the suffix representing the sequence numbers. All variants were tested in multiple in vitro assays to select the best variant that retains the properties of the parent antibody.

The sequences of h17.h01 to h17.h24 are shown below.

4.2 Characterization of humanized variants

4.2.1 Binding Activity

Binding of cAb017 and cAb017 derived humanized variants to cell membrane human NKG2A/CD94 was detected by FACS assay using 293T/hNKG2A/CD94 cells. As shown in FIG. 11, H17.H28, H17.H29 and H17.H30 retained the human NKG2A/CD94 binding activity of parent antibody cAb 017. Table 8 summarizes EC ₅₀ and the highest MFI values analyzed using a four parameter nonlinear fit of GRAPHPAD PRISM 9.0.0.

TABLE 8 binding of cAb017 derived humanized variants to 293T/hNKG2A/CD94

4.2.2 Affinity detection

The binding affinity of the cAb 017-derived humanized variant to human NKG2A/CD94 was determined using biological layer interferometry (Octet). Association and dissociation curves were fitted with a 1:1 binding model and the Ka/Kd values for each antibody were calculated and summarized in table 9. H17.h28, h17.h29, h17.h30 retained the human NKG2A/CD94 binding affinity of parent antibody cAb 017.

TABLE 9 human NKG2A/CD94 binding affinity of cAb 017-derived humanized variants

4.2.3 Blocking Activity

The activity of cAb017 and cAb 017-derived humanized variants H17.H28, H17.H29, H17.H30 blocking the interaction between NKG2A/CD94 and HLA-E was assessed by competitive FACS (FIG. 12, cf. Methods described in example 3.2.4). Table 10 summarizes IC ₅₀ values and highest blocking rate analyzed using a four parameter nonlinear fit of GRAPHPAD PRISM 9.0.0.

TABLE 10 blocking Activity of cAb017 and cAb017 derived humanized variants

4.2.4 Functional verification

4.2.4.1 Binding Activity

Human NKG2A/CD94 binding activity of cAb017 and humanized variants was detected by FACS assay using 293T/hNKG2A/CD94 cells as antigen. All tested mAbs, H17.H28, H17.H68, H17.H69, H17.H70, H17.H71, H17.H72, H17.H73, H17.H74, H17.H75, H17.H76 bound strongly to 293T/hNKG2A/CD94 with similar affinities (FIG. 13).

4.2.4.2 Jurkat-NFAT NKG2A/CD94 reporter assay

In CHO/OKT3/HLA-E stimulated Jurkat cell lines expressing NKG2A/CD94, cAb017 and humanized variants were tested for their functional activity in reversing the inhibition of NFAT signaling. Jurkat effector cells expressing NKG2A were co-cultured with CHO/scOKT3/HLA-E target cells at an effector cell to target cell ratio (E: T) of 6:1. anti-NKG 2A antibodies or hIgG4 isotype antibodies were added to the co-culture at titrated concentrations. After five hours incubation at 37 ℃, one-Glo reagent was used to quantify luciferase activity. Relative Luciferase Unit (RLU) data was plotted using GraphPad software. The NKG2A antibody reversed the NKG2A/HLA-E mediated inhibition of the T-cell response. Specifically, as shown in fig. 14A and 14B, in CHO/OKT3/HLA-E stimulated Jurkat T cell lines expressing NKG2A, cAb017 and all humanized variants reversed inhibition of NFAT signaling with similar potency.

4.2.4.3 Binding specificity:

binding of cAb017 and humanized variants derived from cAb017 to NKG2C was detected by FACS assay using Jurkat-NFAT-NKG2C/CD94 cells (fig. 15) as antigen. As shown in fig. 15, all variants can bind to NKG2C.

4.2.4.4 Jurkat-NFAT NKG2C/CD94 reporter assay

The functional activity of cAb017 and humanized variants to activate NFAT signaling was tested in Jurkat cell lines expressing NKG2C/CD94/DAP 12. The test antibodies or isotype antibodies were mixed with anti-human IgG4 Fc antibodies and added to the co-culture at a titration concentration. After five hours incubation at 37 ℃, one-Glo reagent was used to quantify luciferase activity. Relative Luciferase Unit (RLU) data was plotted using GraphPad software. As shown in fig. 16 and 17, all variants activated NFAT signaling in Jurkat T cell lines expressing NKG 2C.

4.2.5 Hot spot removal

CDR regions of the light and heavy chains of h17.h28 and h17.h73 were analyzed, wherein the amino acid sequence NG in the heavy chain CDR2 was mutated to NA to obtain hotspot removal variants h17.h28.dr02 and h17.h73.dr02. VH and VL of h17.h28.dr02 are shown in SEQ ID nos. 42 and 13, respectively. VH and VL of h17.h73.dr02 are shown in SEQ ID nos. 43 and 29, respectively.

4.2.5.1 Affinity assay

The binding affinity of the hot-spot-derived humanized variants to human NKG2A/CD94 was determined using biological layer interferometry (Octet). Association and dissociation curves were fitted with a 1:1 binding model and the Ka/Kd values for each antibody were calculated and summarized in table 11. H17.h28.dr02 and h17.h73.dr02 retain the human NKG2A/CD94 binding affinity of the parent antibody.

TABLE 11 human NKG2A/CD94 binding affinity of cAb 017-derived humanized variants

4.2.5.2 Jurkat-NFAT NKG2C/CD94 reporter assay

H17.H28.DR02 and H17.H73.DR02 were tested for their functional activity in activating NFAT signaling in Jurkat cell lines expressing NKG2C/CD94/DAP 12. The test antibodies or isotype antibodies were mixed with anti-human IgG4 Fc antibodies and added to the co-culture at a titration concentration. After five hours incubation at 37 ℃, one-Glo reagent was used to quantify luciferase activity. Relative Luciferase Unit (RLU) data was plotted using GraphPad software. As shown in fig. 18, all variants activated NFAT signaling in Jurkat T cell lines expressing NKG 2C.

4.2.5.3 NK92 mediated cytotoxicity assays

The effect of cAb017, h73.Dr02 and h28.dr02 in NK92 mediated cytotoxicity was studied in assay 3.2.5. As shown in fig. 20, all of the test antibodies improved specific lysis of HLA-E expressing tumor cells. H73.dr02 and h28.dr02 show similar efficacy as cAb 017. EC ₅₀ values were analyzed using a GRAPHPAD PRISM 9.0.0 four-parameter nonlinear fit and are shown below:

Span = maximum specific release-minimum specific release

Although particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen to the applicant or others skilled in the art may arise. Accordingly, the appended claims as filed and as may be amended are intended to embrace all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

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

The VL contains complementarity-determining regions LCDR1, LCDR2, and LCDR3, respectively, having amino acid sequences as shown in SEQ ID NO: 39, 40, and 41.

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

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

4. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in claim 1, wherein the VH and the VL are selected from the following:

a) VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11;

b) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13;

c) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15;

d) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17;

e) VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19;

f) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21;

g) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 22, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23;

h) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25;

i) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 26, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27;

j) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 28, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29;

k) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 30, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31;

l) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 33;

m) VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 34, and VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 35;

n) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 42, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13;

o) A VH comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 43, and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29.

5. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in claim 1, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 10, and the VL comprises the amino acid sequence shown in SEQ ID NO: 11;

The VH contains the amino acid sequence shown in SEQ ID NO: 12, and the VL contains the amino acid sequence shown in SEQ ID NO: 13;

The VH contains the amino acid sequence shown in SEQ ID NO: 14, and the VL contains the amino acid sequence shown in SEQ ID NO: 15;

The VH contains the amino acid sequence shown in SEQ ID NO: 16, and the VL contains the amino acid sequence shown in SEQ ID NO: 17;

The VH contains the amino acid sequence shown in SEQ ID NO: 18, and the VL contains the amino acid sequence shown in SEQ ID NO: 19;

The VH contains the amino acid sequence shown in SEQ ID NO: 20, and the VL contains the amino acid sequence shown in SEQ ID NO: 21;

The VH contains the amino acid sequence shown in SEQ ID NO: 22, and the VL contains the amino acid sequence shown in SEQ ID NO: 23;

The VH contains the amino acid sequence shown in SEQ ID NO: 24, and the VL contains the amino acid sequence shown in SEQ ID NO: 25;

The VH contains the amino acid sequence shown in SEQ ID NO: 26, and the VL contains the amino acid sequence shown in SEQ ID NO: 27;

The VH contains the amino acid sequence shown in SEQ ID NO: 28, and the VL contains the amino acid sequence shown in SEQ ID NO: 29;

The VH contains the amino acid sequence shown in SEQ ID NO: 30, and the VL contains the amino acid sequence shown in SEQ ID NO: 31;

The VH contains the amino acid sequence shown in SEQ ID NO: 32, and the VL contains the amino acid sequence shown in SEQ ID NO: 33;

The VH contains the amino acid sequence shown in SEQ ID NO: 34, and the VL contains the amino acid sequence shown in SEQ ID NO: 35;

The VH contains the amino acid sequence shown in SEQ ID NO: 42, and the VL contains the amino acid sequence shown in SEQ ID NO: 13; or

The VH contains the amino acid sequence shown in SEQ ID NO: 43, and the VL contains the amino acid sequence shown in SEQ ID NO: 29, or a conserved substitution thereof.

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

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

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

9. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-8, wherein the antibody or antigen-binding fragment ameliorate the lysis of HLA-E-expressing cells, optionally, the HLA-E-expressing cells include HLA-E-expressing tumor cells.

10. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A increases the response of NK cells, optionally, wherein the NK cells express NKG2A.

11. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A exerts NK-mediated cytotoxicity on tumor cells expressing HLA-E.

12. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A exerts NK-mediated cytotoxicity on tumor cells expressing HLA-E, wherein the _EC50 does not exceed 1 nM, 0.8 nM, 0.5 nM, or 0.3 nM.

13. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A upregulates CD107a (lysosome-associated membrane protein 1).

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

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

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

17. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A binds to NKG2A, wherein the EC ₅₀ does not exceed 1 nM, 0.8 nM or 0.6 nM, or the KD (affinity constant) does not exceed 9.9E-8 nM, 9.9E-9 nM or 9.9E-10 nM.

18. The antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-17, wherein the antibody or antigen-binding fragment thereof that binds to NKG2A blocks the interaction between NKG2A and HLA-E, wherein the _IC50 does not exceed 1 nM, 0.8 nM or 0.6 nM.

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

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

21. An antibody or antigen-binding fragment thereof that binds to NKG2A, wherein the antibody or antigen-binding fragment thereof binds to a peptide as shown in SEQ ID NO: 8.

22. A binding site comprising an amino acid sequence as shown in SEQ ID NO: 8.

23. An isolated polynucleotide, said isolated polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to NKG2A as described in any one of claims 1-21.

24. An isolated vector comprising the isolated polynucleotide as described in claim 23.

25. A host cell comprising the isolated polynucleotide as described in claim 23 or the isolated vector as described in claim 24.

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

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

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

29. The use as claimed in claim 28, wherein the disease includes oncology-related diseases, inflammatory or autoimmune diseases, or infectious diseases.

30. The use as described in claim 28 or 29, wherein the oncology-related diseases include breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies;

The infectious diseases mentioned include viral infectious diseases and bacterial infectious diseases;

The inflammatory or autoimmune diseases mentioned include arthritis, contact dermatitis, hyperIgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory diseases.

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

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

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

34. The method of any one of claims 31-33, wherein the oncology-related diseases include breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies;

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

36. The method of any one of claims 31-35, wherein the method comprises

(1) Increase NK cell response;

(2) Upregulate CD107a;

(3) Activate T cells or increase T cell responses; or

(4) Activate NFAT signal conduction.