Antibodies targeting nkg2a and uses thereof

Abstract

The present invention provides antibodies targeting NKG2A, as well as multispecific antibodies, chimeric receptors, antibody conjugates, pharmaceutical compositions and kits comprising it, and their use in the diagnosis / treatment / prevention of diseases associated with NKG2A expression.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy. More specifically, this invention relates to antibodies targeting NKG2A, and their use in the prevention and / or treatment and / or diagnosis of diseases. Background Technology

[0002] Natural killer (NK) cells are a crucial type of lymphocyte in the body, playing a vital role in both innate and adaptive immunity. NK cells possess two types of surface receptors, classified into inhibitory and activating types based on their function. These receptors mediate different recognition patterns of NK cells, transmitting different activation and inhibition signals. The CD94 / NKG2 family is a well-studied receptor family, mainly including members such as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H. Among them, NKG2A is an inhibitory receptor, and its ligand is the non-classical major histocompatibility complex class I molecule HLA-E. HLA-E molecules expressed on target cells bind to NKG2A, inhibiting the killing function of NK cells. Therefore, antibodies targeting CD94 / NKG2A may enhance the killing activity of tumor-specific lymphocytes against tumor cells.

[0003] Several anti-NKG2A antibodies have been described in the art. For example, Sivori et al. (Eur J Immunol 1996; 26: 2487-92) mentioned mouse anti-NKG2A antibody Z270; Carretero et al. (Eur J Immunol 1997; 27: 563-7) described mouse anti-NKG2A antibody Z199 (now commercially available at Beckman Coulter, Inc., product number IM2750, USA); Vance et al. (J Exp Med 1999; 190: 1801-12) mentioned rat anti-mouse NKG2-antibody 20D5 (now commercially available at BDBiosciences Pharmingen, catalog number .550518, USA); and U.S. Patent Application Publication 20030095965 described mouse antibody 3S9, which is alleged to bind to NKG2A, NKG2C, and NKG2E.

[0004] The present invention aims to provide a novel antibody targeting NKG2A, and its use in disease prevention and / or treatment and / or diagnosis. Summary of the Invention

[0005] In a first aspect, the present invention provides an antibody targeting NKG2A, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6, wherein the 21st amino acid of the light chain variable region is M and the 85th amino acid is T, and the 34th amino acid of the heavy chain variable region is M, the 49th amino acid is A, the 61st amino acid is P, and the 97th amino acid is T.

[0006] In one embodiment, the 22nd amino acid of the light chain variable region is S or T, the 58th amino acid is I or V, and the 104th amino acid is L or V.

[0007] In a preferred embodiment, the light chain variable region of the NKG2A antibody has at least 90% identity with the amino acid sequence selected from SEQ ID NO: 10 and 13, or has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 7, 6, 5, 4, 3, 2) conserved amino acid modifications compared to SEQ ID NO: 10 and 13, and the heavy chain variable region has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 11, or has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 7, 6, 5, 4, 3, 2) conserved amino acid modifications compared to SEQ ID NO: 11. More preferably, the NKG2A antibody comprises a light chain variable region selected from SEQ ID NO: 10 and 13 and a heavy chain variable region as shown in SEQ ID NO: 11.

[0008] In one embodiment, the amino acid sequence of the NKG2A antibody is selected from SEQ ID NO: 12 and 14.

[0009] The present invention also provides a nucleic acid molecule encoding the above-described NKG2A antibody. Therefore, in one embodiment, the nucleic acid molecule encoding the NKG2A antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from SEQ ID NO: 16-17, and the NKG2A antibody encoded therein is capable of specifically binding to NKG2A. Preferably, the nucleic acid molecule encoding the NKG2A antibody is selected from SEQ ID NO: 16-17.

[0010] In another aspect, the present invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising the NKG2A antibody as described above and one or more second antibodies or their antigen-binding portions that specifically bind to other antigens.

[0011] In one embodiment, the second antibody or its antigen-binding portion may be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibody, biantibody, or sdAb.

[0012] The present invention also provides a vector comprising a nucleic acid molecule encoding the above-mentioned NKG2A antibody or multispecific antibody, and a host cell expressing the NKG2A antibody or multispecific antibody.

[0013] In another aspect, the present invention also provides a chimeric receptor comprising one or more NK inhibitory ligands, a transmembrane domain and a signal transduction domain, wherein the NK inhibitory ligand comprises an NKG2A antibody as described above or a multispecific antibody containing the NKG2A antibody, and wherein the signal transduction domain comprises one or more co-stimulatory domains.

[0014] In one embodiment, the chimeric receptor comprises two NK inhibitory ligands, wherein the first NK inhibitory ligand is the NKG2A antibody as described above, and the second NK inhibitory ligand is selected from: (1) antibodies or fragments thereof targeting the following NK inhibitory receptors: LIR1, NKG2B, CD94, LIR2, LIR3, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, and KLRG1; or (2) HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1, PD-L2, CD155, CD112, CD113, Gal-9, FGL1, and the NK inhibitory receptor binding regions contained therein.

[0015] In one embodiment, the signal transduction domain in the chimeric receptor of the present invention comprises one or more co-stimulatory domains. That is, it does not contain primary signal transduction domains, such as those from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0016] In another embodiment, the signal transduction domain of the chimeric receptor of the present invention may further include a primary signal transduction domain, such as the CD3ζ intracellular region.

[0017] The present invention also provides a nucleic acid molecule encoding a chimeric receptor targeting NKG2A as defined above, and a vector comprising said nucleic acid molecule.

[0018] The present invention also provides an engineered immune cell that expresses the chimeric receptor comprising the NKG2A antibody of the present invention, wherein the expression of at least one MHC-related gene is suppressed or silenced.

[0019] In one embodiment, the MHC-related gene is selected from: HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

[0020] In one embodiment, the engineered immune cells expressing the chimeric receptor containing the NKG2A antibody of the present invention further include at least one TCR / CD3 gene whose expression is suppressed or silenced, examples of which include, for example, TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ.

[0021] In one embodiment, the engineered immune cells provided by the present invention also express chimeric antigen receptors that target tumor antigens.

[0022] In one embodiment, the immune cells are selected from T cells, NK cells, NKT cells, macrophages, and dendritic cells.

[0023] In another aspect, the present invention also provides an antibody conjugate comprising an NKG2A antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, radioisotopes, structural portions with extended half-life, detectable markers, and drugs.

[0024] In one embodiment, the structural portion for extending the half-life is selected from: albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind to human serum albumin. In one embodiment, the detectable marker is selected from fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents. In one embodiment, the drug is selected from cytotoxins and immunomodulators.

[0025] In another aspect, the present invention also provides a detection kit comprising the antibodies, multispecific antibodies, antibody-drug conjugates, or chimeric receptors described herein.

[0026] In another aspect, the present invention also provides a pharmaceutical composition comprising the antibody, chimeric receptor, multispecific antibody, engineered cell or antibody conjugate described herein, and one or more pharmaceutically acceptable excipients.

[0027] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing diseases associated with NKG2A expression, comprising administering to a subject an antibody, chimeric receptor, multispecific antibody, antibody-drug conjugate, engineered immune cell, or pharmaceutical composition as described above. Invention Details

[0029] Unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] NKG2A antibody

[0031] In the context of this invention, "Z199 antibody" refers to the mouse anti-NKG2A antibody Z199 as described by Carretero et al. (Eur J Immunol 1997; 27: 563-7), now commercially available through Beckman Coulter, Inc., product number IM2750. "hZ199 antibody" refers to a humanized Z199 antibody comprising a light chain variable region as shown in SEQ ID NO: 7 and a heavy chain variable region as shown in SEQ ID NO: 8, with the full-length amino acid sequence shown in SEQ ID NO: 9.

[0032] The novel NKG2A antibody provided by this invention is obtained by reversing the mutation based on hZ199 to provide higher affinity and better inhibition of NK cell killing.

[0033] As used herein, the term "antibody" has the broadest meaning as understood by those skilled in the art and includes monoclonal antibodies (comprising complete antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences capable of exhibiting desired biological activity. The antibodies described in this invention can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.).

[0034] Typically, a complete antibody consists of two heavy chains and two light chains linked together by disulfide bonds, each light chain being connected to its respective heavy chain via disulfide bonds, forming a "Y"-shaped structure. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain variable region contains three complementation-determining regions (CDRs): CDR-H1, CDR-H2, and CDR-H3, and the heavy chain constant region contains three constant structural domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain variable region contains three CDRs: CDR-L1, CDR-L2, and CDR-L3, and the light chain constant region contains one constant structural domain, CL. Within the heavy / light chain variable regions, the CDRs are separated by more conserved frame regions (FRs). The variable regions of the heavy / light chains are responsible for the recognition and binding of antigens, while the constant regions mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

[0035] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using many numbering schemes well-known in the art, including: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (“Kabat” numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp. Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” JMol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0036] The boundaries of a given CDR or FR can vary depending on the protocol used for identification. For example, the Kabat protocol is based on structure alignment, while the Chothia protocol is based on structural information. Both the Kabat and Chothia protocols number antibodies based on the length of the most common antibody region sequences, placing certain insertions and deletions (“indels”) in different positions, thus producing different numbers. The Contact protocol is based on the analysis of complex crystal structures and is similar to the Chothia numbering protocol in many ways. The AbM protocol is a compromise between the Kabat and Chothia definitions, based on the protocol used by the Oxford Molecular AbM antibody modeling software.

[0037] Therefore, unless otherwise specified, it should be understood that the “CDR” of a given antibody or its region (such as its variable region) encompasses the CDRs defined by any of the above-described schemes or other known schemes. For example, in specifying that a particular CDR (e.g., CDR3) contains a given amino acid sequence, it should be understood that such a CDR may also have the sequence of the corresponding CDR (e.g., CDR3) as defined by any of the above-described schemes or other known schemes. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or its region (such as its variable region) encompasses the FRs defined by any of the above-described schemes or other known schemes. Unless specifically indicated, amino acid numbering herein follows the Chothia scheme.

[0038] As used herein, the term "antibody fragment" or "antigen-binding portion" refers only to a portion of a complete antibody, generally containing the antigen-binding site of the complete antibody and thus retaining the ability to bind antigens. Examples of antibody fragments in this invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragments, Fd', Fv fragments, scFv, disulfide-linked Fv (sdFv), the heavy chain variable region (VH) or light chain variable region (VL) of an antibody, linear antibodies, "dimers" having two antigen-binding sites, single-domain antibodies, nanobodies, natural ligands of said antigens, or functional fragments thereof, etc. Therefore, the term "antibody" in this invention encompasses antibody fragments as defined above.

[0039] The terms “single-chain antibody” and “scFv” are used interchangeably herein and refer to antibodies composed of variable regions (VH) of the heavy chain and variable regions (VL) of the light chain linked by a linker. An optimal linker length and / or amino acid composition can be selected. The linker length significantly affects the folding and interactions of the variable regions of the scFv. In fact, using shorter linkers (e.g., between 5 and 10 amino acids) can prevent intra-chain folding. For information on the selection of linker size and composition, see, for example, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90:6444-6448; U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794; and PCT Publications WO2006 / 020258 and WO2007 / 024715, the entire contents of which are incorporated herein by reference. scFv can contain VH and VL connected in any order, such as VH-connector-VL or VL-connector-VH.

[0040] In a first aspect, the present invention provides an antibody targeting NKG2A, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6, wherein the 21st amino acid of the light chain variable region is M and the 85th amino acid is T, and the 34th amino acid of the heavy chain variable region is M, the 49th amino acid is A, the 61st amino acid is P, and the 97th amino acid is T.

[0041] In one embodiment, the 22nd amino acid of the light chain variable region is S or T, the 58th amino acid is I or V, and the 104th amino acid is L or V.

[0042] In a preferred embodiment, the light chain variable region of the NKG2A antibody has at least 90% identity with the amino acid sequence selected from SEQ ID NO: 10 and 13, or has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 7, 6, 5, 4, 3, 2) conserved amino acid modifications compared to SEQ ID NO: 10 and 13, and the heavy chain variable region has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 11, or has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 7, 6, 5, 4, 3, 2) conserved amino acid modifications compared to SEQ ID NO: 11. More preferably, the NKG2A antibody comprises a light chain variable region selected from SEQ ID NO: 10 and 13 and a heavy chain variable region as shown in SEQ ID NO: 11.

[0043] In one embodiment, the amino acid sequence of the NKG2A antibody is selected from SEQ ID NO: 12 and 14.

[0044] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0045] As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. These conserved modifications include amino acid substitution, addition, and deletion. Modifications can be introduced into the chimeric antigen receptor of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitution of amino acids is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphiphilic properties of the residues involved.

[0046] In one aspect, the present invention also provides a multispecific antibody (preferably a bispecific or trispecific antibody) comprising the NKG2A antibody as described above, which further comprises one or more second antibodies that specifically bind to other antigens.

[0047] As used herein, the term "multispecific" refers to an antigen-binding protein having multiple epitope specificities (i.e., the ability to specifically bind to two, three, or more different epitopes on a single biomolecule or the ability to specifically bind to epitopes on two, three, or more different biomolecules). As used herein, the term "bispecific" indicates that an antigen-binding protein has two different antigen-binding specificities.

[0048] In one implementation, the second antibody may be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibody, biantibody, or sdAb.

[0049] Nucleic acid, vector, host cell

[0050] In another aspect, the present invention relates to nucleic acid molecules encoding the NKG2A antibody or multispecific antibody of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA. According to one embodiment of the present invention, the nucleic acid of the present invention is a substantially isolated nucleic acid.

[0051] In one embodiment, the nucleic acid molecule encoding the NKG2A antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence selected from SEQ ID NO: 16-17, and the NKG2A antibody encoded therein is capable of specifically binding to NKG2A. Preferably, the nucleic acid molecule encoding the NKG2A antibody is as shown in SEQ ID NO: 16-17.

[0052] The nucleic acids of the present invention may also be in the form of a vector, which may be present in and / or part of a vector, such as a plasmid, a sticky-terminal plasmid, or a YAC. The vector may be, in particular, an expression vector, providing a means for expressing the NKG2A antibody in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). This expression vector typically contains at least one nucleic acid molecule of the present invention operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of said regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the NKG2A antibody of the present invention include, but are not limited to, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0053] In another aspect, the present invention also provides host cells expressing the NKG2A antibody, multispecific antibody, and / or containing the nucleic acid or vector of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0054] Suitable bacterial cells include Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0055] Suitable fungal cells include cells from species of the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells from species of the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichiapastoris* and *Pichia methanolica*), and *Hansenula*.

[0056] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0057] However, the present invention may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0058] Chimeric receptor

[0059] In another aspect, the present invention also provides a chimeric receptor comprising the NKG2A antibody as described above. Since NKG2A is an NK inhibitory receptor, the chimeric receptor comprising the NKG2A antibody can be used to inhibit the cytotoxic effects of NK cells.

[0060] In one embodiment, the present invention provides a chimeric receptor comprising one or more NK inhibitory ligands, a transmembrane domain, and a signal transduction domain, wherein the NK inhibitory ligand comprises an NKG2A antibody as described above or a multispecific antibody containing the NKG2A antibody, and wherein the signal transduction domain comprises one or more co-stimulatory domains.

[0061] In one embodiment, the chimeric receptor comprises multiple NK inhibitory ligands, such as two NK inhibitory ligands, wherein the first NK inhibitory ligand is an NKG2A antibody as described above, and the second NK inhibitory ligand is an antibody or fragment thereof targeting other NK inhibitory receptors, and / or a natural ligand of other NK inhibitory receptors or its contained NK inhibitory receptor binding region.

[0062] In one embodiment, the second NK inhibitory ligand is selected from antibodies or fragments thereof that target the following NK inhibitory receptors: NKG2 / CD94 components (e.g., NKG2B, CD94); members of the killer cell Ig-like receptor (KIR) family (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3); members of the leukocyte Ig-like receptor (LIR) family (e.g., LIR1, LIR2, LIR3, LIR5, and LIR8); and members of the NK cell receptor protein 1 (NKR-P1) family. Members (e.g., NKR-P1B and NKR-P1D); immune checkpoint receptors (e.g., PD-1, TIGIT, CD96, TIM3, LAG3); carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1); members of the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family (e.g., SIGLEC7 and SIGLEC9); leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); members of the Ly49 family (e.g., Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4); and cytotoxic cell lectin-like receptor G1 (KLRG1). More preferably, the second NK inhibitory ligand is selected from antibodies or fragments thereof that target the following NK inhibitory receptors: NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, and KLRG1. More preferably, the second NK inhibitory ligand is selected from antibodies or fragments thereof that target the following NK inhibitory receptors: CD94, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LIR1, LAIR1, and KLRG1.

[0063] In one embodiment, the second NK inhibitory ligand is a natural ligand of another NK inhibitory receptor or its contained NK inhibitory receptor binding region, such as HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (e.g., PD-L1 / PD-L2, CD155, CD112, CD113, Gal-9, FGL1, etc.), and their contained NK inhibitory receptor binding regions. Preferably, the second NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, PD-L1, PD-L2, or their contained NK inhibitory receptor binding regions. More preferably, the second NK inhibitory ligand is selected from the extracellular regions of HLA-E, HLA-G, E-cadherin, PD-L1, and PD-L2. More preferably, the second NK inhibitory ligand is the extracellular region of E-cadherin, which includes EC1 and EC2, and more preferably includes EC1, EC2, EC3, EC4 and EC5.

[0064] As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables the expression of a chimeric receptor on the surface of immune cells (e.g., lymphocytes, NK cells, or NKT cells) and guides the cellular response of immune cells against target cells. Transmembrane domains can be natural or synthetic and can be derived from any membrane-binding or transmembrane protein. When the chimeric receptor binds to a target antigen, the transmembrane domain enables signal transduction. Transmembrane domains particularly suitable for use in this invention can be derived from, for example, the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. Alternatively, transmembrane domains can be synthetic and may primarily contain hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the CD8α chain or CD28, and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:17 or 19, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:18 or 20.

[0065] As used herein, the term "co-stimulatory domain" refers to an intracellular functional signaling domain derived from a co-stimulatory molecule that comprises the entire intracellular portion of the co-stimulatory molecule or a functional segment thereof. A "co-stimulatory molecule" refers to a homologous binding partner that specifically binds to a co-stimulatory ligand on a T cell, thereby mediating a co-stimulatory response (e.g., proliferation) of the T cell. Co-stimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Non-limiting embodiments of the co-stimulatory domains of the present invention include, but are not limited to, intracellular regions derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70. Preferably, the co-stimulatory domain of the CAR of the present invention is derived from 4-1BB, CD28, or 4-1BB+CD28. In one embodiment, the 4-1BB co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:23, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:24. In one embodiment, the CD28 co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:21, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:22.

[0066] In one embodiment, the signal transduction domain comprises one or more co-stimulatory domains (i.e., excluding primary signal transduction domains, such as those from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d). In another embodiment, the signal transduction domain of the chimeric receptor of the present invention may further comprise a primary signal transduction domain, such as the CD3ζ intracellular region. In a preferred embodiment, the CD3ζ intracellular region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 25 or 27, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO: 26 or 28.

[0067] In one embodiment, the chimeric receptor of the present invention may further comprise a hinge region located between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antibody. The hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be wholly or partially derived from natural molecules, such as the extracellular regions of CD8, CD4, or CD28, or wholly or partially derived from the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a hinge region portion of CD8α, CD28, FcγRIIIα receptor, IgG4, or IgG1, more preferably a CD8α, CD28, or IgG4 hinge, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:33, 35, or 37, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:34, 36, or 38.

[0068] In one embodiment, the CR of the present invention may further comprise a signal peptide such that, when expressed in cells such as T cells, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long, hydrophobic amino acid segment with a tendency to form a single α-helix. At the terminal end of the signal peptide, there is typically an amino acid segment that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave the peptide during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, etc. In one embodiment, the signal peptide used in this invention is derived from B2M or CD8α, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:29 or 31, or having a coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:30 or 32.

[0069] In one embodiment, the CAR contains an NKG2A antibody as provided herein or a multispecific antibody containing the NKG2A antibody, a CD8α transmembrane region, and a signal transduction domain, the signal transduction domain comprising a co-stimulatory domain selected from CD28 and 4-1BB. Preferably, the signal transduction domain consists of a co-stimulatory domain selected from CD28 and 4-1BB. In another embodiment, the signal transduction domain further comprises a CD3ζ intracellular region. In this embodiment, the CAR may further comprise a signal peptide from B2M, CD8α, IgG1, or GM-CSFRα.

[0070] The present invention also provides a nucleic acid molecule encoding a chimeric receptor targeting NKG2A as defined above, and a vector comprising said nucleic acid molecule.

[0071] As used herein, the term "vector" is a medium nucleic acid molecule used to transfer (exogenous) genetic material into a host cell, in which the nucleic acid molecule may, for example, be replicated and / or expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering isolated nucleic acids into the cell interior by, for example, homologous recombination or using a hybrid recombinase with a specific target site sequence. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences (e.g., those encoding the chimeric receptor polypeptide of the present invention) in a suitable host cell and for the translation of their mRNA. Suitable vectors for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the present invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia virus, Raul's sarcoma virus (RSV, polyomavirus, and adeno-associated virus (AAV) etc.), bacteriophages, phage particles, granules, and artificial chromosomes (including BAC and YAC). The vector itself is typically a nucleic acid molecule, usually consisting of a DNA sequence containing an insert (transgenic) and a larger sequence serving as the vector's "backbone." Engineered vectors typically also include an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selection marker, and a restriction enzyme cleavage site (e.g., a multiple cloning site, MCS). The vector may additionally include elements such as a promoter, polyA tail, 3'UTR, enhancer, terminator, insulator, operon, selection marker, reporter gene, target sequence, and / or protein purification tag. In one specific embodiment, the vector is an in vitro transcription vector.

[0072] Engineered immune cells

[0073] Because NKG2A can bind to non-classical HLA-I molecules, such as HLA-E, it can inhibit the activation of immune cells, such as NK cells. Therefore, introducing exogenous NKG2A antibodies can inhibit NK cell killing by binding to NKG2A, which is particularly useful in certain situations (such as in the absence of HLA-I molecules or in the preparation of universal CAR-T cells).

[0074] Therefore, in one aspect, the present invention also provides an engineered immune cell expressing the chimeric receptor comprising an NKG2A antibody of the present invention, wherein the expression of at least one MHC-related gene is suppressed or silenced. In a preferred embodiment, the engineered immune cell further expresses a second chimeric receptor comprising a second NK inhibitory ligand, wherein the second NK inhibitory ligand is an antibody or fragment thereof targeting another NK inhibitory receptor, and / or a natural ligand of another NK inhibitory receptor or its contained NK inhibitory receptor binding region.

[0075] As used herein, MHC-related genes include MHC genes themselves (e.g., MHC class I and MHC class II molecules), as well as genes that interact with or regulate the expression of MHC genes. Examples of MHC class I molecules include, but are not limited to, HLA-A, HLA-B, HLA-C, and B2M. Examples of MHC class II molecules include, but are not limited to, HLA-DPA1, HLA-DQA1, and HLA-DRA. Examples of genes that interact with or regulate the expression of MHC genes include, but are not limited to, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, and CIITA.

[0076] Therefore, in one embodiment, suppressing or silencing the expression of MHC-related genes means suppressing or silencing the expression of one or more genes selected from the following: HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

[0077] In one embodiment, the engineered immune cells expressing the chimeric receptor containing the NKG2A antibody of the present invention further include at least one TCR / CD3 gene whose expression is suppressed or silenced, examples of which include, for example, TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ.

[0078] In a preferred embodiment, the engineered immune cells expressing the chimeric receptor of the present invention include at least one TCR / CD3 gene and at least one MHC-related gene whose expression is suppressed or silenced, wherein the at least one TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ and combinations thereof; and the at least one MHC-related gene is selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and combinations thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA and combinations thereof.

[0079] In a preferred embodiment, the at least one TCR / CD3 gene is selected from TRAC, TRBC, and combinations thereof, and the at least one MHC-related gene is selected from B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof. In one embodiment, the expression of TRAC or TRBC and B2M in the engineered immune cells is suppressed or silenced. In one embodiment, the expression of TRAC or TRBC and CIITA in the engineered immune cells is suppressed or silenced. In a preferred embodiment, the expression of TRAC or TRBC, B2M, and CIITA in the engineered immune cells is suppressed or silenced. In a preferred embodiment, the expression of TRAC or TRBC, B2M, and RFX5 in the engineered immune cells is suppressed or silenced.

[0080] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art, including but not limited to DNA breaks mediated by a wide range of nucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in the CRISPR system, or gene inactivation through antisense oligonucleotides, RNAi, shRNA and other technologies.

[0081] In one embodiment, the engineered immune cells provided by the present invention also express chimeric antigen receptors that target tumor antigens.

[0082] In one embodiment, the chimeric antigen receptor targets tumor antigens selected from the following: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, CD20, Folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, pod protein, HPV E6, E7, MAGE A1, ETV6-AML, spermin 17, XAGE1, Tie 2. MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, Claudin18.2. NKG2D and any combination thereof. Preferably, the target is selected from: CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2 / neu), MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin18.2, NKG2D, and any combination thereof. Depending on the antigen to be targeted, the chimeric antigen receptor of the present invention can be designed to include antibodies specific to that antigen. For example, if CD19 is the antigen to be targeted, then a CD19 antibody can be used in the chimeric antigen receptor of the present invention.

[0083] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells, and / or NKT cells. In one embodiment, the immune cell is derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Preferably, the immune cell is a T cell. The T cell can be any T cell, such as cultured T cells, such as primary T cells, or T cells derived from cultured T cell lines such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from a variety of sources, including peripheral blood monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified. T cells can be at any developmental stage, including but not limited to CD4+ / CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. Various techniques known to those skilled in the art, such as Ficoll isolation, can be used to obtain T cells from the subject's blood.

[0084] Nucleic acid sequences encoding chimeric receptors can be introduced into immune cells using conventional methods known in the art, such as transduction, transfection, and transformation. "Transfection" is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. An example is RNA transfection, the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. This term is primarily used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of virus-mediated nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow material uptake. Transfection can be performed using calcium phosphate, via electroporation, via cell extrusion, or by mixing cationic lipids with the material to create liposomes that fuse with the cell membrane and deposit their carriers inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and into non-animal eukaryotic cells (including plant cells). Therefore, transformation is a genetic alteration in bacteria or non-animal eukaryotic cells, resulting from direct uptake from their surroundings via the cell membrane and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be achieved artificially. For transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques may include heat shock-mediated uptake, fusion with bacterial protoplasts of intact cells, microinjection, and electroporation. After introducing nucleic acids or vectors into immune cells, those skilled in the art can amplify and activate the resulting immune cells using conventional techniques.

[0085] In one embodiment, the present invention also provides multiple engineered immune cells, wherein one immune cell expresses the chimeric receptor described herein and optionally a chimeric antigen receptor targeting a tumor antigen, and another immune cell expresses a second chimeric receptor targeting another NK inhibitory receptor. In this embodiment, the second chimeric receptor comprises a second NK inhibitory ligand, a transmembrane domain, and a signal transduction domain, wherein the second NK inhibitory ligand, transmembrane domain, and signal transduction domain are defined as described in the section “Chimeric Receptors”. In such embodiments, the multiple engineered immune cells may be administered together or individually. In one embodiment, the multiple immune cells may be in the same composition or in different compositions. Exemplary compositions of the cells include those described in the following sections of this application.

[0086] Antibody conjugates

[0087] In one aspect, the present invention provides an antibody conjugate comprising an NKG2A antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, radioisotopes, structural portions with extended half-life, detectable markers, and drugs.

[0088] In one embodiment, the present invention provides an antibody conjugate comprising an NKG2A antibody and an Fc as defined herein. As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, including native Fc and variant Fc. "Native Fc" refers to a molecule or sequence comprising a non-antigen-binding fragment, whether in monomeric or multimeric form, generated by digesting an intact antibody. The immunoglobulin source for generating native Fc is preferably derived from humans. The native Fc fragment consists of monomeric polypeptides that can be linked covalently (e.g., by disulfide bonds) and non-covalently into dimer or multimer forms. Depending on the class (e.g., IgG, IgA, IgE, IgD, IgM) or subtype (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2), the native Fc molecule has 1-4 intermolecular disulfide bonds between its monomeric subunits. An example of a natural Fc is a disulfide-linked dimer produced by digesting IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). As used herein, the term “natural Fc” generally refers to monomeric, dimeric, and polymeric forms. A “variant Fc” is an amino acid sequence that differs from the amino acid sequence of a “natural” or “wild-type” Fc due to at least one “amino acid modification” as defined herein, also referred to as an “Fc variant.” Therefore, “Fc” also includes single-chain Fc (scFc), i.e., a single-chain Fc consisting of two Fc monomers linked by a polypeptide linker, capable of naturally folding into a functional dimer Fc region. In one embodiment, the Fc is preferably a human immunoglobulin Fc, more preferably a human IgG1 Fc.

[0089] In one embodiment, the present invention provides an antibody conjugate comprising an NKG2A antibody as defined herein and a radioactive isotope. Examples of radioactive isotopes that can be used in the present invention include, but are not limited to, At. 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 , 99m Tc, 123 I, 18 F and 68Ga.

[0090] In one embodiment, the present invention provides an antibody conjugate comprising an NKG2A antibody as defined in the present invention and a structural portion for extending half-life, wherein the structural portion for extending half-life is selected from albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind to human serum albumin.

[0091] In one embodiment, the present invention provides an antibody conjugate comprising an NKG2A antibody as defined herein and a detectable marker. The term "detectable marker" herein refers to a compound that generates a detectable signal. For example, a detectable marker may be an MRI contrast agent, a scintillation scanning contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, or an optical imaging contrast agent. Examples of detectable markers include fluorophores (such as fluorescein, Alexa, or anthocyanins), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as horseradish peroxidase, glucose-6-phosphatase, β-galactosidase), antibiotic resistance genes (such as kanamycin, ampicillin, chloramphenicol, tetracycline, etc.), and contrast agents (such as nanoparticles or gadolinium). Those skilled in the art can select appropriate detectable markers based on the detection system used.

[0092] In one embodiment, the present invention provides an antibody-drug conjugate comprising an NKG2A antibody as defined herein and a drug, such as a cytotoxin or immunomodulator, conjugated to said NKG2A antibody (i.e., an antibody-drug conjugate). Typically, the drug is covalently linked to the antibody and is usually dependent on a linker. In one embodiment, the drug is a cytotoxin. In another embodiment, the drug is an immunomodulator. Examples of cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin, cis-dichlorodiamineplatin(II) (DDP), cisplatin, carboplatin, zolrubicin, doxorubicin, detoxin, carminoxetine, idarubicin, epirubicin, mitoxantrone, and actinomycin. Bleomycin D, bleomycin, salinomycin, sclerosomycin, atrazocin (AMC), vincristine, vinblastine, paclitaxel, ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, bacitracin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, dihydroxyanthradinone, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), interferon, and combinations thereof. Examples of immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, vorciclosporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogues, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., G-CSF and (GM-CSF), interferons (e.g., interferon-α, interferon-β, and interferon-γ), stem cell growth factors named "S1 factor", erythropoietin, and thrombopoietin, or combinations thereof.

[0093] reagent kits and pharmaceutical compositions

[0094] In another aspect, the present invention also provides a detection kit comprising the antibodies, multispecific antibodies, chimeric receptors, or antibody-drug conjugates described herein.

[0095] In another aspect, the present invention also provides a pharmaceutical composition comprising the antibody, chimeric receptor, multispecific antibody or antibody-drug conjugate described herein, and one or more pharmaceutically acceptable excipients.

[0096] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adhesion agents, flow aids, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers, and tension modifiers. Those skilled in the art know how to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients used in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. Typically, the selection of a suitable excipient depends in particular on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

[0097] The pharmaceutical compositions according to the invention are suitable for administration via a variety of routes. Typically, administration is performed via parenteral delivery. Parenteral delivery methods include local, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0098] The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, particularly as ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures, or fluid extracts, or in forms particularly suitable for the desired method of administration. Processes known in this invention for manufacturing pharmaceuticals may include, for example, conventional mixing, dissolving, granulation, coating, grinding, emulsification, encapsulation, embedding, or lyophilization processes. Pharmaceutical compositions containing, for example, immune cells as described herein, are generally provided in solution form and preferably contain pharmaceutically acceptable buffers.

[0099] The pharmaceutical compositions according to the invention can also be administered in combination with one or more other pharmaceutical agents suitable for treating and / or preventing the disease to be treated. Preferred examples of pharmaceutical agents suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, and orlistatine E. E) vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNases and RNases; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides. Furthermore, the pharmaceutical compositions of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0100] Therapeutic / Preventive / Diagnostic Uses

[0101] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing diseases associated with NKG2A expression, comprising administering to a subject an antibody, multispecific antibody, antibody-drug conjugate, or pharmaceutical composition as described above.

[0102] In one implementation, diseases associated with NKG2A expression include cancer, infectious diseases, inflammatory diseases, and autoimmune diseases. Examples of cancers treatable with the antibodies of this invention include, but are not limited to: solid cancers, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, and skin cancer, including squamous cell carcinoma; lymphoid hematopoietic tumors, including leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, piloblastic lymphoma, and Burkitt lymphoma, and multiple myeloma; myeloid hematopoietic tumors, including acute... Sexual and chronic myeloid leukemia, promyelocytic leukemia, and myelodysplastic syndromes; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous systems, including astrocytoma, neuroblastoma, glioma, and schwannoma; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratoma. Examples of infectious diseases that can be treated with the antibodies of this invention include, but are not limited to, infections caused by viruses, bacteria, protozoa, or fungi. Viruses include, for example, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, varicella virus, adenovirus, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, cytomegalovirus, arbovirus, Coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, and human immunodeficiency virus type 1 or 2 (HIV-1, HIV-2). Bacteria include, for example, Staphylococcus spp., Streptococcus spp., Bacillus spp., Lactobacillus spp., Listeria spp., Corynebacterium diphtheriae, etc. Examples of inflammatory diseases that can be treated with the antibodies of this invention include, but are not limited to: adrenalitis, alveolar ulceration, cholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, laryngitis, cochlear inflammation, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachian tube inflammation, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatitis and spleen inflammation, keratitis, inner otitis media, laryngitis, lymphangitis, mastitis, otitis media, meningitis, endometritis, and mucositis.Examples of autoimmune diseases that can be treated with the antibodies of this invention include, but are not limited to: hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Wegener's granulomatosis, autoimmune hepatitis, Behcet's disease, Crohn's disease, primary biliary cirrhosis, scleroderma, ulcerative colitis, Sjogren's syndrome, type 1 diabetes, uveitis, Graves' disease, Alzheimer's disease, psoriasis, vitiligo, etc.

[0103] In another embodiment, when the chimeric receptor comprising an anti-NKG2A antibody of the present invention is co-expressed with a chimeric antigen receptor targeting a tumor antigen in immune cells, the treatable diseases depend on the tumor antigen targeted by the chimeric antigen receptor. For example, when the chimeric receptor comprising an anti-NKG2A antibody of the present invention is co-expressed or administered with a chimeric antigen receptor targeting CD19, the treatable diseases are those related to CD19 expression, such as B-cell malignancies, including acute lymphoblastic leukemia (B-ALL), chronic B-lymphoblastic leukemia (B-CLL), B-cell Hodgkin's lymphoma (B-HL), and non-Hodgkin's lymphoma (B-NHL). In this embodiment, the chimeric receptor comprising an anti-NKG2A antibody is used to inhibit the killing of reinfused engineered immune cells by NK cells, while the chimeric antigen receptor is used to target cells by binding to tumor antigens.

[0104] The present invention will now be described in detail with reference to the accompanying drawings and examples. It should be noted that those skilled in the art should understand that the drawings and embodiments of the present invention are merely illustrative and do not constitute any limitation on the present invention. Where there is no contradiction, the embodiments and features described in this application can be combined with each other. Attached Figure Description

[0105] Figure 1 The image shows the sequence alignment results of the light chain variable region and heavy chain variable region of hZ199 and its reversion mutant antibodies hZ199-V1 and hZ199-V2. The amino acid sites of the reversion mutations are highlighted in gray, and the numbers indicate the positions of these mutation sites in the light chain variable region and heavy chain variable region.

[0106] Figure 2 The image shows the scFv expression level in UNKi-T cells containing the NKG2A antibody.

[0107] Figure 3 The study demonstrated the inhibitory effect of UNKi-T cells containing NKG2A antibody on NK cell killing. Two-way ANOVA was used for analysis, and statistical analysis was performed using the t-test. ** indicates a p-value less than 0.01, indicating statistical significance. Detailed Implementation

[0108] Example 1. Preparation of NKG2A antibody

[0109] To enhance the affinity and specificity of the antibody, a novel NKG2A antibody was prepared by reverse mutation based on the humanized Z199 antibody (hZ199). hZ199 contains CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, CDR-L3 as shown in SEQ ID NO: 3, CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6. The amino acid sequence of its light chain variable region is shown in SEQ ID NO: 7, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 8, and the full-length amino acid sequence is shown in SEQ ID NO: 9. Methods for reverse mutation are known in the art. For example, primers for reverse mutation are designed for some amino acids (mainly the framework region) in the hZ199 sequence that may affect antibody affinity. Five mutation sites are designed in the light chain variable region and four mutation sites are designed in the heavy chain variable region. Finally, two antibodies with reverse mutations are obtained by combining them and named hZ199-V1 and hZ199-V2. Their sequences are shown in Table 1 below.

[0110] Table 1. Sequences of hZ199 and its reversion mutant antibody

[0111] hZ199 hZ199-V1 hZ199-V2 VL SEQ ID NO:7 SEQ ID NO:10 SEQ ID NO:13 VH SEQ ID NO:8 SEQ ID NO:11 SEQ ID NO:11 full length SEQ ID NO:9 SEQ ID NO:12 SEQ ID NO:14

[0112] Example 2. Preparation of UNKi-T cells expressing chimeric receptors containing NKG2A antibody and verification of their function.

[0113] The sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), catalog number: PPL00157-4a): B2m signal peptide (SEQ ID NO: 29), hZ199 (SEQ ID NO: 9) or hZ199-V1 (SEQ ID NO: 12), CD28 hinge region (SEQ ID NO: 35), CD8α transmembrane region (SEQ ID NO: 17), and CD28 co-stimulatory domain (SEQ ID NO: 21). The hZ199 plasmid and hZ199-V1 plasmid were obtained, and the correct insertion of the target sequence into the plasmid was confirmed by sequencing.

[0114] The sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), catalog number: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 31), hZ199-V2 (SEQ ID NO: 14), IgG4 hinge region (SEQ ID NO: 37), CD28 transmembrane region (SEQ ID NO: 19), and 4-1BB co-stimulatory domain (SEQ ID NO: 23) to obtain the hZ199-V2 plasmid. The correct insertion of the target sequence into the plasmid was confirmed by sequencing.

[0115] Add 3 ml of Opti-MEM (Gibco, catalog number 31985-070) to a sterile tube to dilute the plasmid. Then, add the packaging vector psPAX2 (Addgene, catalog number 12260) and the envelope vector pMD2.G (Addgene, catalog number 12259) in a plasmid:viral packaging vector:viral envelope vector ratio of 4:2:1. Next, add 120 μL of X-treme GENE HP DNA transfection reagent (Roche, catalog number 06366236001), mix immediately, and incubate at room temperature for 15 min. Then, add the plasmid / vector / transfection reagent mixture dropwise to a culture flask of 293T cells. Collect the virus at 24 and 48 hours, combine them, and then ultracentrifuge (25000g, 4℃, 2.5 h) to obtain concentrated lentivirus.

[0116] T cells were activated using DynaBeads CD3 / CD28 CTSTM (Gibco, catalog number 40203D) and cultured at 37°C and 5% CO2 for 1 day. Then, concentrated lentivirus was added, and the cells were cultured for another 3 days to obtain T cells expressing a chimeric receptor containing an NKG2A antibody.

[0117] Then, the TCR / CD3 component (specifically the TRAC gene) and MHC-related genes (specifically B2M and RFX5) were knocked out in wild-type T cells (i.e., NT cells) and the chimeric receptor T cells expressing the NKG2A antibody. Mock T cells, UNKi-hZ99-T cells (containing the hZ199 plasmid), UNKi-V1-T cells (containing the hZ199-V1 plasmid), and UNKi-V2-T cells (containing the hZ199-V2 plasmid) were obtained, respectively. The expression efficiency of CD3 / HLA-I / HLA-II in UNKi-T cells, Mock T cells, and NT cells was detected by flow cytometry using FITC mouse anti-human CD3 (BD ​​Pharmingen, catalog number 555916), PE mouse anti-human HLA-I (R&D catalog number FAB7098P), and APC anti-human DR, DP, DQ (biolegend, catalog number 361714) antibodies. The results are shown in Table 2 below.

[0118] Table 2. Gene expression efficiency in UNKi-T cells

[0119]

[0120]

[0121] As can be seen from Table 2, the expression of CD3 / HLA-I / HLA-II in the UNKi-T cells and Mock T cells prepared in this invention was effectively inhibited or silenced.

[0122] The expression of anti-NKG2A scFv in UNKi-T cells and Mock T cells was detected using Biotin-SP (long spacer) Affini Pure Goat Anti-Human IgG, F(ab') fragment-specific antibody (Jackson ImmunoResearch, catalog number 109-065-097) and APC Streptavidin (BD, catalog number 554067). Figure 2 ).

[0123] It can be seen that scFv is effectively expressed in the UNKi-T cells prepared by this invention.

[0124] The inhibitory effect of UNKi-T cells prepared according to this invention on NK cell killing was detected according to the following method: UNKi-T cells and Mock-T cells prepared according to this invention were labeled with Far-Red (invitrogen, catalog number C34564). Then, at a ratio of 1x10... 4 Labeled UNKi-T cells and Mock T cells were seeded into 96-well plates at a concentration of cells / well, and NK92 cells were added at an effector-to-target ratio of 2:1 for co-culture. After 16-18 hours, the proportion of T cells in the culture was detected by flow cytometry, and the NK cell killing rate against T cells was calculated. The results are shown below. Figure 3 As shown.

[0125] from Figure 3 As can be seen, compared with NT cells that do not express chimeric receptors, the UNKi-T cells of the present invention can significantly reduce the cytotoxic effect of NK cells on T cells. Furthermore, compared with hZ199 antibodies, the antibodies of the present invention (i.e., the reversion mutant hZ199-V1 and hZ199-V2 antibodies) show better inhibitory effects on NK cell cytotoxicity, indicating that the antibodies of the present invention have greater affinity than hZ199 antibodies.

[0126] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> Nanjing Beiheng Biotechnology Co., Ltd. <120> Antibodies targeting NKG2A and their applications <130> BHCN31 <160> 38 <170> SIPOSequenceListing 1.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR-L1 <400> 1 Ser Ala Ser Ser Ser Val Ser Ser Tyr Ile Tyr 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR‑L2 <400> 2 Leu Thr Ser Asn Leu Ala Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR‑L3 <400> 3 Gln Gln Trp Ser Gly Asn Pro Tyr Thr 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR‑H1 <400> 4 Gly Phe Thr Phe Ser Ser Tyr 1 5 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR‑H2 <400> 5 Ser Ser Gly Gly Ser Tyr 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CDR‑H3 <400> 6 His Gly Asp Tyr Pro Arg Phe Phe Asp Val 1 5 10 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199 VL <400> 7 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Tyr 20 25 30 Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 8 <211> 119 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199 VH <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Lys Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Gly Asp Tyr Pro Arg Phe Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 9 <211> 244 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199 scFv <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Tyr 20 25 30 Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Lys Ser 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Glu Ile 165 170 175 Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His 210 215 220 Gly Asp Tyr Pro Arg Phe Phe Asp Val Trp Gly Gln Gly Thr Thr Val 225 230 235 240 Thr Val Ser Ser <210> 10 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199‑V1 VL <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Tyr 20 25 30 Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 119 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199‑V1 VH <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg His Gly Asp Tyr Pro Arg Phe Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 12 <211> 244 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199‑V1 scFv <400> 12 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Tyr 20 25 30 Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Glu Ile 165 170 175 Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg His 210 215 220 Gly Asp Tyr Pro Arg Phe Phe Asp Val Trp Gly Gln Gly Thr Thr Val 225 230 235 240 Thr Val Ser Ser <210> 13 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199‑V2 VL <400> 13 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Tyr 20 25 30 Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 14 <211> 244 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199‑V2 scFv <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Tyr 20 25 30 Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Glu Ile 165 170 175 Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg His 210 215 220 Gly Asp Tyr Pro Arg Phe Phe Asp Val Trp Gly Gln Gly Thr Thr Val 225 230 235 240 Thr Val Ser Ser <210> 15 <211> 732 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199‑V1 <400> 15 gaaatagtcc tgactcagtc tccggcaact ttgtctttgt cacctggaga acgcgctaca 60 atgtcctgct ccgcgtcctc tagcgtttca agctacatat actggtacca acaaaagccg 120 ggtcaagccc cgcgcctgct gatctatctc accagcaacc tcgcgtccgg tgtgcctgct 180 cgattcagcg gttctgggtc tgggacggac ttcactctca ctatttcaag cctcgaacca 240 gaggacttcg ctacttacta ttgtcagcaa tggagtggga acccgtacac gttcggacaa 300 ggaactaagc tcgagattaa aggtagtacc agcggatctg ggaagcctgg tagtggagag 360 ggcagcacaa agggtgaggt gcagttggta gaatcaggtg ggggactcgt gaaacccggc 420 ggatcattga ggctcagttg cgccgcatct ggattcacgt ttagttccta cgccatgtca 480 tgggtcaggc aagcccctgg aaaaggcctt gagtgggttg ctgaaatttc cagcgggggt 540 agctacactt actacccgga tagcgttaaa ggcagattca ctattagtag ggataacgcc 600 aagaacagtc tttacctcca gatgaattcc cttagggctg aagatacagc ggtctactac 660 tgtacaaggc acggggacta tccccgcttc ttcgatgtat ggggacaagg cacgcttgtg 720 acagtgagca gc 732 <210> 16 <211> 732 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> hZ199‑V2 <400> 16 gaaatagtct tgacccagtc tcccgctacc ctcagtcttt cacccggtga acgcgctacg 60 atgacgtgtt cagcatccag ctctgtgtct tcctacattt attggtacca gcagaaacca 120 ggccaagcgc cgcgcctcct catttacctt acttcaaacc ttgcatccgg tattcccgcg 180 agattcagcg gtagtggttc tggaacggat tttacactta cgattagttc acttgaaccc 240 gaagatttcg ccacgtatta ctgtcagcaa tggtcaggaa atccttacac tttcggccaa 300 ggtaccaaag tcgagatcaa agggtccact tctggttcag gtaagccagg tagtggcgag 360 gggagtacaa agggggaagt gcagctcgtc gagtcaggtg gcggacttgt aaaaccgggg 420 gggtcactcc ggctgtcctg cgcagcgagt ggatttacct ttagcagtta tgcaatgtcc 480 tgggtaagac aagctccggg caagggactg gaatgggtgg ctgaaatctc tagtggtgga 540 agttatacct actaccctga tagcgtgaag ggtagattta ctattagtcg ggacaacgct 600 aagaatagct tgtaccttca aatgaacagt ttgcgagcag aggacactgc agtttattat 660 tgcacccggc acggagatta tcctcgcttt tttgacgtat ggggacaggg aactctcgtg 720 actgttagta gc 732 <210> 17 <211> 25 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α <400> 17 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys Lys 20 25 <210> 18 <211> 75 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α <400> 18 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gcaaa 75 <210> 19 <211> 27 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 transmembrane domain <400> 19 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 20 <211> 81 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 transmembrane domain <400> 20 ttttgggtcc tcgtcgtagt tggaggggta cttgcctgtt atagcctcct ggttaccgta 60 gcatttatta tattctgggt g 81 <210> twenty one <211> 41 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 costimulatory domain <400> twenty one Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 22 <211> 123 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> The CD28 <400> 22 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <210> 23 <211> 40 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> 4‑1BB <400> 23 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 1 5 10 15 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 20 25 30 Glue Glue Glue Gly Cys Glue 35 40 <210> 24 <211> 120 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> 4‑1BB is not covered <400> 24 cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga tttccagaag aagaagagg aggatgtgaa <210> 25 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD3ζ is also a slightly different type <400> 25 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 1 5 10 15 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 20 25 30 Tyrian Asp Valleu Asp Lys Arg Arg Gly Arg Asp Pro Glu Meth Gly Gly 35 40 45 Light Pro Arg Arg Light Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 100 105 110 Arg <210> 26 <211> 339 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD3ζ is the source of the <400> 26 ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 60 ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 120 ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 180 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300 acctacgacg cccttcacat gcaggccctg ccccctcgc 339 <210> 27 <211> 114 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD3ζ signal transduction domain <400> 27 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 1 5 10 15 Gly Gln Asn Gln Leu Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 20 25 30 Phe Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 35 40 45 Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 50 55 60 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 65 70 75 80 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser 85 90 95 Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu Pro 100 105 110 Pro Arg <210> 28 <211> 342 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD3ζ signaling domain <400> 28 ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 60 ctctttaacg agctcaatct aggacgaaga gaggagttcg atgttttgga caagagacgt 120 ggccgggacc ctgagatggg gggaaagccg cagagaagga agaaccctca ggaaggcctg 180 tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 240 gagcgccgga ggggcaaggg gcacgatggc cttttccagg gtctcagtac agccaccaag 300 gacacctttg acgcccttca catgcaggcc ctgccccctc gc 342 <210> 29 <211> 20 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> B2M signal peptide <400> 29 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala 20 <210> 30 <211> 60 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> B2M signal peptide <400> 30 atgtcccgct ctgttgcttt ggctgtgctg gcccttttgt cccttagcgg actggaggcc 60 <210> 31 <211> 21 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α signal peptide <400> 31 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 32 <211> 63 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α signal peptide <400> 32 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 33 <211> 45 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α hinge region <400> 33 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 34 <211> 135 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD8α hinge region <400> 34 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 35 <211> 39 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 hinge region <400> 35 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro 35 <210> 36 <211> 117 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> CD28 hinge region <400> 36 attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60 catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagccc 117 <210> 37 <211> 12 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> IgG4 hinge region <400> 37 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 38 <211> 36 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> IgG4 hinge region <400> 38 gaaagcaaat acgggccgcc gtgtccaccc tgtccg 36

Claims

1. An antibody targeting NKG2A, comprising a light chain variable region and a heavy chain variable region, wherein, The light chain variable region is selected from SEQ ID NO: 10 or 13 and the heavy chain variable region is as set forth in SEQ ID NO:

11.

2. The antibody of claim 1, wherein the amino acid sequence of the antibody is selected from SEQ ID NO: 12 or 14.

3. A nucleic acid molecule encoding the antibody of claim 1 or 2.

4. A multispecific antibody comprising the antibody of claim 1 or 2 and one or more second antibodies or antigen binding portions thereof that specifically bind to other antigens.

5. The multispecific antibody of claim 4, wherein the second antibody or antigen binding portion thereof is selected from a full-length antibody, a Fab, a Fab', a (Fab')2, a Fv, a scFv, a scFv-scFv, a minibody, a diabody, or a sdAb.

6. A vector comprising a nucleic acid molecule encoding the antibody of claim 1 or 2 or the multispecific antibody of claim 4 or 5.

7. A host cell expressing the antibody of claim 1 or 2 or the multispecific antibody of claim 4 or 5.

8. A chimeric receptor comprising one or more NK inhibitory ligands, a transmembrane domain, and a signaling domain, wherein the NK inhibitory ligand comprises the antibody of claim 1 or 2 or the multispecific antibody of claim 4 or 5 and the signaling domain comprises one or more costimulatory domains.

9. The chimeric receptor of claim 8, comprising two NK inhibitory ligands, wherein the first NK inhibitory ligand comprises the antibody of claim 1 or 2 or the multispecific antibody of claim 4 or 5 and the second NK inhibitory ligand is selected from: (1) an antibody or fragment thereof that targets the following NK inhibitory receptors: NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, and KLRG1; and / or (2) HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1, PD-L2, CD155, CD112, CD113, Gal-9, FGL1, and a binding region thereof comprising an NK inhibitory receptor.

10. The chimeric receptor of claim 8, wherein the signaling domain consists of one or more costimulatory domains.

11. The chimeric receptor of claim 8, wherein the signaling domain further comprises a CD3 zeta endodomain.

12. The chimeric receptor of any one of claims 8-11, wherein the costimulatory domain is selected from a CD28 or 4-1BB endodomain.

13. An engineered immune cell expressing the chimeric receptor of any one of claims 8-12 and wherein expression of at least one MHC-associated gene is inhibited or silenced.

14. The engineered immune cell of claim 13, wherein the MHC-associated gene is selected from the group consisting of: HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

15. The engineered immune cell of claim 13, wherein the engineered immune cell further comprises at least one TCR / CD3 gene selected from the group consisting of TRAC, TRBC, CD3y, CD35, CD3s, and CD3z is inhibited or silenced.

16. The engineered immune cell of claim 13, wherein the engineered immune cell further expresses a chimeric antigen receptor targeting a tumor antigen.

17. The engineered immune cell of claim 13, selected from the group consisting of T cells, NK cells, NKT cells, macrophages, dendritic cells.

18. An antibody conjugate comprising the antibody of claim 1 or 2 or the multispecific antibody of claim 4 or 5, and a second functional moiety, wherein the second functional moiety is selected from the group consisting of a half-life extending moiety and a detectable label.

19. A test kit comprising the antibody of claim 1 or 2, the multispecific antibody of claim 4 or 5, the chimeric receptor of any one of claims 8-12, or the antibody conjugate of claim 18.

20. A pharmaceutical composition comprising the antibody of claim 1 or 2, the multispecific antibody of claim 4 or 5, the chimeric receptor of any one of claims 8-12, the engineered immune cell of any one of claims 13-17, or the antibody conjugate of claim 18, and one or more pharmaceutically acceptable excipients.

21. Use of the antibody of claim 1 or 2, the multispecific antibody of claim 4 or 5, the chimeric receptor of any one of claims 8-12, the engineered immune cell of any one of claims 13-17, or the antibody conjugate of claim 18, or the pharmaceutical composition of claim 20, for the manufacture of a medicament for inhibiting killing of the engineered immune cell by NK cells upon reinfusion.

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