Antibody targeting NKG2A and its uses

Abstract

The present invention provides an antibody targeting NKG2A, a multispecific antibody containing the same, a chimeric receptor, an antibody conjugate, a pharmaceutical composition and kit, and uses thereof in the diagnosis / treatment / prevention of diseases related to NKG2A expression.

Description

Technology Field

[0001] The present invention belongs to the field of immunotherapy. More specifically, the present invention relates to an antibody targeting NKG2A and its use in the prevention and / or treatment and / or diagnosis of disease. Background Technology

[0002] Natural killer (NK) cells are a highly important type of lymphocyte in the body, playing a crucial role in both innate and adaptive immunity. NK cells possess two types of surface receptors, which can be classified into inhibitory and activating types based on their function. These receptors mediate different recognition modes of NK cells and transmit distinct activation and inhibitory signals. The CD94 / NKG2 family is a relatively well-studied receptor family, primarily comprising members such as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H. Among these, NKG2A is an inhibitory receptor, and its ligand is the non-classical Major Histocompatibility Complex (MAC) Class I molecule HLA-E. HLA-E molecules expressed on target cells can inhibit the cytotoxic function of NK cells after binding to NKG2A. Therefore, antibodies targeting CD94 / NKG2A can enhance the cytotoxic activity of tumor-specific lymphocytes in tumor cells.

[0003] Various anti-NKG2A antibodies are disclosed in the industry. For example, Sivori et al. (Eur J Immunol 1996; 26:2487-92) mentioned the mouse anti-NKG2A antibody Z270, Carretero et al. (Eur J Immunol 1997; 27:563-7) described the mouse anti-NKG2A antibody Z199 (currently commercially available Beckman Coulter, Inc., Cat. No. IM2750, USA), Vance et al. (J Exp Med 1999; 190:1801-12) mentioned the rat anti-mouse NKG2 antibody 20D5 (currently commercially available BD Biosciences Pharmingen, Cat. No. 550518, USA), and U.S. Patent Application Publication 20030095965 described the mouse antibody 3S9, known to bind to NKG2A, NKG2C, and NKG2E.

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

[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. The light chain variable region comprises CDR-L1 represented by SEQ ID NO: 1, CDR-L2 represented by SEQ ID NO: 2, and CDR-L3 represented by SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 represented by SEQ ID NO: 4, CDR-H2 represented by SEQ ID NO: 5, and CDR-H3 represented by SEQ ID NO: 6. 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, max. 7, max. 6, max. 5, max. 4, max. 3, max. 2) conserved modifications of amino acids compared to SEQ ID NO: 10 and 13, and the heavy chain variable region has at least 90% identity with the amino acid sequence represented by SEQ ID NO: 11, or has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, max. 7, max. 6, max. 5, max. 4, max. 3, max. 2) conserved modifications of amino acids compared to SEQ ID NO: 11 It has. 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 represented by 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 further provides a nucleic acid molecule encoding the NKG2A antibody. Accordingly, in one embodiment, the nucleic acid molecule encoding the NKG2A antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with a nucleotide sequence selected from SEQ ID NO: 16 to 17, and the NKG2A antibody encoded therein can specifically bind to NKG2A. Preferably, the nucleic acid molecule encoding the NKG2A antibody is selected from SEQ ID NO: 16 to 17.

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

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

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

[0013] In another aspect, the present invention further provides a chimeric receptor comprising one or more NK inhibitory ligands, a transmembrane domain, and a signaling domain. The NK inhibitory ligand comprises the NKG2A antibody or a multispecific antibody comprising the NKG2A antibody, and the signaling domain comprises one or more co-stimulatory domains.

[0014] In one embodiment, the chimeric receptor comprises two NK inhibitory ligands. Herein, the first NK inhibitory ligand is an NKG2A antibody as described above, and the second NK inhibitory ligand is selected from (1) an antibody or fragment 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 NK inhibitory receptor binding domains contained therein.

[0015] In one embodiment, the signaling domain within the chimeric receptor of the present invention is composed of one or more co-stimulatory domains. That is, it does not include primary signaling domains such as FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

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

[0017] The present invention further 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 further provides engineered immune cells that express a chimeric receptor comprising the NKG2A antibody of the present invention and in which the expression of at least one MHC-related gene is suppressed or silenced.

[0019] In one embodiment, the MHC-associated 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, it is selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

[0020] In one embodiment, the engineered immune cell expressing a chimeric receptor comprising the NKG2A antibody of the present invention further comprises having the expression of at least one TCR / CD3 gene suppressed or silenced, wherein examples of said TCR / CD3 genes include, for example, TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ.

[0021] In one embodiment, the engineered immune cell provided by the present invention further expresses a chimeric antigen receptor that targets a tumor antigen.

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

[0023] In another aspect, the present invention further provides an antibody conjugate comprising the NKG2A antibody and a second functional structure as defined in the present invention. The second functional structure is selected from Fc, a radioisotope, a structural portion that extends the half-life, a detectable marker, and a drug.

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

[0025] In another aspect, the present invention further provides a detection kit, comprising an antibody according to the present invention, a multispecific antibody, an antibody conjugate, or a chimeric receptor.

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

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

[0028] Details of the invention

[0029] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains.

[0030] NKG2A antibody

[0031] In the context of the present invention, "Z199 antibody" refers to the mouse anti-NKG2A antibody Z199 described by Carretero et al. (Eur JImmunol 1997; 27:563-7), which is currently commercially available through Beckman Coulter, Inc., and Cat. No. is IM2750. "hZ199 antibody" refers to a humanized Z199 antibody comprising a light chain variable region indicated by SEQ ID NO: 7 and a heavy chain variable region indicated by SEQ ID NO: 8, the full-length amino acid sequence of which is indicated by SEQ ID NO: 9.

[0032] The novel NKG2A antibody provided in the present invention is obtained by performing a reverse mutation based on hZ199, and provides higher affinity and better NK cell killing inhibitory effect.

[0033] In this specification, the term “antibody” has the broadest possible meaning as understood by those skilled in the art, including monoclonal antibodies (including whole antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides having one or more CDR sequences capable of exhibiting expected biological activity. Antibodies according to the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subtype (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.).

[0034] Typically, an intact antibody contains two heavy chains and two light chains linked together by disulfide bonds, with each light chain connected to each heavy chain by a disulfide bond, exhibiting a "Y"-shaped structure. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region, wherein the heavy chain variable region contains three complementarity determining regions (CDR): CDR-H1, CDR-H2, and CDR-H3, and the heavy chain constant region contains three constant domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region, wherein the light chain variable region contains three CDRs: CDR-L1, CDR-L2, and CDR-L3, and the light chain constant region contains one constant domain CL. In the heavy / light chain variable regions, the CDRs are separated by a more conserved framework region (FR). The variable regions of the heavy and light chains are responsible for the recognition and binding of antigens, whereas the constant regions can mediate the binding of antibodies to host tissues or factors, and include various cells of the immune system (e.g., effector cells) and the first components of the classical complement system.

[0035] The precise amino acid sequence boundaries for a given CDR or FR can be easily determined using several numbering schemes well known in the art. These schemes include Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme); Al-Lazikaniet et al. (1997) JMB273, 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); Lefranc MP et al. "IMGTunique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.", Dev Comp Immunol, Jan. 2003; 27(1):55-77 ("IMGT" numbering); Honegger A and Pl Includes ckthun 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 may vary depending on the method used for identification. For example, the Kabat method is based on structural comparison, whereas the Chothia method is based on structural information. Both Kabat and Chothia numbering methods are based on the most common antibody region sequence lengths and generate different numbers by placing specific insertions and deletions ("indels") at different locations. The Contact method is based on the analysis of complex crystal structures and is similar to the Chothia numbering method in many respects. The AbM method is a compromise between the Kabat and Chothia definitions and is based on the method used by Oxford Molecular's AbM antibody modeling software.

[0037] Accordingly, unless otherwise specified, the “CDR” of a given antibody or its region (e.g., its variable region) is understood to include a CDR defined by any of the above methods or other known methods. For example, if it is specified that a given amino acid sequence is included in a specific CDR (e.g., CDR3), such CDR may further have the sequence of a corresponding CDR (e.g., CDR3) defined by any of the above methods or other known methods. Likewise, unless otherwise specified, the “FR” of a given antibody or its region (e.g., its variable region) is understood to include an FR defined by any of the above methods or other known methods. Unless otherwise specified, the amino acid numbering of the present invention follows the Chothia method.

[0038] The terms "antibody fragment" or "antigen binding site" as used herein refer to a portion of an intact antibody, and generally possess the ability to bind an antigen as they include the antigen binding site of the intact antibody. Examples of antibody fragments of the present invention include, but are not limited to, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, disulfide-linked Fv (sdFv), the heavy chain variable region (VH) or light chain variable region (VL) of an antibody, linear antibodies, "diabodies" having two antigen binding sites, single-domain antibodies, nanobodies, natural ligands of the antigens, or functional fragments thereof. Accordingly, the "antibody" of the present invention includes antibody fragments as defined above.

[0039] "Single-chain antibody" or "scFv" may be used interchangeably herein and refers to an antibody in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a linker. The optimal length and / or amino acid composition of the linker may be selected. The length of the linker can significantly affect the folding and interaction of the variable domains of the scFv. In fact, using a relatively short linker (e.g., between 5 and 10 amino acids) can prevent in-chain folding. Regarding the selection of the size and composition of the linker, see, for example, Hollinger et al., 1993 Proc Natl Acad.Sci.USA90:6444-6448; U.S. Patent Application Publications Nos. 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 may comprise VH and VL connected in any order, e.g., VH-linker-VL or VL-linker-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. The light chain variable region comprises CDR-L1 represented by SEQ ID NO: 1, CDR-L2 represented by SEQ ID NO: 2, and CDR-L3 represented by SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 represented by SEQ ID NO: 4, CDR-H2 represented by SEQ ID NO: 5, and CDR-H3 represented by SEQ ID NO: 6. 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, max. 7, max. 6, max. 5, max. 4, max. 3, max. 2) conserved modifications of amino acids compared to SEQ ID NO: 10 and 13, and the heavy chain variable region has at least 90% identity with the amino acid sequence represented by SEQ ID NO: 11, or has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, max. 7, max. 6, max. 5, max. 4, max. 3, max. 2) conserved modifications of amino acids compared to SEQ ID NO: 11 It has. 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 represented by 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 sequences (nucleotides or amino acids) have the same residue at the same position in comparison, typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of completely identical sequences have 100% identity. Those skilled in the art know that sequence identity can be determined using some algorithms, 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 ClustalW.

[0045] As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody or antibody fragment containing the corresponding amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications may be introduced into the chimeric antigen receptor of the present invention by standard techniques known in the art, e.g., site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is the replacement 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 and include 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 may be selected based on similarity of, for example, polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the associated residues.

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

[0047] As used herein, the term “multispecificity” means that an antigen-binding protein has multiple epitope specificities (i.e., can specifically bind to two, three, or more different epitopes on a single biomolecule, or can specifically bind to two, three, or more different epitopes on a biomolecule). As used herein, the term “bispecificity” indicates that an antigen-binding protein has two different antigen-binding specificities.

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

[0049] Nucleic acid, vector, host cell

[0050] In another aspect, the present invention relates to a nucleic acid molecule encoding the NKG2A antibody or the multispecific antibody of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA. In one embodiment of the present invention, the nucleic acid of the present invention is basically an 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%, and 100% sequence identity with a nucleotide sequence selected from SEQ ID NO: 16 to 17, and the NKG2A antibody encoded therein can specifically bind to NKG2A. Preferably, the nucleic acid molecule encoding the NKG2A antibody is represented by SEQ ID NO: 16 to 17.

[0052] The nucleic acid of the present invention may be in the form of a vector, or may be present in and / or part of a vector. Such a vector is, for example, a plasmid, cosmid, or YAC. The vector may be, in particular, an expression vector, that is, it may provide a vector in which the NKG2A antibody is expressed in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such an expression vector typically comprises at least one nucleic acid molecule of the present invention, which may be operably linked to one or more suitable expression control elements (e.g., a promoter, enhancer, terminator, etc.). It is well known to those skilled in the art that the control elements and their sequences are selected to facilitate expression in a specific host. Specific examples of control 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 further provides a host cell comprising the NKG2A antibody or multispecific antibody of the present invention and / or the nucleic acid or vector of the present invention. A preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

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

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

[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 for expressing heterologous proteins.

[0058] Chimeric receptors

[0059] In another aspect, the present invention further provides a chimeric receptor comprising an NKG2A antibody as described above. Since NKG2A is an NK inhibitory receptor, the chimeric receptor comprising an NKG2A antibody can be used to inhibit the killing action 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 signaling domain. The NK inhibitory ligand comprises the NKG2A antibody or a multispecific antibody comprising the NKG2A antibody, and the signaling domain comprises one or more co-stimulatory domains.

[0061] In one embodiment, the chimeric receptor comprises various NK inhibitory ligands, for example, two NK inhibitory ligands. Herein, 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 ligands, and / or a natural ligand of other NK inhibitory receptors or an NK inhibitory receptor binding region contained therein.

[0062] In one embodiment, the second NK inhibitory ligand is selected from antibodies or fragments thereof targeting the following NK inhibitory receptors: NKG2 / CD94 components (e.g., NKG2A, CD94), killer cell Ig-like receptor (KIR) family members (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3), leukocyte Ig-like receptor (LIR) family members (e.g., LIR1, LIR2, LIR3, LIR5, and LIR8), NK cell receptor protein 1 (NKR-P1) family members (e.g., NKR-P1B and NKR-P1D), immune checkpoint receptors (e.g., PD-1, TIGIT, and CD96, TIM3, LAG3), carcinogen antigen-associated cell adhesion molecule 1 (CEACAM1), and sialic acid binding. Members of the 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 killer cell lectin-like receptor G1 (KLRG1). More preferably, the second NK inhibitory ligand is selected from antibodies or fragments thereof targeting 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 an NK inhibitory receptor binding region included therein, e.g., 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 an NK inhibitory receptor binding region including these. Preferably, the second NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, PD-L1, PD-L2, or an NK inhibitory receptor binding region including these. More preferably, the second NK inhibitory ligand is selected from an HLA-E extracellular region, an HLA-G extracellular region, an E-cadherin extracellular region, a PD-L1 extracellular region, and a PD-L2 extracellular region. More preferably, the second NK inhibitory ligand is an E-cadherin extracellular region and comprises EC1 and EC2, and more preferably comprises EC1, EC2, EC3, EC4 and EC5.

[0064] As used herein, the term "transmembrane domain" refers to a polypeptide structure capable of expressing a chimeric receptor on the surface of immune cells (e.g., lymphocytes, NK cells, or NKT cells) and guiding the cellular response of the immune cells toward target cells. The transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein. When the chimeric receptor binds to a target antigen, the transmembrane domain may perform signal transduction. In particular, the transmembrane domain to which the present invention applies may 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, the transmembrane domain may be synthetic and may primarily contain hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from a CD8α chain or CD28. It 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 represented by SEQ ID NO: 17 or 19, or its encoding 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 represented by SEQ ID NO: 18 or 20.

[0065] As used herein, the term “co-stimulator domain” refers to an intracellular functional signaling domain from the co-stimulator molecule, which includes the entire intracellular portion of the co-stimulator molecule or a functional fragment thereof. “Co-stimulator molecule” refers to a homologous binding gametophyte that mediates the co-stimulator response (e.g., proliferation) of a T cell by specifically binding to a co-stimulator ligand on a T cell. Co-stimulator molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of the co-stimulatory domain 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 and the amino acid sequence represented by SEQ ID NO: 23 have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity, or the coding sequence thereof 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 represented by SEQ ID NO: 24.In one embodiment, the CD28 co-stimulatory domain and the amino acid sequence represented by SEQ ID NO: 21 have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity, or the coding sequence thereof has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the nucleic acid molecule represented by SEQ ID NO: 22.

[0066] In one embodiment, the signaling domain is composed of one or more co-stimulatory domains (i.e., does not include primary signaling domains derived from, for example, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d). In another embodiment, the signaling domain of the chimeric receptor of the present invention may further include a primary signaling domain, such as an intracellular region of CD3ζ. In a preferred embodiment, the CD3ζ intracellular region and the amino acid sequence represented by SEQ ID NO: 25 or 27 have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity, or the coding sequence thereof has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the nucleic acid sequence represented by 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 acts to connect the transmembrane domain to the antibody. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antibody. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids. The hinge region may be derived wholly or partially from a natural molecule, such as wholly or partially from the extracellular region of CD8, CD4, or CD28, or wholly or partially from the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a portion of the hinge region of CD8α, CD28, FcγRIIIα receptor, IgG4, or IgG1, more preferably a CD8α, CD28, or IgG4 hinge, which 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 represented by SEQ ID NO: 33, 35, or 37, or its encoding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleotide sequence represented by SEQ ID NO: 34, 36, or 38.

[0068] In one embodiment, the CR of the present invention may comprise a signal peptide such that when expressed in cells such as T cells, the nascent protein is directed to the cell surface following the endoplasmic reticulum. The core of the signal peptide may comprise a long length of hydrophobic amino acids that tend to form a single α-helix. The end of the signal peptide typically has an amino acid region that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave during or after translocation to produce a free signal peptide and a mature protein. Subsequently, the free signal peptide is digested by a specific protease. The signal peptides that may be used in the present invention are well known to those skilled in the art and are signal peptides derived from, for example, B2M, CD8α, IgG1, GM-CSFRα, etc. In one embodiment, the signal peptide of the present invention is derived from B2M or CD8α, which 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 represented by SEQ ID NO: 29 or 31, or its encoding 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 represented by SEQ ID NO: 30 or 32.

[0069] In one embodiment, the CR comprises the NKG2A antibody provided herein or a multispecific antibody comprising the NKG2A antibody, a CD8α transmembrane region, and a signaling domain, wherein the signaling domain comprises a co-stimulatory domain selected from CD28 and 4-1BB. Preferably, the signaling domain consists of a co-stimulatory domain selected from CD28 and 4-1BB. In another embodiment, the signaling domain further comprises a CD3ζ intracellular region. In the above embodiment, the CAR may further comprise a signaling peptide derived from B2M, CD8α, IgG1, or GM-CSFRα.

[0070] The present invention further 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" refers to a nucleic acid molecule used as a vehicle for delivering (exogenous) genetic material into a host cell, said nucleic acid molecule may be replicated and / or expressed, for example, in the host cell. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering isolated nucleic acids into a cell by homologous recombination or hybrid recombination enzymes, for example, using the sequence of a specific targeting site. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding the chimeric receptor polypeptide of the present invention, and for the translation of their mRNA in a suitable host cell. Vectors suitable for use in the present invention are known in the art and many are commercially available. In one embodiment, the vector of the present invention comprises plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vacciniaviruses, Rouss sarcoma virus (RSV), polyomaviruses, and adeno-associated viruses (AAV), etc.), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is typically a nucleic acid molecule, typically a DNA sequence containing an insert (transgene) and a relatively large sequence serving as the "backbone" of the vector. The engineered vector also typically includes a host cell autogenous replication origin (where stable expression of polynucleotides is required), a selection marker, and a restriction enzyme cleavage site (e.g., a multiclonal site, MCS, etc.). The vector further includes elements such as a promoter, a polyA tail (polyA), a 3' UTR, an enhancer, a terminator, an insulator, an operator, a selection marker, a reporter gene, a targeting sequence, and / or a protein purification tag. In a specific embodiment, the vector is an in vitro transcribed vector.

[0072] engineered immune cells

[0073] NKG2A can bind to non-classical HLA-I class molecules such as HLA-E and then inhibit the activation of immune cells such as NK cells. Therefore, the introduction of exogenous NKG2A antibodies can inhibit the killing action of NK cells by binding to NKG2A, which is particularly useful in some cases (e.g., HLA-I class molecule deletion or universal CAR-T cell production).

[0074] Accordingly, in one aspect, the present invention further provides an engineered immune cell that expresses a chimeric receptor comprising the NKG2A antibody of the present invention and in which 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 inhibitor ligand, wherein the second NK inhibitor ligand is an antibody or fragment thereof targeting another NK inhibitor receptor and / or a natural ligand of another NK inhibitor receptor or an NK inhibitor receptor binding region contained therein.

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

[0076] Accordingly, in one embodiment, suppressing or silencing the expression of MHC-related genes means that the expression of one or more of the following genes is suppressed or silencing: 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 HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

[0077] In one embodiment, the engineered immune cell expressing a chimeric receptor comprising the NKG2A antibody of the present invention further comprises having the expression of at least one TCR / CD3 gene suppressed or silenced, wherein examples of said TCR / CD3 genes include, for example, TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ.

[0078] In a preferred embodiment, the engineered immune cell expressing the chimeric receptor of the present invention comprises having the expression of at least one TCR / CD3 gene and at least one MHC-associated gene suppressed or silenced. Herein, 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-associated 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 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-associated 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 and B2M and CIITA in the engineered immune cells is suppressed or silenced. In a preferred embodiment, the expression of TRAC or TRBC and B2M and RFX5 in the engineered immune cells is suppressed or silenced.

[0080] Methods for suppressing gene expression or silencing genes are well known to those skilled in the art, and include, but are not limited to, mediating DNA cleavage by, for example, meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system, or inactivating genes by techniques such as antisense oligonucleotides, RNAi, shRNA, etc.

[0081] In one embodiment, the engineered immune cell provided by the present invention further expresses a chimeric antigen receptor that targets a tumor antigen.

[0082] In one embodiment, the chimeric antigen receptor targets a tumor antigen selected from the following: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, 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 GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumein, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate-specific protein, servibin and telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, 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 carboxyesterase, 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 may be designed to include an antibody specific to the corresponding antigen. For example, if CD19 is the antigen to be targeted, a CD19 antibody may 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 having one or more effector functions (e.g., cytotoxic apoptotic activity, cytokine secretion, ADCC and / or CDC induction). For example, immune cells may be T cells, macrophages, dendritic cells, monocytes, NK cells, and / or NKT cells. In one embodiment, immune cells are 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, pluripotent stem cells, or hematopoietic stem cells. Preferably, immune cells are T cells. T cells may be any T cells, such as T cells cultured in vitro like primary T cells, or T cells from T cell lines cultured in vitro like Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and their transgene species. T cells can be obtained from various sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. T cells may be concentrated or purified. T cells may include, but are not limited to, CD4+ / CD8+ T cells at any stage of development, 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. T cells may be obtained from a subject's blood using various techniques known to those skilled in the art, such as Ficoll isolation.

[0084] A nucleic acid sequence encoding a chimeric receptor can be introduced into immune cells by employing conventional methods known in the art (e.g., transduction, transfection, transformation, etc.). "Transfection" is the process of introducing a nucleic acid molecule or polynucleotide (including vectors) into a target cell. One example is RNA transfection, that is, the process of introducing RNA (e.g., ex vivo transcribed RNA, ivtRNA) into a host cell. The term is primarily used for non-viral methods in eukaryotic cells. The term "transduction" is typically used to describe the delivery of a virus-mediated nucleic acid molecule or polynucleotide. Transfection in animal cells typically involves opening temporary pores or "holes" in the cell membrane to allow for the uptake of a substance. Transfection can be performed using calcium phosphate, electroporation, cell extrusion, or by fusing a substance with a cationic lipid and depositing the carrier into an internal liposome. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) to bacteria as well as to non-animal eukaryotic cells (including plant cells). Thus, transformation is a genetic modification of bacteria or non-animal eukaryotic cells, which is produced by direct uptake of the surrounding environment via the cell membrane and the subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be performed by manual means. For transformation to occur, the cell or bacterium must be in a receptive state. In the case of prokaryotic transformation, techniques may include heat shock-mediated uptake, fusion of a complete cell and a bacterial protoplast, microinjection, and electroporation. After the nucleic acid or vector is introduced into the immune cell, a person skilled in the art may amplify and activate the immune cell obtained by conventional techniques.

[0085] In one embodiment, the present invention further provides various engineered immune cells. Herein, one immune cell expresses a chimeric antigen receptor targeting a chimeric receptor according to the present invention and any tumor antigen, and another immune cell expresses a second chimeric receptor targeting another NK inhibitory receptor. In such an embodiment, the second chimeric receptor comprises a second NK inhibitory ligand, a transmembrane domain, and a signaling domain, wherein the second NK inhibitory ligand, the transmembrane domain, and the signaling domain are defined as described in the "Chimeric Receptor" section. In such an embodiment, the various engineered immune cells may be administered together or individually. In one embodiment, the various immune cells may be in the same composition or different compositions. Exemplary compositions of the cells include compositions described below in this application.

[0086] antibody conjugate

[0087] In one aspect, the present invention provides an antibody conjugate comprising the NKG2A antibody defined in the present invention and a second functional structure. The second functional structure is selected from Fc, a radioisotope, a structural portion that extends the half-life, a detectable marker, and a drug.

[0088] In one embodiment, the present invention provides an antibody conjugate comprising the NKG2A antibody and Fc as defined in the present invention. As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, comprising natural Fc and variant Fc. "Natural Fc" refers to a molecule or sequence comprising a non-antigen binding fragment, whether monomeric or polymeric, generated by the degradation of an intact antibody. The source of the immunoglobulin from which natural Fc is produced is preferably human. The natural Fc fragment consists of monomeric polypeptides that can be linked in a dimeric or polymeric form by covalent bonds (e.g., disulfide bonds) and non-covalent bonds. Depending on the type (e.g., IgG, IgA, IgE, IgD, IgM) or subtype (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2), there are 1 to 4 intermolecular disulfide bonds between the monomeric subunits of the natural Fc molecule. An example of natural Fc is a dimer linked via a disulfide bond generated by the papain degradation of IgG (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). As used herein, the term “natural Fc” generally refers to monomeric, dimeric, and multimeric forms. “Variant Fc” refers to an amino acid sequence that differs from the amino acid sequence of “natural” or “wild-type” Fc by at least one “amino acid modification” as defined herein, and is also referred to as “Fc variant.” Thus, “Fc” comprises single-strand Fc (scFc), i.e., a single-strand Fc composed of two Fc monomers linked by a polypeptide linker, which can naturally fold into a functional dimeric Fc region. In one embodiment, said Fc is preferably Fc of human immunoglobulin, and more preferably Fc of human IgG1.

[0089] In one embodiment, the present invention provides an antibody conjugate comprising the NKG2A antibody defined in the present invention and a radioisotope. Examples of radioisotopes that may be used in the present invention include 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 68 Ga is included but not limited to this.

[0090] In one embodiment, the present invention provides an antibody conjugate comprising an NKG2A antibody defined in the present invention and a structural portion that extends the half-life, said structural portion that extends the half-life is selected from a binding structure of albumin, a binding structure of transferrin, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and a polypeptide (including an antibody) that binds to human serum albumin.

[0091] In one embodiment, the present invention provides an antibody conjugate comprising the NKG2A antibody defined in the present invention and a detectable marker. The term "detectable marker" refers to a compound capable of signal detection as described herein. For example, a detectable marker may be an MRI contrast agent, a scintigraphy contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, or an optical imaging contrast agent. Examples of detectable markers include fluorescent dyes (e.g., fluorescein, Alexa, or cyanine), chemiluminescent compounds (e.g., luminol), bioluminescent compounds (e.g., luciferase or alkaline phosphatase), enzymes (e.g., mustard peroxidase, glucose-6-phosphatase, β-galactosidase), antibiotic resistance genes (e.g., kanamycin, ampicillin, chloramphenicol, tetracycline, etc.), and contrast agents (e.g., nanoparticles or gadolinium). A person skilled in the art may select a suitable detectable marker depending on the detection system used.

[0092] In one embodiment, the present invention provides an antibody conjugate comprising the NKG2A antibody defined in the present invention and a drug conjugated to said NKG2A antibody, such as a cytotoxin or an immunomodulator (i.e., an antibody-drug conjugate). Typically, the drug is linked to the antibody via a covalent bond and typically relies on a linker. In one embodiment, said drug is a cytotoxin. In another embodiment, said drug is an immunomodulator. Examples of cytotoxins include 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, streptozosin, mitomycin, cis-dichlorodiamine platinum(II) (DDP), cisplatin, carboplatin, zorubicin, doxorubicin, detorubicin, caminomycin, idarubicin, epirubicin, mitoxantrone, actinomycin D, bleomycin, caliceamycin, mitromycin, antramycin (AMC), vincristine, vinblastine, Paclitaxel, lysine, Pseudomonas exotoxin, gemcitabine, cytocalcin B, gramicidin D, ethidium bromide, emetine, etoposide, tenifoside, colchicine, dihydroxyanthrasedione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitothan (O,P'-(DDD)), interferon, and combinations thereof are included but not limited thereto.Examples of immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate retinoid, glucocorticoids and analogs, 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 designated as “Factor S1”, erythropoietin and thrombopoietin, or combinations thereof.

[0093] Kit and pharmaceutical composition

[0094] In another aspect, the present invention further provides a detection kit, comprising an antibody according to the present invention, a multispecific antibody, a chimeric receptor, or an antibody conjugate.

[0095] In another aspect, the present invention further provides a pharmaceutical composition, comprising an antibody according to the present invention, a chimeric receptor, a multispecific antibody or antibody conjugate, and one or more pharmaceutically acceptable excipients.

[0096] As used herein, the term “pharmaceuticalally acceptable excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically suitable for the subject and the active ingredient (i.e., capable of producing the desired therapeutic effect without causing unwanted 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, coating agents, 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 retardants, stabilizers, and tension modifiers. The selection of suitable excipients is known to those skilled in the art for the preparation of the desired pharmaceutical composition of the present invention. Exemplary excipients for use in the pharmaceutical composition of the present invention include saline solution, buffered saline solution, dextrose, and water. Typically, the selection of suitable excipients depends particularly on the active agent used, the disease to be treated, and the desired formulation of the pharmaceutical composition.

[0097] The pharmaceutical composition according to the present invention is suitable for various routes of administration. Typically, administration is performed parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, vaginal, sublingual, or nasal administration.

[0098] The pharmaceutical composition according to the present invention may be prepared in various forms, e.g., solid, liquid, gas, or lyophilized, and in particular may be in the form of an ointment, cream, transdermal patch, gel, powder, tablet, solution, gaseous aerosol, granule, pill, suspension, emulsion, capsule, syrup, elixir, extract, tincture, or liquid extract, or in a form particularly suitable for use in a desired method of administration. Processes known in the present invention for the preparation of the pharmaceutical composition may include, for example, conventional mixing, dissolution, granulation, preparation of coated tablets, milling, emulsification, encapsulation, embedding, or lyophilization processes. A pharmaceutical composition comprising immune cells such as those described herein is typically provided in the form of a solution and preferably comprises a pharmaceutically acceptable buffer.

[0099] The pharmaceutical composition according to the present invention may be administered in combination with one or more other agents suitable for the treatment and / or prevention of a disease to be treated. Preferred examples of agents suitable for combination include anticancer drugs such as cisplatin, metansin derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine, and doxorubicin; and peptide cytotoxins such as lysine, diphtheria toxin, Pseudomonas exotoxin A, DNase, and RNase. Radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatin 213; prodrugs such as antibody-designated enzyme prodrugs; immunostimulators such as platelet factor 4, melanoma growth-promoting 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 are included. Additionally, the pharmaceutical composition of the present invention may be used in combination with one or more other therapeutic methods, such as chemotherapy and radiation therapy.

[0100] For treatment / prevention / diagnosis purposes

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

[0102] In one embodiment, diseases associated with NKG2A expression include cancer, infectious diseases, inflammatory diseases, and autoimmune diseases. Examples of cancers that can be treated with the antibody of the present invention include solid tumors 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 lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, blastocyst lymphoma, and Burkitt lymphoma, and multiple myeloma; myeloid hematopoietic tumors including acute and chronic myeloid leukemia, promyelocytic leukemia, and myelodysplastic syndrome; and mesenchymal tumors including fibrosarcoma and rhabdomyosarcoma. Other tumors including melanoma, seminoma, teratoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors including, but not limited to, melanoma, xerosis pigmentosa, keratospinata, seminoma, follicular carcinoma of the thyroid, and teratoma. Examples of infectious diseases that can be treated with the antibodies of the present invention include, but are not limited to, infections caused by viruses, bacteria, protozoa, or fungi.Here, viruses include, for example, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, varicella virus, adenovirus, herpes simplex type 1 (HSV-1), herpes simplex type 2 (HSV-2), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, cytomegalovirus, arbovirus, coxsackievirus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type 1 or 2 (HIV-1, HIV-2), and bacteria include, for example, Staphylococcus, Streptococcus, Bacillus, Lactobacillus, Listeria, Corynebacterium diphtheria, etc. Examples of inflammatory diseases that can be treated with the antibody of the present invention include, but are not limited to, adrenalitis, alveolar inflammation, cholecystocholinc, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, vocal cord inflammation, cochlear inflammation, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, Eustachian tube inflammation, fibrous tissue inflammation, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenic inflammation, keratitis, otitis interna, laryngitis, lymphangitis, mastitis, otitis media, meningitis, endometritis, mucositis, etc. Examples of autoimmune diseases that can be treated with the antibody of the present invention include, but are not limited to, hemolytic anemia, pernicious anemia, polyarteritis nodosum, systemic lupus erythematosus, Wegener granulomatous disease, autoimmune hepatitis, Behcet's disease, Crohn's disease, primary biliary cirrhosis, scleroderma, ulcerative colitis, Sjogren's syndrome, type 1 diabetes mellitus, uveitis, Graves' disease, Alzheimer's disease, psoriasis, vitiligo, etc.

[0103] In another embodiment, when a chimeric receptor comprising the anti-NKG2A antibody of the present invention and a chimeric antigen receptor targeting a tumor antigen are co-expressed in immune cells, the disease that can be treated depends on the tumor antigen targeted by the chimeric antigen receptor. For example, when a chimeric receptor comprising the anti-NKG2A antibody of the present invention and a chimeric antigen receptor targeting CD19 are co-expressed or administered, the disease that can be treated is a disease associated with CD19 expression, e.g., B-cell malignancies including acute lymphoblastic leukemia (B-ALL), chronic B-lymphoblastic leukemia (B-CLL), B-cell Hodgkin lymphoma (B-HL), and non-Hodgkin lymphoma (B-NHL). In such an embodiment, the chimeric receptor comprising the anti-NKG2A antibody is used to inhibit the killing of NK cells by re-injected engineered immune cells, and the chimeric antigen receptor is used to direct the killing of target cells by binding to the tumor antigen.

[0104] The present invention will be described in detail below with reference to the attached drawings and embodiments. Those skilled in the art should note that the drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the invention. Unless contradictory, the embodiments and features of the embodiments may be combined with one another. Brief explanation of the drawing

[0105] Figure 1 shows the results of a sequence comparison of the light chain variable region and heavy chain variable region of hZ199 and its reversal mutant antibodies, hZ199-V1 and hZ199-V2. The amino acid sites of the reversal mutant are highlighted in gray, and the numbers indicate the locations of the corresponding mutant sites in the light chain variable region and heavy chain variable region. Figure 2 shows the scFv expression level of UNKi-T cells containing NKG2A antibodies. Figure 3 shows the inhibitory effect of UNKi-T cells containing NKG2A antibodies on NK cell killing activity. Analysis was performed using two-way ANOVA, and statistical analysis was conducted using a T-test. ** indicates that the P-value was less than 0.01, indicating that it reached a significant level. Specific details for implementing the invention

[0106] Example 1. Preparation of NKG2A antibody

[0107] To increase the affinity and specificity of the antibody, a new NKG2A antibody was produced based on the humanized Z199 antibody (hZ199) through a reversal mutation method. hZ199 includes CDR-L1 represented by SEQ ID NO: 1, CDR-L2 represented by SEQ ID NO: 2, CDR-L3 represented by SEQ ID NO: 3, CDR-H1 represented by SEQ ID NO: 4, CDR-H2 represented by SEQ ID NO: 5, and CDR-H3 represented by SEQ ID NO: 6, and the amino acid sequence of its light chain variable region is represented by SEQ ID NO: 7, the amino acid sequence of its heavy chain variable region is represented by SEQ ID NO: 8, and the full-length amino acid sequence is represented by SEQ ID NO: 9. The method of reversal mutation is known in the art. For example: primers for reversal mutations were designed for amino acids (mainly in the framework region) in the hZ199 sequence that may affect antibody affinity, five mutation sites were designed in the light chain variable region, and four mutation sites were designed in the heavy chain variable region, and two antibodies for reversal mutations were obtained by final combination. These were named hZ199-V1 and hZ199-V2, and their sequences are as shown in Table 1 below.

[0108] Table 1. Sequences of hZ199 and its reversal mutant antibodies

[0109]

[0110] Example 2. Preparation of UNKi-T cells expressing a chimeric receptor containing an NKG2A antibody and verification of their function

[0111] Sequences encoding proteins such as 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-stimulation domain (SEQ ID NO: 21) were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Cat. No.: PPL00157-4a) to obtain the hZ199 plasmid and hZ199-V1 plasmid, and the correct insertion of the target sequences into the plasmids was confirmed through sequencing.

[0112] Sequences encoding the proteins CD8α signal peptide (SEQ ID NO: 31), hZ199-V2 (SEQ ID NO: 14), IgG4 hinge region (SEQ ID NO: 37), CD828 transmembrane region (SEQ ID NO: 19), and 4-1BB co-stimulation domain (SEQ ID NO: 23) were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Cat. No.: PPL00157-4a) to obtain the hZ199-V2 plasmid, and the correct insertion of the target sequences into the plasmid was confirmed through sequencing.

[0113] Dilute the plasmid by adding 3 ml of Opti-MEM (Gibco, Cat. No. 31985-070) to a sterile tube, then add psPAX2 (Addgene, Cat. No. 12260) and envelope vector pMD2.G (Addgene, Cat. No. 12259) according to a ratio of plasmid:virus packaging vector:virus envelope vector = 4:2:1. After that, add 120 µl of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately, and incubate at room temperature for 15 minutes, then drop the plasmid / vector / transfection reagent mixture into a culture flask for 293T cells. Viruses were collected at 24 and 48 hours and combined, and then concentrated lentiviruses were obtained by ultra-high velocity centrifugation (25,000 g, 4°C, 2.5 hours).

[0114] T cells were activated with DynaBeads CD3 / CD28 CTS™ (Gibco, Cat. No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Then, concentrated lentivirus was added and cultured for 3 days, after which T cells expressing a chimeric receptor containing an NKG2A antibody were obtained.

[0115] Then, by employing the CRISPR / Cas9 system, the TCR / CD3 component (specifically the TRAC gene) and MHC-related genes (specifically B2M and RFX5) of wild-type T cells (i.e., NT cells) and T cells expressing a chimeric receptor containing the NKG2A antibody were knocked out, thereby obtaining 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), respectively. The expression efficiency of CD3 / HLA-I / HLA-II in UNKi-T cells, Mock T cells, and NT cells was detected using a flow cytometer with FITC Mouse Anti-Human CD3 (BD ​​Pharmingen, Cat. No. 555916) antibody, PE mouse anti-human HLA-I (R&D Cat. No. FAB7098P) antibody, and APC anti-human DR, DP, DQ (biolegend, Cat. No. 361714) antibody, and the results are shown in Table 2.

[0116] Table 2. Gene expression efficiency in UNKi-T cells

[0117]

[0118] Table 2 shows that the expression of CD3 / HLA-I / HLA-II in UNKi-T cells and Mock T cells produced by the present invention is effectively suppressed or silenced.

[0119] Anti-NKG2A scFv expression was detected in UNKi-T cells and Mock T cells using Biotin-SP (long spacer) AffiniPure Goat Anti-Human IgG, F(ab')fragment specific antibody (Jackson ImmunoResearch, Cat. No. 109-065-097) and APC Streptavidin (BD, Cat. No. 554067) (Fig. 2).

[0120] It can be seen that all scFvs in the UNKi-T cells produced in the present invention are effectively expressed.

[0121] The inhibitory effect of UNKi-T cells prepared according to the present invention on NK cell apoptosis was detected according to the following method. UNKi-T cells and Mock-T cells prepared according to the present invention were labeled with Far-Red (Invitrogen, Cat. No. C34564). Then, 1 x 10⁶ labeled UNKi-T cells and Mock-T cells were used. 4 The cells were plated in a 96-well plate at a cell / well concentration and co-cultured with NK92 cells added at an effect target ratio of 2:1. After 16 to 18 hours, the proportion of T cells in the culture was measured using a flow cytometer, and the killing rate of NK cells against T cells was calculated, as shown in Figure 3.

[0122] As can be seen in Figure 3, compared to NT cells that do not express a chimeric receptor, the UNKi-T cells of the present invention can significantly reduce the killing activity of NK cells against T cells. In addition, compared to the hZ199 antibody, the antibodies of the present invention (i.e., reverse mutant hZ199-V1 and hZ199-V2 antibodies) showed a superior inhibitory effect on the killing activity of NK cells, indicating that the affinity of the antibodies of the present invention is greater than that of the hZ199 antibody.

[0123] It should be noted that the foregoing is merely a preferred embodiment of the present invention and is not intended to limit the invention, and that various modifications and changes to the present invention are possible for those skilled in the art to which the present invention pertains. Those skilled in the art understand that all modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

Claim 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 represented by SEQ ID NO:

11. Claim 2 An antibody according to claim 1, characterized in that the amino acid sequence of the antibody is selected from SEQ ID NO: 12 or 14. Claim 3 A nucleic acid molecule characterized by encoding an antibody according to claim 1. Claim 4 A multispecific antibody characterized by comprising one or more second antibodies or antigen-binding sites thereof that specifically bind to the antibody according to claim 1 and other antigens. Claim 5 A multispecific antibody according to claim 4, wherein the second antibody or its antigen-binding site is selected from full-length antibodies, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, minibody, diabody, or sdAb. Claim 6 A vector characterized by comprising a nucleic acid molecule encoding an antibody according to claim 1 or a multispecific antibody according to claim 4. Claim 7 A separated host cell characterized by expressing an antibody according to claim 1 or a multispecific antibody according to claim 4. Claim 8 A chimeric receptor comprising one or more NK inhibitory ligands, a transmembrane domain, and a signaling domain, wherein the NK inhibitory ligand comprises an antibody according to claim 1 or a multispecific antibody according to claim 4, and the signaling domain comprises one or more co-stimulatory domains. Claim 9 In claim 8, the chimeric receptor comprises two NK inhibitory ligands, wherein the first NK inhibitory ligand comprises an antibody according to claim 1 or a multispecific antibody according to claim 4, and the second NK inhibitory ligand is selected from (1) an antibody or fragment thereof targeting the following NK inhibitory receptors: NKG2A, NKG2B, CD94, LIR1, 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 NK inhibitory receptor binding domains containing them. Claim 10 A chimeric receptor according to claim 8, wherein the signal transduction domain comprises one or more co-stimulation domains. Claim 11 A chimeric receptor according to claim 8, wherein the signaling domain further comprises an intracellular CD3ζ region. Claim 12 A chimeric receptor according to claim 8, characterized in that the co-stimulatory domain is selected from the CD28 or 4-1BB intracellular region. Claim 13 Isolated engineered immune cells, characterized in that they express a chimeric receptor according to claim 8, wherein the expression of at least one MHC-related gene is suppressed or silenced. Claim 14 In claim 13, the isolated engineered immune cell is characterized in that the MHC-associated 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. Claim 15 In claim 13, the isolated engineered immune cell further comprises having at least one TCR / CD3 gene expression suppressed or silenced, wherein the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ. Claim 16 In paragraph 13, the isolated engineered immune cell is characterized by further expressing a chimeric antigen receptor that targets a tumor antigen. Claim 17 In claim 13, isolated engineered immune cells characterized by being selected from T cells, NK cells, NKT cells, macrophages, and dendritic cells. Claim 18 An antibody conjugate comprising an antibody according to claim 1 or a multispecific antibody according to claim 4 and a second functional structure, wherein the second functional structure is selected from Fc, a radioisotope and a structural part that extends the half-life, a detectable marker and a drug. Claim 19 A detection kit for NKG2A expression-related diseases, wherein the kit comprises an antibody according to claim 1 or a multispecific antibody according to claim 4, wherein the NKG2A expression-related diseases include cancer, infectious diseases, inflammatory diseases, and autoimmune diseases. Claim 20 A pharmaceutical composition for the treatment, prevention, or diagnosis of NKG2A expression-related diseases, wherein the composition comprises an antibody according to claim 1 or a multispecific antibody according to claim 4, and one or more pharmaceutically acceptable excipients, wherein the NKG2A expression-related diseases include cancer, infectious diseases, inflammatory diseases, and autoimmune diseases. Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete

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