TCR constant region with introduction of exogenous disulfide bonds and its products and applications

Abstract

The present invention provides a TCR constant region for introducing exogenous disulfide bonds, and products and applications thereof, belonging to the field of biomedicine, wherein the TCR constant region includes a point mutation for introducing exogenous disulfide bonds, which includes a point mutation 1 located in the α chain and an amino acid site involved in the formation of endogenous disulfide bonds separated by a specific number of amino acids, or a point mutation 2 located in the β chain and an amino acid site involved in the formation of endogenous disulfide bonds separated by a specific number of amino acids. The present invention provides a new solution strategy for improving the stability, expression rate, efficacy, and safety of engineered TCRs, and is of great significance to biomedicine fields such as TCR-T cell therapy, TCR chimeric receptor-modified cell therapy, TCR antibodies, and bispecific antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a TCR constant region into which exogenous disulfide bonds are introduced, and products and applications thereof. Background Art

[0002] Tumor immunotherapy has become another important anti-tumor treatment method after surgery, radiotherapy and chemotherapy. Innovative immunotherapy based on T cells has shown great potential in the field of tumor immunotherapy. This type of therapy modifies T cells through genetic engineering technology to give them the ability to recognize and eliminate tumor cells. Among them, chimeric antigen receptor T (CAR-T) cells and T cell receptor (TCR) edited T cells are the most representative cell therapies.

[0003] TCR is an immunoglobulin-like receptor expressed on the surface of T cells. It is composed of two peptide chains (α, β or γ, δ), which can specifically recognize antigen epitopes presented by MHC and mediate immune responses. There are currently two known types of TCRs, of which the TCR composed of two peptide chains of α and β is called αβTCR, and the TCR composed of two peptide chains of γ and δ is called γδTCR. In peripheral blood, T cells expressing αβTCR account for 90%-95% of total T cells. Each peptide chain of αβTCR is composed of a variable region (V region) that can bind to antigens and a highly conserved constant region (C region), wherein the constant region includes three parts: the extracellular region, the transmembrane region, and the intracellular region. The αβTCR expressed on the cell membrane needs to bind to six CD3 subunits to form a TCR-CD3 complex, thereby maintaining the stability of its membrane display and transmitting signals to the cell through the intracellular domain of the CD3 molecule.

[0004] Most of the TCR-T cells involved in TCR-T therapy are T cells into which exogenous αβTCR is transferred. One of the important problems in TCR-T cell therapy at present is the mismatch between the exogenously introduced TCR and the endogenous TCR, that is, the mismatch between the α or β chain of the transferred TCR and the β or α chain of the endogenous TCR. This TCR mismatch phenomenon is believed to be widely present in TCR-T therapy. This will lead to, on the one hand, a decrease in the expression rate of the introduced functional TCR, which will affect the efficacy of TCR-T; on the other hand, the TCR heterodimers formed by mismatching may produce the ability to recognize self-antigens, thereby leading to autoimmune (on-target) or cross-reaction (off-target) toxicity, that is, attacks and killing of healthy tissues of the body, causing unknown safety risks.

[0005] Currently, the commonly used methods to solve expression and mismatch include: (1) replacing the constant region of the transferred exogenous TCR molecule with the constant region of the mouse TCR. However, the introduction of the mouse TCR constant region protein fragment will cause the reinfused TCR-T cells to induce host-versus-graft disease (HvGD) in the patient's body, resulting in the rejection and elimination of TCR-T cells, greatly weakening the therapeutic effect and the feasibility of secondary reinfusion. (2) Site-directed mutation of the corresponding pair of amino acids in the constant region of the two chains of the TCR, thereby introducing a mortise and tenon structure or an additional disulfide bond, but this strategy has limited effect when used alone. (3) Changing the TCR structure, creating TCR-CD3 chimeric molecules or single-chain TCRs to avoid mismatching between the transferred TCR and the endogenous TCR, but because the interaction between the peptides is unclear and can easily lead to the loss of the original structure and function of the TCR, this strategy is basically no longer used.

[0006] In addition to TCR-T therapy, some TCR chimeric receptor-edited T cell therapies have also been reported. The forms of TCR chimeric receptors include: fusing the constant region of TCR with the variable region of alpaca, mouse or human antibodies; fusing the complete TCR containing the constant region and variable region with the variable region of alpaca, mouse or human antibodies.

[0007] In terms of enhancing the inter-chain pairing of exogenous TCR and improving the efficiency of TCR expression in cell staining, existing technologies have many shortcomings and still need to be optimized and improved. Summary of the invention

[0008] In order to solve the various deficiencies in the prior art, the present invention provides a new technical solution for optimizing the TCR structure. Specifically, amino acid mutations that can form interchain exogenous disulfide bonds are screened for TCR modification, and combined with other optimization methods disclosed in the prior art to improve the application effect of TCR.

[0009] Based on the overall concept of the present invention, the aspects protected by the present invention are diverse, including but not limited to: TCR constant region point mutations for introducing exogenous disulfide bonds and the applications and products of TCR constant regions, TCRs, TCR chimeric receptors, and TCR fusion proteins constructed by their combination, corresponding genetic engineering products, clinical applications and products, etc.

[0010] In the present invention, "TCR constant region" includes TCR α chain constant region (TRAC) and β chain constant region (TRBC), which can be human constant region or derived from another species, such as mouse. The prior art discloses that adding disulfide bonds to the constant region can promote the correct pairing of TCR α and β chains (Kuball J et al. Blood. 2007 Mar 15; 109 (6): 2331-8). The positions of the amino acid sequence of the wild-type TCR in the present invention are numbered according to the naming rules of the International Immunogenetics Information System (IMGT). For example, an amino acid in the TCR α chain constant region (TRAC) is numbered as 48 in the position listed in IMGT, and it is described herein as the 48th amino acid of TRAC; an amino acid in the TCR β chain constant region (TRBC) is numbered as 57 in the position listed in IMGT, and it is described herein as the 57th amino acid of TRBC; and the same applies to others. In the present invention, if there are special instructions for the sequence position numbering of other amino acids, they shall be in accordance with the special instructions.

[0011] In the present invention, "TCR" refers to "T cell receptor", including natural TCR and TCR variants, fragments and constructs. Therefore, the term includes heterodimers and multimers and single-chain constructs comprising TCR α chain and TCR β chain; optionally including other domains and / or parts, as long as the TCR retains its ability to recognize antigen targets. In the natural form of TCR, it exists as a complex of several proteins on the surface of T cells. The T cell receptor consists of two (separate) protein chains, which are produced by independent T cell receptor α and β (TCR α and TCR β) genes and are referred to as α chain and β chain. Each chain of TCR has an N-terminal immunoglobulin-like (Ig)-variable (V) region / domain, an Ig-constant (C) region / domain, a transmembrane / cell membrane spanning region that anchors the chain in the plasma membrane, and a short cytoplasmic tail at the C-terminus. Antigen specificity is conferred by the variable regions (TRAV and TRBV) of the α and β chains.

[0012] The "TCR chimeric receptor" of the present invention has a basic structure comprising a combination of the aforementioned TCR constant region and antigen recognition region, wherein the antigen recognition domain can be derived from functional molecules that can bind to target molecules, such as antibodies, receptors, ligands, polypeptide antigens, etc., wherein antibodies include but are not limited to single-chain antibody variable regions (scFv), nanobodies (VHH), TCR-like antibodies that recognize pMHC complexes, etc. TCR chimeric receptors can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens on immune cells (such as T cells), allowing immune cells to recognize tumor antigens or viral antigens and kill tumor cells or virus-infected cells.The antigen recognition domain in the TCR chimeric receptor is derived from an antibody and can target one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1, β1,4-acetyl-aminogalactosyltransferase 1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL premelanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13Rα2, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family protein, BAGE (B melanoma antigen family) family protein, GAGE ​​(growth hormone releasing factor) family protein, AFP (α-fetoprotein), MUC1 (mucin 1, cadherin 1), CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11Rα, EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1, ST8α-N-acetyl-ceramide α-2,8-sialic acid converting enzyme 1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3, kallikrein-related peptidase 3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16, cadherin 16), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25-dihydrovitamin D3) receptor), β2-MG (β-2-microglobulin) and PROGRP (GRP gastrin-releasing peptide), NY-ESO-1, MAGE-A1 / 3 / 4 / 6, MAGE-C2, MART-1, WT1, PR AME, gp100, MCPyV, hTG, TRAIL, HERV-E, HA-1, Tyrosinase, TGFβRII, HBV, HPV, CMV, EBV, HIV, AFP; the antigen recognition domain in the TCR chimeric receptor is derived from the receptor or ligand and can be one or more of the following: APRIL, TACI, FLT3, NKG2D, etc.; the antigen recognition domain in the TCR chimeric receptor is derived from the polypeptide antigen and can be one or more of the following polypeptide antigens: DSG1 / 3, DARPins, adnectin peptides, antigen peptides recognized by autoreactive BCR, etc.

[0013] The "fusion protein" of the present invention refers to a new type of multi-domain artificial protein produced by fusing a certain biologically active functional protein molecule with other natural proteins (fusion partners) by genetic engineering, chemical modification and other methods. The protein performance can be optimized. The functional protein molecule is generally an endogenous ligand or its receptor, mainly including active substances such as cytokines, growth factors, hormones, enzymes or peptides. Common fusion partners include immunoglobulins (Ig), albumin or transferrin. Among them, fusion proteins based on antibody Fc fragments (Fc crystallizable) are the most widely used.

[0014] The "TCR fusion protein" of the present invention refers to a novel multi-domain artificial protein produced by fusing a certain biologically active functional protein molecule with other natural proteins (fusion partners) by genetic engineering, chemical modification and other methods, which comprises the TCR constant region of the present invention alone or in combination with an antigen recognition domain or a functional domain. The TCR constant region is the TCR modified structure described in the present invention, which may or may not include the transmembrane region and the intracellular region of the TCR constant region and its derived truncations; the antigen recognition domain is a functional molecule that binds to the target molecule, including but not limited to antibodies, receptors, ligands, polypeptide antigens, etc.; the functional domain can be a domain that can activate related functional cells, such as antibodies targeting CD3 for activating T cells, IgG1 or anti-CD16 antibodies combined with NK cells for inducing ADCC effects, or PD1 / TIM3 / LAG3 and other immunosuppressive molecule antibodies or ligands to improve the inhibitory effect of the tumor microenvironment.

[0015] In the present invention, "nucleic acid molecule" is used with the same meaning as polynucleotide and nucleic acid, and also includes DNA, RNA, probes, oligonucleotides and primers.

[0016] The "vector" of the present invention refers to a nucleic acid delivery vehicle into which a polynucleotide is inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; CRISPR / CAS plasmids; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0017] The "cell" of the present invention includes cells in individual animals and cultured cells. It can be understood that cells are the basic units of life activities. Generally speaking, most microorganisms such as bacteria and protozoa are composed of one cell, that is, single-cell organisms; higher plants and higher animals are multicellular organisms. Cells can be divided into two categories: prokaryotic cells and eukaryotic cells.

[0018] In the present invention, "INESS-TCR" means introducing screened exogenous disulfide bonds based on the constant region of human or mouse TCR, or introducing exogenous disulfide bonds combined with other mutations to increase the pairing and membrane display efficiency of TCR.

[0019] In the present invention, the "truncate" is a kind of protein or polypeptide variant, and the variant can be a mutation with replacement, deletion or insertion of amino acids. Specifically, the truncate can be a one or more amino acids removed from one or both ends of the polypeptide. Therefore, in theory, variant peptides can be generated by manipulating the gene encoding the polypeptide. Variants can be generated by changing the basic composition or characteristics of the polypeptide, but not changing at least some of its pharmacological activities. In some embodiments, the truncate has no more than 20 missing amino acids compared to the peptide or part thereof before mutation. In some embodiments, the truncate has no more than 15 missing amino acids. In some embodiments, the truncate has no more than 10 missing amino acids. In some embodiments, the truncate has no more than 8 missing amino acids. In some embodiments, the truncate has no more than 5 missing amino acids. In some embodiments, the truncate has no more than 3 missing amino acids. In some embodiments, the truncate has no more than 2 missing amino acids. In some embodiments, the truncate has no more than 1 missing amino acid. In other embodiments, the truncate is constructed using the methods and techniques disclosed herein.

[0020] Based on the TCR constant region from various sources, as known to those skilled in the art, it may be possible to truncate 1, 2, 3, 4, 5 or more amino acid residues at its C-terminus and / or N-terminus without significantly affecting the binding properties of the TCR, which is obvious to those skilled in the art. Such truncations include, but are not limited to, truncating the intracellular region of the TCR constant region, retaining only the extracellular region and the transmembrane region; or truncating the intracellular region and the transmembrane region of the TCR constant region, retaining only the extracellular region, etc. For example, the constant region still has a function after the N-terminus of the constant region is truncated as disclosed in the prior art patent number CN116034113A; for example, the intracellular serine residues of the prior art α chain constant region disclosed in Shuhei Ishikura et al. (doi.org / 10.1074 / jbc.M110.127936) can be changed and still have the corresponding function. The present invention includes all such variants.

[0021] First, The present invention provides:

[0022] The TCR constant region with exogenous disulfide bonds introduced includes:

[0023] (1) A point mutation 1 located in the α chain, selected from:

[0024] Any one or more of the amino acid sites that are 3, 5, 41, 42, 78, or 82 amino acids away from the amino acid sites that participate in endogenous disulfide bond formation in the amino acid sequence in the N-terminal direction, or 4, 6, 24, 31, 38, or 39 amino acids away from the amino acid sites in the C-terminal direction, are mutated to C;

[0025] and / or;

[0026] (2) Point mutation 2 located in the β strand, selected from:

[0027] The amino acid sites participating in endogenous disulfide bond formation are mutated to C in any one or more of the sites separated by 1, 45, 72, 76, 104, 111, 112, or 116 amino acids in the N-terminal direction, or the sites separated by 17, 18, 24, or 31 amino acids in the C-terminal direction.

[0028] The sources of the TCR constant region can be diverse, including but not limited to: human, mouse, rabbit, horse, monkey, pig, chicken, cat, dog, sheep, etc.

[0029] Preferably, the TCR constant region can be selected from any one of the following (A, B, C):

[0030] A. The TCR constant region is a human TCR constant region, and the human TCR constant region comprises:

[0031] hTRAC includes mutation 1, and, hTRBC includes mutation 2;

[0032] The mutation 1 is selected from any one or more of P89C, M134C, F20C, V127C, V100C, L12C, R53C, and K102C;

[0033] The mutation 2 is selected from any one or more of A19C, V163C, Y149C, A156C, A129C, F14C, S54C, and T85C;

[0034] The amino acid sequence of hTRAC is the 135-255th position of the sequence with GenBank number AAO72258.1;

[0035] The hTRBC is selected from hTRBC1 or hTRBC2;

[0036] The hTRBC1 has a GenBank number of AAA60713.1 and a sequence number of 61 to 237;

[0037] The UniProtKB number of hTRBC2 is C25777;

[0038] B. The TCR constant region is a mouse TCR constant region, and the mouse TCR constant region comprises:

[0039] The mTRAC basis includes mutation 3, and the mTRBC basis includes mutation 4;

[0040] The mutation 3 is selected from any one or more of S87C, T131C, L116C, V123C, T96C, L12C, K53C, and K98C;

[0041] The mutation 4 is selected from any one or more of A22C, V159C, Y145C, A152C, A125C, F14C, S54C, and T81C;

[0042] The amino acid sequence of mTRAC is the 136-272th position of the sequence with GenBank number AAP88970.1;

[0043] The mTRBC is selected from mTRBC1 or mTRBC2;

[0044] The UniProtKB number of the mTRBC1 is P01852;

[0045] The UniProtKB number of the mTRBC2 is P01851;

[0046] C, truncations of the TCR constant regions in A or B.

[0047] The pre-mutation sequence of the TCR constant region described above (the sequence indicated by the database number) is sometimes also referred to as the wild-type sequence in the present invention.

[0048] In some specific embodiments, the hTRBC is selected from hTRBC1, and the mTRBC is selected from mTRBC1.

[0049] In the TCR constant region described above, the amino acid site involved in the formation of endogenous disulfide bonds in hTRAC is 95C; the amino acid site involved in the formation of endogenous disulfide bonds in hTRBC is 131C; the amino acid site involved in the formation of endogenous disulfide bonds in mTRAC is 91C; and the amino acid site involved in the formation of endogenous disulfide bonds in mTRBC is 127C.

[0050] More preferably, the TCR constant region can be selected from any one of the following:

[0051] 1) In the human TCR constant region:

[0052] The mutation 1 is selected from any one of CYS1-P89C, CYS2-M134C, CYS3-F20C, CYS4-V127C, CYS5-V100C, CYS7-L12C, CYS8-R53C, and CYS9-K102C;

[0053] The mutation 2 is selected from any one of CYS1-A19C, CYS2-V163C, CYS3-Y149C, CYS4-A156C, CYS5-A129C, CYS7-F14C, CYS8-S54C, and CYS9-T85C8;

[0054] The same number in the mutation 1 and mutation 2 is a paired exogenous disulfide bond forming site;

[0055] The human TCR constant region includes any one or more exogenous disulfide bond forming sites;

[0056] 2) In the murine TCR constant region:

[0057] The mutation 3 is selected from any one of: mCYS1-S87C, mCYS2-T131C, mCYS3-L116C, mCYS4-V123C, mCYS5-T96C, mCYS7-L12C, mCYS8-K53C, and mCYS9-K98C8;

[0058] The mutation 4 is selected from any one of: mCYS1-A22C, mCYS2-V159C, mCYS3-Y145C, mCYS4-A152C, mCYS5-A125C, mCYS7-F14C, mCYS8-S54C, and mCYS9-T81C8;

[0059] The same number in the mutation 3 and mutation 4 is a paired exogenous disulfide bond forming site;

[0060] The murine TCR constant region includes any one or more exogenous disulfide bond forming sites;

[0061] 3) A truncated version of the human TCR constant region described in 1) or the mouse TCR constant region described in 2).

[0062] In some specific embodiments, the amino acid residue sites in the human and mouse TCR constant regions that can be mutated to form disulfide bonds are listed in Table 1:

[0063] Table 1:

[0064]

[0065] According to any of the above described TCR constant regions, mutations of endogenous disulfide bonds may be selectively included thereon, and the endogenous disulfide bonds do not form disulfide bonds after mutation. The TCR constant region under this description, which contains the aforementioned amino acid site mutations, selectively includes or does not include mutations of amino acid sites involved in the formation of endogenous disulfide bonds. The amino acid sites involved in the formation of endogenous disulfide bonds may be sites that have been disclosed or undisclosed in the prior art, and are not limited to the specific amino acid sites disclosed in the present invention. In some embodiments, as in hTRAC, the amino acid involved in the formation of endogenous disulfide bonds is 95C, and in mTRAC, the amino acid involved in the formation of endogenous disulfide bonds is, the amino acid sites involved in the formation of endogenous disulfide bonds in hTRBC1 and hTRBC2 are 131C, and the amino acid sites involved in the formation of endogenous disulfide bonds in mTRBC1 and mTRBC1 are 127C, so these sites can be mutated accordingly to introduce mutations of endogenous disulfide bonds.

[0066] The requirement for the mutation of the endogenous disulfide bond is that no disulfide bond is formed after the mutation. In some cases, it can be understood that the amino acid position corresponding to the disulfide bond is mutated from an amino acid that can form a disulfide bond to an amino acid that cannot form a disulfide bond. In general understanding, the amino acid that can form a disulfide bond generally refers to C (cysteine), but other possibilities that have been disclosed or not disclosed in the prior art are also within the scope of protection of the present invention; the amino acid that cannot form a disulfide bond does not have a unique directionality, and any mutation type that can achieve the purpose of destroying endogenous disulfide bonds is acceptable. In some specific embodiments, the mutation can be from C to A. More specifically, it can be any one or more of C91A, C95A, C127A or C131A.

[0067] In some specific embodiments, the mutation of the endogenous disulfide bond site is selected from any of the following:

[0068] (1) mTRAC includes C91A, and mTRBC includes C127A;

[0069] (2) hTRAC includes C95A, and hTRBC includes C131A.

[0070] According to any of the above described TCR constant regions, other amino acid mutations disclosed or not disclosed in the prior art may be selectively included therein, and as an example, any one or more of the following may also be included:

[0071] (1) hTRAC includes T48C and hTRBC includes S57C; or mTRAC includes T48C and mTRBC includes S57C; for forming an exogenous disulfide bond;

[0072] (2) hTRAC includes S61R, and hTRBC includes R79G; or mTRAC includes G61R, and mTRBC includes R75G; for forming a Knob-into-hole structure;

[0073] (3) hTRAC includes any one or more of P91S, E92D, S93V, and S94P; and / or, hTRBC includes any one or more of E18R, S22A, F133I, V136A, and Q139H;

[0074] (4) hTRAC includes any one or more of S115L, G119V, and F120L; or mTRAC includes any one or more of S112L, M114I, G115V, and G61R.

[0075] According to the description of the present invention, a person skilled in the art can also design such a combined mutation based on the wild-type sequence of the TCR constant region:

[0076] Including: any one or more mutations of endogenous disulfide bond sites, and / or, any one or more mutations of forming exogenous disulfide bonds disclosed in the prior art; and / or, any one or more other mutations disclosed in the prior art.

[0077] The above-mentioned combined mutations include but are not limited to the following forms:

[0078] mTRAC includes C91A, and, mTRBC includes C127A, and, mTRAC includes T48C, and, mTRBC includes S57C;

[0079] mTRAC includes C91A, and, mTRBC includes C127A, and, mTRAC includes G61R, and, mTRBC includes R75G;

[0080] mTRAC includes C91A, and mTRBC includes C127A, and mTRAC includes any one or more of S112L, M114I, G115V, and G61R;

[0081] hTRAC includes C95A, and hTRBC includes C131A, and hTRAC includes T48C, and hTRBC includes S57C

[0082] hTRAC includes C95A, and hTRBC includes C131A, and hTRAC includes any one or more of P91S, E92D, S93V, S94P; and / or hTRBC includes any one or more of E18R, S22A, F133I, V136A, Q139H;

[0083] hTRAC includes C95A, and hTRBC includes C131A, and hTRAC includes any one or more of S115L, G119V, and F120L;

[0084] hTRAC includes T48C, and hTRBC includes S57C, and hTRAC includes S61R, and hTRBC includes R79G, and / or hTRAC includes any one or more of P91S, E92D, S93V, S94P, and / or hTRBC includes any one or more of E18R, S22A, F133I, V136A, Q139H;

[0085] hTRAC includes S61R, and hTRBC includes R79G, and hTRAC includes any one or more of P91S, E92D, S93V, S94P, and / or hTRBC includes any one or more of E18R, S22A, F133I, V136A, Q139H;

[0086] hTRAC includes T48C, and hTRBC includes S57C, and hTRAC includes any one or more of S115L, G119V, and F120L;

[0087] hTRAC includes S61R, and hTRBC includes R79G, and hTRAC includes any one or more of S115L, G119V, and F120L;

[0088] hTRAC includes any one or more of P91S, E92D, S93V, S94P, and / or, hTRBC includes any one or more of E18R, S22A, F133I, V136A, Q139H, and hTRAC includes any one or more of S115L, G119V, F120L.

[0089] A person skilled in the art can, based on the above mutations, combine the TCR constant regions presented by A, B, and C to obtain a TCR constant region, and such a combination is also within the scope of protection of the present invention.

[0090] Second, The present invention provides:

[0091] The application of the TCR constant region in constructing TCR, TCR chimeric receptor or TCR fusion protein and corresponding products.

[0092] The product is TCR, TCR chimeric receptor or TCR fusion protein.

[0093] The product at least includes the aforementioned TCR constant region or its truncated form.

[0094] The product may also selectively include an antigen recognition region: the antigen recognition region is a wild-type or artificially synthesized complete protein or a truncated version thereof that can bind to other proteins, polypeptides, and small molecule compounds. As known to those skilled in the art, the antigen recognition region includes but is not limited to TCR variable regions, antibody variable regions, receptors or ligands; the selection of the antigen recognition region can be determined by those skilled in the art according to actual needs. Preferably, the types of antigens targeted by the antigen recognition region include but are not limited to: CD19, CD20, CD22, CD23, BCMA, CD79A, TRBC1, TRBC2, GPRC5D, CD123, CD33, CLL1, Siglec6, CD70, GCC, GPC3, Claudin18.2, Claudin6, MSLN, GD2, IL-13Rα2, CD7, Gucy2c, B7H3, EpCAM, CEA, CEACAM-1, EGFR, EGFRVIII, DLL3, Nectin4, NY-ESO-1, MAGE-A 1 / 3 / 4 / 6, MAGE-C2, MART-1, WT1, PRAME, gp100, MCPyV, hTG, TRAIL, HERV-E, HA-1, Tyrosinase, TGFβRII, HBV, HPV, CMV, EBV, HIV, AFP, HER2, MSLN, KK-LC-1, KRAS mutant, P53 mutant, myeloproliferative leukemia protein (MPL), CD30, CD32, CD99, CD138, CD179b, CD200R, CD276, CD324, Fc receptor-like 5 (FcRH5), CD171, CS 1 (signaling lymphocyte activation molecule family 7, SLAMF7), C-type lectin-like molecule 1 (CLL1), cadherin 1, cadherin 6, cadherin 16, cadherin 17, cadherin 19, epidermal growth factor receptor variant III (EGFRviii), ganglioside GD2, ganglioside GD3, human leukocyte antigen A2 (HLA A2), B cell maturation antigen (BCMA), Tn antigen, prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), FMS-like tyrosine kinase 3 (FLT3), fibroblast activation protein (FAP), tumor-associated glycoprotein (TAG) 72, CD38, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), B7 H3 (CD276), KIT, interleukin 13 receptor subunit α2 (IL13Ra2), interleukin 11 receptor subunit α (IL11Ra), mesothelin (MSLN), prostate stem cell antigen (PSCA), vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, platelet-derived growth factor receptor beta (PDGFRβ), protease serine 21 (PRSS21), sialoglycolipid stage-specific embryonic antigen 4 (SSEA 4), CD20, immunoglobulin Fc region, tissue factor, folate receptor alpha, epidermal growth factor receptor 2 (ERBB2), cadherin 1 (MUC1), epidermal growth factor receptor (EGFR), neural small adhesion molecule (NCAM), protease, prostatic acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), Ephrin B2, insulin-like growth factor I receptor (IGF 1 receptor), carbonic anhydrase IX (CAIX), latent membrane protein 2 (LMP2), melanocyte protein gpl00, bcr abl, tyrosinase, erythropoietin-producing hepatocellular carcinoma A2 (EphA2), fucosylated monosialoganglioside (fucosylated GM1), sialyl Lewis a(sLea), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl GD2 ganglioside, folate receptor β, TEM1 / CD248, tumor endothelial marker-associated protein 7 (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), T cell receptor (TCR) β1 constant chain, TCRβ2 constant chain, TCRγδ, G protein-coupled receptor class C group 5 member D (GPRC5D), CXORF61 protein, CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), carbohydrate antigen GloboH, breast differentiation antigen NY BR 1, uroplakin2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), pan-linked protein 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 family member K (LY6K), olfactory receptor family 51 subfamily E member 2 (OR51E2), T cell receptor gamma chain variable reading frame protein (TARP), Wilms tumor antigen 1 protein (WT1), tumor testis antigen NY ESO 1, tumor testis antigen LAGE 1a, legumain, human papillomavirus (HPV) E6, HPV E7, human T lymphotrophic virus (HTLV1) Tax, Kaposi's sarcoma-associated herpes virus glycoprotein (KSHV) K8.1 protein, Epstein-Barr virus (EBV) encoded glycoprotein 350 (EBB gp350), HIV1 envelope glycoprotein gp120, multiplex automated genome engineering (MAGE) A1, translocation Ets leukemia virus (ETV) protein 6 AML, sperm protein 17, X antigen family member (XAGE) 1, transmembrane tyrosine protein kinase receptor Tie2. Melanoma tumor testis antigen MAD CT 1, melanoma tumor testis antigen MAD CT 2, Fos-related antigen 1, p53, p53 mutant, prostein, survival rate and telomerase, prostate cancer tumor antigen 1 (PCTA 1) / galectin 8, MelanA / MART1, Ras mutant, human telomerase reverse transcriptase (hTERT), delta-like 3 (DLL3), trophoblast cell surface antigen 2 (TROP2), protein tyrosine kinase 7 (PTK7), guanylate cyclase C (GCC), alpha-fetoprotein (AFP), sarcoma translocation breakpoint, adrenergic receptor, melanoma apoptosis inhibitor (ML IAP), ERG (TMPRSS2 ETS fusion gene), N-acetylglucosyltransferase V (NA17), paired box protein Pax 3 (PAX3), androgen receptor, cell cycle protein B1, v myc avian myelocytoma viral oncogene neuroblastoma derived homolog (MYCN), Ras homology family member C (RhoC), tyrosinase-related protein 2 (TRP 2), cytochrome P4501B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like (BORIS or Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), PAX5, pre-acrosomal protein binding protein sp32 (OY TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP4), synovial sarcoma, X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE 1), RU1, RU2, intestinal carboxylesterase, heat shock protein 70 2 mutant (mut hsp70 2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), IgA receptor Fc fragment (FCAR), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), dopamine receptor, EGF like module containingmucin like hormone receptor like2, EMR2), lymphocyte antigen 75 (LY75), glypican 3 (GPC3), Fc receptor-like 5 (FCRL5), immunoglobulin lambda-like polypeptide 1 (IGLL1), FITC, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), chorionic gonadotropin receptor (CGHR), CC chemokine receptor 4 (CCR4), ganglioside GD3, signaling lymphocyte activation molecule (SLAM) family member 6 (SLAMF6), SLAMF4, or any combination of more than one thereof.

[0095] Specifically, the antigen recognition region can be selected from the following:

[0096] (1) The antigen recognition region can be a TCR variable region. The TCR variable region is used to target all feasible antigen types including but not limited to the aforementioned ones; the source of the TCR can be human, mouse, rabbit, horse, monkey, pig, chicken, cat, dog, sheep, etc., and after fusion expression with any TCR constant region or its truncated form in the present invention, a functional TCR can be formed. The selection of the TCR variable region can be determined by those skilled in the art according to actual needs.

[0097] (2) The antigen recognition region may be an antibody variable region, and the antibody variable region is used to target all feasible antigen types including but not limited to the aforementioned ones; the antibody may be derived from human, mouse, rabbit, horse, monkey, pig, chicken, cat, dog, sheep, etc., and the selection of the antibody variable region may be determined by a person skilled in the art according to actual needs.

[0098] (3) The antigen recognition region is a receptor or ligand, and the receptor includes but is not limited to: VEGFR2, ERBB2, PDGFRβ, EGFR, TSHR, HAVCR1, GPR20, ADRB3, RAGE 1, LAIR1, FCAR, LHR, FSHR, CGHR, CCR4, PD-1 and other receptor types disclosed or not disclosed in the present invention, and the ligand includes but is not limited to: PD-L1, acetylcholine, epidermal growth factor, platelet growth factor, erythropoietin, growth hormone and other polypeptide hormones, steroid hormones, vitamin D3, thyroxine, retinoic acid, epinephrine, dopamine, amphetamine, diazepam, aripiprazole and other ligand types disclosed or not disclosed in the present invention. The selection of ligands and receptors can be determined by those skilled in the art according to actual needs.

[0099] The product may also selectively include a transmembrane region. The transmembrane region generally refers to the region in the protein sequence that spans the cell membrane, and sometimes refers to the portion of the transmembrane protein that is embedded in the phospholipid bilayer of the cell membrane. It can usually be an α-helical structure with about 20-25 amino acid residues. Most of the amino acids that make up its protein are hydrophobic amino acids. The choice of the transmembrane region can be determined by a person skilled in the art according to actual needs. Preferably, the source protein of the transmembrane region includes but is not limited to: CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD5, CD 2. Any one or more of the transmembrane regions of CD22, CD27, CD28, CD40, CD64, CD66, CD69, CD16, CD79, CD89, CD40L, HVEM, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, PD-1, and FcR, or mutants thereof that retain their transmembrane function.

[0100] In some specific embodiments, the transmembrane region is a transmembrane region of TCR, characterized in that it can interact with the transmembrane region of the CD3 molecule to form a TCR-CD3 complex, and the main function of the two polymorphic subunits in the TCR-CD3 complex is to recognize antigens bound to MHC molecules; the main function of the CD3 molecule is to participate in the assembly and stability of the TCR-CD3 complex and signal transduction. Optionally, the transmembrane region is a transmembrane region of a human TCRα chain or a mutant sequence thereof; the mutant sequence may include any one or more of S115L, G119V, and F120L on the basis of the transmembrane region of the TCRα chain, and the mutation site is within hTRAC. Optionally, the transmembrane region is a transmembrane region of a mouse TCRα chain or a mutant sequence thereof; the mutant sequence may include any one or more of S112L, M114I, G115V, and G61R on the basis of the transmembrane region of the TCRα chain, and the mutation site is within mTRAC. The scope of protection of the present invention also includes other unstated transmembrane region mutation types.

[0101] The product may also selectively include an intracellular signal transduction domain. The intracellular signal transduction domain is a part of the intracellular domain that can play a T cell signal transduction function, which can usually be a T cell receptor TCR / CD3ζ chain or an immunoglobulin Fc receptor FcεRIγ chain, containing an immunoreceptor tyrosine-based activation motif (ITAMs). In fact, those skilled in the art can select the following sources as needed: CD3, CD28, 4-1BB, CD27, ICOS, OX40, γ C , IL-2RA, IL-2RB, IL-4R, IL-7RA, IL-9R, IL-15RA, IL-21R, TSLPR, ZAP-70, PLCγ, etc.

[0102] In some specific experiments of the present invention, as an example, the above-mentioned various TCR constant region sequences are applied by taking TCR targeting NY-ESO-1 and TCR targeting p53 (R175H) as examples, that is, the variable region of TCR targeting NY-ESO-1 (V region, TRBV coding sequence is SEQ ID NO.85, TRAV coding sequence is SEQ ID NO.86) or the variable region of TCR targeting p53 (R175H) (V region, TRBV coding sequence is SEQ ID NO.87, TRAV coding sequence is SEQ ID NO.88) is spliced ​​with the above-mentioned various TCR constant region sequences, and the spliced ​​complete TCR gene is transferred into cells, so as to analyze the pairing and membrane display efficiency of various TCRs and the killing function of tumor cells. The involved TCR constant regions include 7 groups, which are specifically described as follows:

[0103] (1) INESS-hTCR: Based on the constant region of natural human TCR (hTCR), 9 pairs of exogenous mutated disulfide bonds corresponding to the human sequences in Table 1 are introduced respectively (each TCR contains only 1 exogenous disulfide bond, and there are 9 TCR sequences in total). The 9 TCRs included are selected from the following:

[0104] ① The mutation site contained in TRBC is: CYS1B (A19C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 1; the mutation site contained in TRAC is: CYS1A (P89C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 2.

[0105] ②The mutation site contained in TRBC is: CYS2B (V163C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 3; the mutation site contained in TRAC is: CYS2A (M134C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 4.

[0106] ③The mutation site contained in TRBC is: CYS3B (Y149C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 5; the mutation site contained in TRAC is: CYS3A (F20C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 6.

[0107] ④The mutation site contained in TRBC is: CYS4B (A156C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 7; the mutation site contained in TRAC is: CYS4A (V127C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 8.

[0108] ⑤The mutation site contained in TRBC is: CYS5B (A129C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 9; the mutation site contained in TRAC is: CYS5A (V100C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 10.

[0109] ⑥The mutation site contained in TRBC is: CYS6B (S57C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 11; the mutation site contained in TRAC is: CYS6A (T48C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 12.

[0110] ⑦The mutation site contained in TRBC is: CYS7B (F14C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 13; the mutation site contained in TRAC is: CYS7A (L12C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 14.

[0111] ⑧The mutation site contained in TRBC is: CYS8B (S54C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 15; the mutation site contained in TRAC is: CYS8A (R53C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 16.

[0112] ⑨The mutation site contained in TRBC is: CYS9B (T85C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 17; the mutation site contained in TRAC is: CYS9A (K102C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 18.

[0113] (2) INESS-sTCR: Based on (1), other mutations were introduced. Eight of the TCRs were as follows: based on the human TCR constant region, the selected exogenous mutated disulfide bonds were introduced, and then the 4857 mutation, or the mm mutation, or the TMa mutation, or the 4857, mm, and TMa mutations were introduced. The specific information is as follows:

[0114] ① The mutation sites contained in TRBC include CYS5B (A129C) and S57C, and preferably, the corresponding nucleotide sequence is SEQID NO: 19; the mutation sites contained in TRAC include CYS5A (V100C) and T48C, and preferably, the corresponding nucleotide sequence is SEQID NO: 20.

[0115] ②The mutation sites contained in TRBC include CYS5B (A129C), mmB (E18R, S22A, F133I, V136A, Q139H), preferably, the corresponding nucleotide sequence is SEQ ID NO: 21; the mutation sites contained in TRAC include CYS5A (V100C), mmA (P91S, E92D, S93V, S94P), preferably, the corresponding nucleotide sequence is SEQ ID NO: 22.

[0116] ③The mutation sites contained in TRBC are: CYS5B (A129C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 23; the mutation sites contained in TRAC are: CYS5A (V100C), hTMa (S115L, G119V, F120L), preferably, the corresponding nucleotide sequence is SEQ ID NO: 24.

[0117] ④The mutation sites contained in TRBC are: CYS5B (A129C), S57C, mmB (E18R, S22A, F133I, V136A, Q139H), preferably, the corresponding nucleotide sequence is SEQ ID NO: 25; the mutation sites contained in TRAC are: CYS5A (V100C), T48C, mmA (P91S, E92D, S93V, S94P), hTMa (S115L, G119V, F120L), preferably, the corresponding nucleotide sequence is SEQ ID NO: 26.

[0118] ⑤The mutation sites contained in TRBC are: CYS9B (T85C), S57C, preferably, the corresponding nucleotide sequence is SEQID NO: 27; the mutation sites contained in TRAC are: CYS9A (K102C), T48C, preferably, the corresponding nucleotide sequence is SEQID NO: 28.

[0119] ⑥The mutation sites contained in TRBC are: CYS9B (T85C), mmB (E18R, S22A, F133I, V136A, Q139H), preferably, the corresponding nucleotide sequence is SEQ ID NO: 29; the mutation sites contained in TRAC are: CYS9A (K102C), mmA (P91S, E92D, S93V, S94P), preferably, the corresponding nucleotide sequence is SEQ ID NO: 30.

[0120] ⑦The mutation sites contained in TRBC are: CYS9B (T85C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 31; the mutation sites contained in TRAC are: CYS9A (K102C), hTMa (S115L, G119V, F120L), preferably, the corresponding nucleotide sequence is SEQ ID NO: 32.

[0121] ⑧The mutation sites contained in TRBC are: CYS9B (T85C), S57C, mmB (E18R, S22A, F133I, V136A, Q139H), preferably, the corresponding nucleotide sequence is SEQ ID NO: 33; the mutation sites contained in TRAC are: CYS9A (K102C), T48C, mmA (P91S, E92D, S93V, S94P), hTMa (S115L, G119V, F120L), preferably, the corresponding nucleotide sequence is SEQ ID NO: 34.

[0122] (3) INESS-mutEC-hTCR: Based on (1), endogenous disulfide bond site mutations are introduced. It contains 9 TCRs: based on the human TCR constant region, the selected exogenous mutated disulfide bonds are introduced, and then the endogenous disulfide bond site mutations are introduced to remove the natural endogenous disulfide bonds of the TCR. The specific information is as follows:

[0123] ① The mutation sites contained in TRBC are: CYS1B (A19C), C131A, preferably, the corresponding nucleotide sequence is SEQID NO: 35; the mutation sites contained in TRAC are: CYS1A (P89C), C95A, preferably, the corresponding nucleotide sequence is SEQID NO: 36.

[0124] ②The mutation sites contained in TRBC are: CYS2B (V163C), C131A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 37; the mutation sites contained in TRAC are: CYS2A (M134C), C95A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 38.

[0125] ③The mutation sites contained in TRBC are: CYS3B (Y149C), C131A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 39; the mutation sites contained in TRAC are: CYS3A (F20C), C95A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 40.

[0126] ④The mutation sites contained in TRBC are: CYS4B (A156C), C131A, preferably, the corresponding nucleotide sequence is SEQ ID NO:41; the mutation sites contained in TRAC are: CYS4A (V127C), C95A, preferably, the corresponding nucleotide sequence is SEQ ID NO:42.

[0127] ⑤The mutation sites contained in TRBC are: CYS5B (A129C), C131A, preferably, the corresponding nucleotide sequence is SEQ ID NO:43; the mutation sites contained in TRAC are: CYS5A (V100C), C95A, preferably, the corresponding nucleotide sequence is SEQ ID NO:44.

[0128] ⑥The mutation sites contained in TRBC are: CYS6B (S57C), C131A, preferably, the corresponding nucleotide sequence is SEQID NO:45; the mutation sites contained in TRAC are: CYS6A (T48C), C95A, preferably, the corresponding nucleotide sequence is SEQID NO:46.

[0129] ⑦The mutation sites contained in TRBC are: CYS7B (F14C), C131A, preferably, the corresponding nucleotide sequence is SEQID NO:47; the mutation sites contained in TRAC are: CYS7A (L12C), C95A, preferably, the corresponding nucleotide sequence is SEQID NO:48.

[0130] ⑧The mutation sites contained in TRBC are: CYS8B (S54C), C131A, preferably, the corresponding nucleotide sequence is SEQID NO:49; the mutation sites contained in TRAC are: CYS8A (R53C), C95A, preferably, the corresponding nucleotide sequence is SEQID NO:50.

[0131] ⑨The mutation sites contained in TRBC are: CYS9B (T85C), C131A, preferably, the corresponding nucleotide sequence is SEQID NO:51; the mutation sites contained in TRAC are: CYS9A (K102C), C95A, preferably, the corresponding nucleotide sequence is SEQID NO:52.

[0132] (4) INESS-mutEC-sTCR: Based on (3), other mutations were introduced. Eight of the TCRs were as follows: based on the human TCR constant region, the selected exogenous mutated disulfide bonds and endogenous disulfide bond site mutations were introduced, and then 4857 mutations, or mm mutations, or TMa mutations were introduced, or 4857, mm, and TMa mutations were included at the same time. The specific information is as follows:

[0133] ①The mutation sites contained in TRBC are: CYS5B (A129C), C131A, S57C, preferably, the corresponding nucleotide sequence is SEQ ID NO: 53; the mutation sites contained in TRAC are: CYS5A (V100C), C95A, T48C, preferably, the corresponding nucleotide sequence is SEQ ID NO: 54.

[0134] ②The mutation sites contained in TRBC are: CYS5B (A129C), C131A, mmB (E18R, S22A, F133I, V136A, Q139H), preferably, the corresponding nucleotide sequence is SEQ ID NO: 55; the mutation sites contained in TRAC are: CYS5A (V100C), C95A, mmA (P91S, E92D, S93V, S94P), preferably, the corresponding nucleotide sequence is SEQ ID NO: 56.

[0135] ③The mutation sites contained in TRBC are: CYS5B (A129C), C131A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 57; the mutation sites contained in TRAC are: CYS5A (V100C), C95A, hTMa (S115L, G119V, F120L), preferably, the corresponding nucleotide sequence is SEQ ID NO: 58.

[0136] ④The mutation sites contained in TRBC are: CYS5B (A129C), C131A, S57C, mmB (E18R, S22A, F133I, V136A, Q139H), preferably, the corresponding nucleotide sequence is SEQ ID NO: 59; the mutation sites contained in TRAC are: CYS5A (V100C), C95A, T48C, mmA (P91S, E92D, S93V, S94P), hTMa (S115L, G119V, F120L), preferably, the corresponding nucleotide sequence is SEQ ID NO: 60.

[0137] ⑤The mutation sites contained in TRBC are: CYS9B (T85C), C131A, S57C, preferably, the corresponding nucleotide sequence is SEQ ID NO: 61; the mutation sites contained in TRAC are: CYS9A (K102C), C95A, T48C, preferably, the corresponding nucleotide sequence is SEQ ID NO: 62.

[0138] ⑥The mutation sites contained in TRBC are: CYS9B (T85C), C131A, mmB (E18R, S22A, F133I, V136A, Q139H), preferably, the corresponding nucleotide sequence is SEQ ID NO: 63; the mutation sites contained in TRAC are: CYS9A (K102C), C95A, mmA (P91S, E92D, S93V, S94P), preferably, the corresponding nucleotide sequence is SEQ ID NO: 64.

[0139] ⑦The mutation sites contained in TRBC are: CYS9B (T85C), C131A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 65; the mutation sites contained in TRAC are: CYS9A (K102C), C95A, hTMa (S115L, G119V, F120L), preferably, the corresponding nucleotide sequence is SEQ ID NO: 66.

[0140] ⑧The mutation sites contained in TRBC are: CYS9B (T85C), C131A, S57C, mmB (E18R, S22A, F133I, V136A, Q139H), preferably, the corresponding nucleotide sequence is SEQ ID NO: 67; the mutation sites contained in TRAC are: CYS9A (K102C), C95A, T48C, mmA (P91S, E92D, S93V, S94P), hTMa (S115L, G119V, F120L), preferably, the corresponding nucleotide sequence is SEQ ID NO: 68.

[0141] (5) INESS-mul-TCR: Based on (3), three pairs of disulfide bonds (CYS5, CYS6, CYS9) were selected for combined mutation. Four of the TCRs were as follows: based on the human TCR constant region, endogenous disulfide bond site mutations were introduced, and then two pairs of disulfide bond site mutations of CYS5+CYS6 were introduced, or two pairs of disulfide bond site mutations of CYS9+CYS6 were introduced, or two pairs of disulfide bond site mutations of CYS5+CYS9 were introduced, or three pairs of disulfide bond site mutations of CYS5+CYS6+CYS9 were introduced. The specific information is as follows:

[0142] ①The mutation sites contained in TRBC are: C131A, CYS5B (A129C), CYS6B (S57C); the mutation sites contained in TRAC are: C95A, CYS5A (V100C), CYS6A (T48C).

[0143] This item is the same as ① in (4).

[0144] ②The mutation sites contained in TRBC are: C131A, CYS6B (S57C), CYS9B (T85C); the mutation sites contained in TRAC are: C95A, CYS6A (T48C), CYS9A (K102C).

[0145] This item is the same as ⑤ in (4).

[0146] ③The mutation sites contained in TRBC are: C131A, CYS5B (A129C), CYS9B (T85C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 69; the mutation sites contained in TRAC are: C95A, CYS5A (V100C), CYS9A (K102C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 70.

[0147] ④The mutation sites contained in TRBC are: C131A, CYS5B (A129C), CYS6B (S57C), CYS9B (T85C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 71; the mutation sites contained in TRAC are: C95A, CYS5A (V100C), CYS6A (T48C), CYS9A (K102C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 72.

[0148] (6) INESS-mTCR: Based on the constant region of natural mouse TCR (mTCR), 9 pairs of exogenous mutant disulfide bonds corresponding to the mouse sequences in Table 1 were introduced (each TCR contains only 1 exogenous disulfide bond, and there are 9 TCR sequences in total). The specific information of 2 TCRs is as follows:

[0149] ① The mutation sites contained in TRBC are: mCYS5B (A125C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 73; the mutation sites contained in TRAC are: mCYS5A (T96C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 74.

[0150] ②The mutation sites contained in TRBC are: mCYS9B (T81C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 75; the mutation sites contained in TRAC are: mCYS9A (K98C), preferably, the corresponding nucleotide sequence is SEQ ID NO: 76.

[0151] (7) INESS-mutEC-mTCR: Based on (6), the endogenous disulfide bond sites were mutated and the endogenous disulfide bond sites of mouse TCR were removed. The specific information of the two TCRs is as follows:

[0152] ① The mutation sites contained in TRBC are: mCYS5B (A125C), C127A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 77; the mutation sites contained in TRAC are: mCYS5A (T96C), C91A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 78.

[0153] ②The mutation sites contained in TRBC are: mCYS9B (T81C), C127A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 79; the mutation sites contained in TRAC are: mCYS9A (K98C), C91A, preferably, the corresponding nucleotide sequence is SEQ ID NO: 80.

[0154] The amino acid sequence of the TCR described in the present invention may also be any one or more of the amino acid sequences having more than 80% similarity to the above specific point mutation sequences and having the same disulfide bond positions.

[0155] Figure 1 Schematic diagram of the T cell receptor INESS-TCR with introduced exogenous disulfide bonds. Figure 1 A shows the schematic diagram of the INESS-TCR structure. INESS-TCR is a TCR with an exogenous disulfide bond introduced. It is divided into 7 groups according to whether the endogenous disulfide bond is mutated, whether it is combined with other optimizations of the TCR constant region, and the different sources of the TCR constant region. Figure 1B shows the sequence diagram of INESS-TCR. The gene sequences of the α and β polypeptide chains of TCR contained in INESS-TCR are connected through the furin and P2A protease cleavage site polypeptide segments. The two polypeptide chains will be transcribed and translated into proteins together, and then cut into two independent peptide chains by the proteases corresponding to furin and p2A. The two peptide chains can be assembled into a complete TCR by forming disulfide bonds between the chains. TCR and the CD3 molecule subunits on the T cell membrane are assembled into a TCR-CD3 complex, which is stably expressed on the cell membrane. INESS-TCR mutates the adjacent amino acid residues in the spatial structure of the two peptide chains of TCR into cysteine, and a disulfide bond is formed between the two cysteines, thereby promoting the binding between the two chains of TCR and the expression efficiency of TCR on the cell membrane.

[0156] Based on the aforementioned TCR constant region and TCR, the present invention also provides a chimeric antibody comprising the aforementioned TCR constant region or TCR.

[0157] As is generally known in the art, the chimeric antibody can be a chimeric monoclonal antibody or a chimeric bispecific antibody.

[0158] The present invention also provides a fusion protein comprising the aforementioned TCR constant region, TCR or TCR chimeric antibody.

[0159] In a third aspect, the present invention provides the corresponding genetic engineering products, clinical applications and products of the aforementioned TCR constant region, TCR or TCR chimeric antibody or fusion protein.

[0160] The genetic engineering product is selected from any one of the following:

[0161] (1) a nucleic acid molecule encoding: the aforementioned TCR constant region or TCR constant region truncations, or a product containing the corresponding TCR constant region or truncations;

[0162] (2) an expression vector comprising the nucleic acid molecule described in (1), or a product expressing the aforementioned TCR constant region or a product containing the corresponding TCR constant region;

[0163] (3) Cells: transferred with the expression vector described in (2), or expressing or secreting the aforementioned TCR constant region or a product containing the corresponding TCR constant region;

[0164] Preferably, the cells are selected from: T cells, NK cells, NKT cells, macrophages, dendritic cells, neutrophils; the cells are derived from the patient's own body, healthy volunteers, umbilical cord blood or in vitro induced pluripotent stem cells.

[0165] Based on codon degeneracy, knowing the amino acid sequence, a person skilled in the art can design a variety of nucleic acid molecules to express it, so the nucleic acid molecules protected by the present invention cover all possibilities.

[0166] The type of expression vector can be selected from any disclosed or undisclosed type in the prior art, as long as it can achieve the expression of the corresponding product. Preferably, it can be a viral vector or an animal expression vector, and more preferably, it can be a lentiviral expression vector or a mammalian expression vector.

[0167] The cell comprises the aforementioned vector or expresses the aforementioned TCR constant region, TCR or TCR chimeric antibody or fusion protein.

[0168] The cells may be of a cell type disclosed or undisclosed in the prior art.

[0169] The clinical application may be an application in the preparation of downstream products.

[0170] The downstream product is selected from any of the following:

[0171] (1) Detection reagents, used to bind to antigens to make judgments or obtain the antigens they bind to;

[0172] (2) Drugs for preventing or treating diseases, preferably, the diseases include tumors, autoimmune diseases, viral infections, fungal infections or bacterial infections.

[0173] The present invention also provides downstream products based on the aforementioned applications.

[0174] The downstream product is selected from any of the following:

[0175] (1) Detection reagents, including or excluding excipients;

[0176] (2) Drugs, with or without excipients.

[0177] The excipients in the medicine are generally pharmaceutical excipients.

[0178] The pharmaceutical excipients are excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated in terms of safety and are included in drug preparations in addition to active ingredients. In addition to excipients, carriers, and stability enhancement, pharmaceutical excipients also have important functions such as solubilization, dissolution assistance, and sustained and controlled release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. According to their sources, they can be divided into natural products, semi-synthetic products, and fully synthetic products. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc. According to their administration routes, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and ocular administration, etc. The same pharmaceutical excipients can be used in drug preparations with different administration routes and have different functions and uses.

[0179] The diagnostic reagent is used to make a diagnosis in combination with an antigen; specifically, it can be used to detect or diagnose: NY-ESO-1, MAGE-A1 / 3 / 4 / 6, MAGE-C2, MART-1, WT1, PRAME, gp100, MCPyV, hTG, TRAIL, HERV-E, HA-1, Tyrosinase, TGFβRII, HBV, HPV, CMV, EBV, HIV, AFP, HER2, MSLN, KK-LC-1, KRAS, P53, CD19, CD20, CD22, BCMA, CD79A, TRBC1, TRBC2, GPRC5D, CD123, CD33, CLL1, Siglec6, CD70, GCC, GPC3, Claudin18.2, Claudin6, MSLN, GD2, IL13Rα2, CD7, Gucy2c, B7H3, EpCAM, CEA, CEACAM-1, EGFR, EGFRVIII, DLL3, Nectin4 or any one or more of their related diseases.

[0180] In some specific embodiments, the method can be used to detect or diagnose NY-ESO-1 or mutant p53 related diseases.

[0181] The drug is preferably used to prevent or treat tumors, autoimmune diseases, viral infections, fungal infections or bacterial infections.

[0182] More preferably, the drug is used for the treatment or prevention of: NY-ESO-1, MAGE-A1 / 3 / 4 / 6, MAGE-C2, MART-1, WT1, PRAME, gp100, MCPyV, hTG, TRAIL, HERV-E, HA-1, Tyrosinase, TGFβRII, HBV, HPV, CMV, EBV, HIV, AFP, HER2, MSLN, KK-LC-1, KRAS, P53, CD19, CD20, CD 22. Diseases in which any one or more of BCMA, CD79A, TRBC1, TRBC2, GPRC5D, CD123, CD33, CLL1, Siglec6, CD70, GCC, GPC3, Claudin18.2, Claudin6, MSLN, GD2, IL13Rα2, CD7, Gucy2c, B7H3, EpCAM, CEA, CEACAM-1, EGFR, EGFRVIII, DLL3, and Nectin4 are used as therapeutic targets.

[0183] In some specific embodiments, the drug is used to treat or prevent NY-ESO-1 or mutant p53 related diseases.

[0184] The NY-ESO-1-related diseases include, but are not limited to, breast cancer, bladder cancer, prostate cancer, melanoma, non-small cell lung cancer, hepatocellular carcinoma, ovarian cancer, myxoid / round cell liposarcoma, neuroblastoma, synovial sarcoma, etc. The mutant p53-related diseases are widely present in various tumors, including, but not limited to, ovarian cancer, breast cancer, small cell lung cancer, pancreatic cancer, bladder cancer, prostate cancer, melanoma, non-small cell lung cancer, hepatocellular carcinoma, acute myeloid leukemia, lymphoma, etc.

[0185] The present invention also protects a detection method or a treatment method based on the above product.

[0186] Specifically, a method for detecting an antigen is provided, comprising: using the aforementioned detection reagent to contact with a test object for detection.

[0187] Specifically, a method for treating a disease is provided, comprising: administering the aforementioned drug to a patient in need thereof.

[0188] As used herein, the term "treat or prevent" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or side effects resulting from a disease. As used herein, "treat or prevent" includes, but is not limited to: (a) preventing a disease or symptom from occurring in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) inhibiting the symptoms of a disease, i.e., preventing its development; or (c) alleviating the symptoms of a disease, i.e., causing the disease or symptom to regress.

[0189] Beneficial effects of the present invention:

[0190] The present invention screens out nine pairs of amino acid residues that are spatially adjacent by analyzing the protein structure of the constant regions of the TCR α and β chains, and mutates them into cysteine, thereby introducing new disulfide bonds in the TCR α and β chains. Experiments have verified that four of the nine pairs of cysteines can improve the pairing of exogenous TCRs and the expression efficiency on T cells, and can enhance the killing function of TCR-T cells against tumors. And when combined with the currently reported optimization strategies, the upper membrane expression efficiency of TCR and the killing function of TCR-T can be significantly improved.

[0191] In addition, there is a natural disulfide bond between the two chains of TCR, which plays an important role in forming dimers for the α and β chains, thereby maintaining the stability of the natural TCR structure. The exogenously introduced TCR chain can also form this natural disulfide bond with the endogenous TCR chain, which is also the main cause of mismatching. Therefore, in the present invention, the amino acids involved in the formation of natural disulfide bonds in the constant regions of the TCR α and β chains are mutated, and then the newly screened 9 pairs of amino acid residues are introduced by gene editing to promote the formation of new disulfide bonds between the two chains of TCR, thereby enhancing the independent pairing of exogenous TCRs and reducing mismatches. Experimental verification shows that after deleting the endogenous disulfide bonds, the expression rate of TCR on the cell surface is significantly reduced. The 4 pairs of amino acid residues introduced can promote the formation of new disulfide bonds between the two chains of TCR and restore the expression level of TCR on the cell surface, of which the introduction of 2 pairs of amino acid residues can make the pairing and upper membrane expression efficiency of TCR significantly higher than the original TCR without deleting the endogenous disulfide bonds. And when combined with the currently reported optimization strategies, it can significantly improve the efficiency of TCR expression on the membrane and the killing function of TCR-T. Compared with the reported optimization strategies, mutating the amino acids that form natural disulfide bonds and then introducing new disulfide bonds can more effectively solve the mismatch between exogenous and endogenous TCR chains, thereby improving safety.

[0192] In addition to the modification of the human TCR constant region, the mouse TCR constant region was also modified. The screened exogenous disulfide bond amino acid mutations were introduced into the wild-type TCR constant region and the TCR constant region with mutations in the amino acid sites involved in the formation of endogenous disulfide bonds, thereby improving the membrane expression efficiency and tumor killing function of TCR using the mouse TCR constant region.

[0193] Finally, based on the human TCR constant region with mutated endogenous disulfide bonds, we constructed and introduced different combinations of exogenous disulfide bonds, proving that the combination of the new disulfide bonds we screened can better promote the pairing, membrane binding and function of TCR.

[0194] The present invention provides a new solution strategy for improving the stability, expression rate, efficacy and safety of engineered TCR, and is of great significance to biomedical fields such as TCR-T cell therapy, TCR chimeric receptor modified cell therapy, TCR antibodies, and bispecific antibodies. The TCR-T cell immunotherapy involved in the present invention is expected to provide a safer and more effective option for many solid tumor patients in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0195] Figure 1 Schematic diagram of the T cell receptor INESS-TCR with introduced exogenous disulfide bonds.

[0196] Figure 2 A is a pseudo-color flow cytometric image of the expression efficiency and upper membrane level of INESS-hTCR in human primary T cells. A is a pseudo-color flow cytometric image of the expression efficiency and upper membrane level of NY-ESO-1 TCR in human primary T cells, and B is a pseudo-color flow cytometric image of the expression efficiency and upper membrane level of p53 (R175H) TCR in human primary T cells.

[0197] Figure 3 A is a flow cytometric histogram of the expression efficiency and upper membrane level detection of INESS-hTCR in human primary T cells. A is a flow cytometric histogram of the expression efficiency and upper membrane level detection of NY-ESO-1 TCR targeting in human primary T cells, and B is a flow cytometric histogram of the expression efficiency and upper membrane level detection of p53 (R175H) TCR targeting in human primary T cells.

[0198] Figure 4 are the functional test results of INESS-hTCR in human primary T cells, A is the death percentage of target cells carrying NY-ESO-1 antigen, B is the content of cytokine IL-2 in the co-culture supernatant, C is the content of cytokine IFN-γ in the co-culture supernatant, and D is the death percentage of target cells carrying p53 (R175H) antigen.

[0199] Figure 5This is a pseudo-color flow cytometry image showing the expression efficiency and membrane level detection of INESS-sTCR in primary human T cells.

[0200] Figure 6 Flow cytometry histogram showing the expression efficiency and membrane level detection of INESS-sTCR in primary human T cells.

[0201] Figure 7 are the functional detection results of INESS-sTCR in human primary T cells, A is the percentage of target cell death, B is the content of cytokine IL-2 in the co-culture supernatant, and C is the content of cytokine IFN-γ in the co-culture supernatant.

[0202] Figure 8 This is a pseudo-color flow cytometry image showing the expression efficiency and membrane level detection of INESS-mutEC-hTCR in primary human T cells.

[0203] Fig. 9 Flow cytometry histogram showing the expression efficiency and membrane level detection of INESS-mutEC-hTCR in primary human T cells.

[0204] Fig.10 are the functional detection results of INESS-mutEC-hTCR in human primary T cells, A is the percentage of target cell death, B is the content of cytokine IL-2 in the co-culture supernatant, and C is the content of cytokine IFN-γ in the co-culture supernatant.

[0205] Fig.11 This is a pseudo-color flow cytometry image showing the expression efficiency and membrane level detection of INESS-mutEC-sTCR in primary human T cells.

[0206] Fig.12 Flow cytometry histogram showing the expression efficiency and membrane level detection of INESS-mutEC-sTCR in primary human T cells.

[0207] Fig.13 are the functional detection results of INESS-mutEC-sTCR in human primary T cells, A is the percentage of target cell death, B is the content of cytokine IL-2 in the co-culture supernatant, and C is the content of cytokine IFN-γ in the co-culture supernatant.

[0208] Fig.14 This is a pseudo-color flow cytometric image showing the expression efficiency and membrane level detection of INESS-mul-TCR in primary human T cells.

[0209] Fig.15 Flow cytometry histogram showing the expression efficiency and membrane level detection of INESS-mul-TCR in primary human T cells.

[0210] Fig.16are the functional detection results of INESS-mul-TCR in human primary T cells, A is the percentage of target cell death, B is the content of cytokine IL-2 in the co-culture supernatant, and C is the content of cytokine IFN-γ in the co-culture supernatant.

[0211] Fig.17 This is a pseudo-color flow cytometry image showing the expression efficiency and membrane level detection of INESS-mTCR in primary human T cells.

[0212] Fig.18 The figure shows the flow cytometry histogram of the expression efficiency and membrane level of INESS-mTCR in primary human T cells.

[0213] Fig.19 are the functional detection results of INESS-mTCR in human primary T cells, A is the percentage of target cell death, B is the content of cytokine IL-2 in the co-culture supernatant, and C is the content of cytokine IFN-γ in the co-culture supernatant.

[0214] Fig. 20 This is a pseudo-color flow cytometry image showing the expression efficiency and membrane level detection of INESS-mutEC-mTCR in primary human T cells.

[0215] Fig.21 Flow cytometry histogram showing the expression efficiency and membrane level detection of INESS-mutEC-mTCR in primary human T cells.

[0216] Fig. 22 are the functional detection results of INESS-mutEC-mTCR in human primary T cells, A is the percentage of target cell death, B is the content of cytokine IL-2 in the co-culture supernatant, and C is the content of cytokine IFN-γ in the co-culture supernatant. DETAILED DESCRIPTION

[0217] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0218] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" may be used interchangeably. Similarly, the terms "comprising," "including," and "having" may be used interchangeably.

[0219] Unless otherwise stated or defined, the term "comprising" and variations thereof such as "including" and "containing" should be understood to mean including the stated elements or steps but not excluding any other elements or steps. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising or containing at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists of only these embodiments.

[0220] As used herein, the term "T cell receptor" or "TCR" includes natural TCRs as well as TCR variants, fragments and constructs. The term thus includes heterodimers and multimers and single-chain constructs comprising TCR alpha chains and TCR beta chains; optionally comprising other domains and / or portions, as long as the TCR retains its ability to recognize antigen targets.

[0221] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the scope of those skilled in the art.

[0222] The embodiments of the present invention are described in detail below to provide a person skilled in the art with a full disclosure and description of how to conduct and utilize the tests, screening and treatment methods of the present invention. It should be noted that these embodiments are merely illustrative and are not to be construed as limitations of the present invention.

[0223] In the examples, various TCR constant regions of the present invention were combined with TCR variable regions targeting NY-ESO-1 or p53 (R175H) to obtain a complete TCR, and the TCR was functionally verified.

[0224] In the human TCR (hTCR) related experiments, TCRs: TP-2 and TP-66 containing fully human TCR constant regions and TCR: TP-9 containing fully human TCR constant regions with endogenous disulfide bonds mutated were used as controls. TP-9 is based on the TP-2 sequence, with the 95th amino acid position (cysteine) of the constant region of the α chain mutated to arginine, and the 131st amino acid position (cysteine) of the constant region of the β chain mutated to arginine, to help remove the naturally formed disulfide bonds between the α chain and the β chain.

[0225] In experiments related to mouse TCR (mTCR), TCR: TP-16M containing the full mouse TCR constant region and TCR: TP-17M containing the full mouse TCR constant region with endogenous disulfide bonds mutated were used as controls.

[0226] The TCR involved in the embodiment contains two polypeptide chains, and the gene sequence is connected by the furin and p2A protease cleavage site polypeptide segments. The two polypeptide chains will be transcribed and translated into a fusion polypeptide together, and then cut into two independent protein subunits by the proteases corresponding to furin and p2A, and the two subunits are covalently bound by disulfide bonds. The entire TCR gene is inserted into the lentiviral expression vector pCDH through the restriction endonuclease cleavage sites XbaI and BamHI. The vector carries ampicillin resistance, EF-1α promoter and RFP fluorescent reporter gene.

[0227] Example 1 Construction of INESS-hTCR

[0228] The specific construction method is as follows:

[0229] 1. TCR constant region sequence determination

[0230] The TCR constant region contained in INESS-hTCR is based on the original human TCR (hTCR) constant region sequence, and the amino acid residues at the CYS1-9 sites of the constant region of the α chain and β chain are mutated to cysteine. The gene sequences of the constant region (C region) of the α chain and β chain of hTCR and the variable region (V region) of the TCR targeting NY-ESO-1 and p53 (R175H) were synthesized at Qingke Biotechnology Co., Ltd. and the plasmids containing the TCR V region targeting NY-ESO-1 were named TP-WTHC1-TP-WTHC9, and the plasmids containing the TCR V region targeting p53 (R175H) were named P53-WTHC1-P53-WTHC9.

[0231] 2. Construction of INESS-hTCR Plasmid

[0232] The fragments constituting each plasmid were obtained from the synthesized gene fragments by PCR cloning, and the corresponding site mutations were introduced by primers. All PCR primers carried 15-20 bp bases homologous to the previous and next sequence fragments or vectors. The pCDH vector was digested by XbaI and BamHI. The PCR product and the linearized vector were subjected to agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The recovered fragments and the linearized vector were recombinantly connected by pEASY*-Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant connection product was transformed into the DH5α strain and spread on an LB plate containing ampicillin for overnight growth. Single clones were selected for sequencing. The cloned bacterial solution with correct sequencing was inoculated in LB liquid culture medium, cultured overnight, and the plasmid was extracted. After the sequencing was correct again, subsequent experiments were carried out.

[0233] Example 2 Functional verification of INESS-hTCR

[0234] 1. Lentiviral packaging

[0235] The pCDH plasmid carrying the target gene and the packaging plasmids pMDLg, pRSV-Rev, and pMD2.G (plasmids purchased from Addgene) were transfected into 293T cells (transfected using PEI) at a ratio of 4:2:1:1. The cell culture supernatant was collected at 48 hours and 72 hours, and the virus was concentrated 20 times by PEG8000. The concentrated virus was used immediately or stored in a -80°C refrigerator.

[0236] 2. Isolation, culture and lentiviral infection of primary human T cells.

[0237] Obtain human peripheral blood cells, separate the mononuclear cells (PBMC), culture them in RPMI1640 medium containing 10% inactivated serum for 4 hours, take the cells suspended in the supernatant and plate them in a well plate coated with CD3 (5μg / mL) antibody and Retronectin (5μg / mL) for 14 hours, activate them for 24 hours, and add the lentivirus solution for infection. The infection method is 32°C, 1500rpm centrifugation for 1.5 hours. After infection, culture them in RPMI 1640 medium containing 10% inactivated serum and 200IU / mL IL-2 to a sufficient number.

[0238] 3. Detection of the expression efficiency and membrane level of INESS-hTCR in primary human T cells.

[0239] The cells were taken 5 days after infection and stained with MHC antigen peptide complex tetramer NY-ESO-1 Tetramer or p53 (R175H) Tetramer, and then flow cytometry was performed to analyze the expression efficiency and membrane level. Figure 2-Figure 3 .

[0240] 4. Functional verification of INESS-hTCR in primary human T cells.

[0241] NY-ESO-1 positive target cells are Huh7-0201-NY-ESO-1-luc / GFP cells (Huh7 cells are human hepatoma cell lines purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-526; stably transfected with NY-ESO-1 (Uniprotpk number: P78358-1), Luciferase (Uniprotpk number: P08659) and GFP genes (Uniprotpk number: P42212) , thus constructed), p53 (R175H) positive target cells are KLE-luc / GFP cells (KLE cells are derived from human endometrial adenocarcinoma tumor cell lines, in which the 175th amino acid of the p53 protein in the cells is mutated from R to H, purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CL-0133; Luciferase (Uniprotpk number: P08659) and GFP gene (Uniprotpk number: P42212) were stably transferred into the cells, thus constructed). The T cells obtained above were co-cultured with the corresponding target cells according to a certain effector-target ratio (NY-ESO-1 related effector-target ratio is 1:9; p53 (R175H) related effector-target ratio is 4:1). After 24 hours, the amount of surviving target cells was detected using a firefly luciferase kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and the percentage of target cell death was calculated ( Figure 4 A, D), ELISA kits (purchased from Thermo Fisher Scientific) were used to detect the cytokines IL-2 and IFN-γ ( Figure 4 (B, C).

[0242] Example 3 Construction of INESS-sTCR

[0243] 1. TCR constant region sequence determination

[0244] INESS-sTCR is based on the TP-WTHC5 sequence in Example 1, and the constant regions of the α chain and β chain are subjected to 4857 mutation, mm mutation, TMa mutation or all three mutations at the same time, and are named TP-WTHC5-4857, TP-WTHC5-mm, TP-WTHC5-TMa and TP-WTC5, respectively; based on the TP-WTHC9 sequence in Example 1, the constant regions of the α chain and β chain are subjected to 4857 mutation, mm mutation, TMa mutation or all three mutations at the same time, and are named TP-WTHC9-4857, TP-WTHC9-mm, TP-WTHC9-TMa and TP-WTC9, respectively.

[0245] 2. Construction of INESS-sTCR Plasmid

[0246] The fragments constituting each plasmid were obtained by PCR cloning from TP-WTHC5 or TP-WTHC9 plasmid. The corresponding site mutations were introduced by primers, and all PCR primers carried 15-20 bp bases homologous to the previous and next sequence fragments or vectors. The pCDH vector was digested by XbaI and BamHI. The PCR product and the linearized vector were subjected to agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The recovered fragments and the linearized vector were recombinantly connected using a pEASY*-Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant connection product was transformed into the DH5α strain and spread on an LB plate containing ampicillin for overnight growth. Single clones were selected for sequencing. The cloned bacterial solution with correct sequencing was inoculated in LB liquid culture medium, cultured overnight, and the plasmid was extracted. After the sequencing was correct again, subsequent experiments were carried out.

[0247] Example 4 Functional verification of INESS-sTCR

[0248] The same as in Example 2. Figure 5-Figure 7 .

[0249] Example 5 Construction of INESS-mutEC-hTCR

[0250] 1. TCR constant region sequence determination

[0251] INESS-mutEC-hTCR is based on the 9 TCR sequences in Example 1: TP-WTHC1 to TP-WTHC9, with the 95th amino acid site (cysteine) of the constant region of the α chain mutated to arginine, and the 131st amino acid site (cysteine) of the constant region of the β chain mutated to arginine, respectively, to release the naturally formed disulfide bond between the α chain and the β chain, and named TP-HC1 to TP-HC19;

[0252] 2. Construction of INESS-mutEC-hTCR Plasmid

[0253] The fragments constituting each plasmid were obtained by PCR cloning from TP-WTHC1~9 plasmids. The corresponding site mutations were introduced by primers, and all PCR primers carried 15~20bp bases homologous to the previous and next sequence fragments or vectors. The pCDH vector was digested by XbaI and BamHI. The PCR product and the linearized vector were subjected to agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The recovered fragments and the linearized vector were recombined and connected by pEASY*-Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant connection product was transformed into the DH5α strain and spread on an LB plate containing ampicillin for overnight growth. Single clones were selected for sequencing. The cloned bacterial solution with correct sequencing was inoculated in LB liquid culture medium, cultured overnight, and the plasmid was extracted. After the sequencing was correct again, subsequent experiments were carried out.

[0254] Example 6 Functional verification of INESS-mutEC-hTCR

[0255] The same as in Example 2. Figure 8-Figure 10 .

[0256] Example 7 Construction of INESS-mutEC-sTCR

[0257] 1. TCR constant region sequence determination

[0258] INESS-mutEC-sTCR is based on the 8 TCR sequences in Example 2: TP-WTHC5-4857, TP-WTHC5-mm, TP-WTHC5-TMa, TP-WTC5, TP-WTHC9-4857, TP-WTHC9-mm, TP-WTHC9-TMa, and TP-WTC9, while the 95th amino acid site (cysteine) of the constant region of the α chain is mutated to arginine, and the 131st amino acid site (cysteine) of the constant region of the β chain is mutated to arginine to release the naturally formed disulfide bond between the α chain and the β chain, and they are named TP-HC5-4857, TP-HC5-mm, TP-HC5-TMa, TP-C5, TP-HC9-4857, TP-HC9-mm, TP-HC9-TMa, and TP-C9, respectively.

[0259] 2. Construction of INESS-mutEC-sTCR Plasmid

[0260] The fragments constituting each plasmid were obtained by PCR cloning from the 8 plasmids in Example 2. The corresponding site mutations were introduced by primers, and all PCR primers carried 15 to 20 bp bases homologous to the previous and next sequence fragments or vectors. The pCDH vector was digested by XbaI and BamHI. The PCR product and the linearized vector were subjected to agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the recovered fragments and the linearized vector were recombinantly connected by pEASY*-Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant connection product was transformed into the DH5α strain and spread on an LB plate containing ampicillin for overnight growth. Single clones were selected for sequencing. The cloned bacterial solution with correct sequencing was inoculated in LB liquid culture medium, cultured overnight, and the plasmid was extracted. After the sequencing was correct again, subsequent experiments were carried out.

[0261] Example 8 Functional verification of INESS-mutEC-sTCR

[0262] The same as in Example 2. Figure 11-13 .

[0263] Example 9 Construction of INESS-mul-TCR

[0264] 1. TCR constant region sequence determination

[0265] INESS-mul-TCR includes 4 types of TCR: TP-HC56, TP-HC69, TP-HC59, and TP-HC569. TP-HC56 is TP-HC5-4857 in Example 7; TP-HC69 is TP-HC6-4857 in Example 7; based on the TP-HC5 sequence, CYS9 mutation was performed and named TP-HC59; based on the TP-HC56 sequence, CYS9 mutation was performed and named TP-HC569.

[0266] 2. Construction of INESS-mul-TCR Plasmid

[0267] The fragments constituting each plasmid were cloned from the TP-HC5 plasmid. The corresponding site mutations were introduced by primers, and all PCR primers carried 15-20 bp bases homologous to the previous and next sequence fragments or vectors. The pCDH vector was digested by XbaI and BamHI. The PCR product and the linearized vector were subjected to agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The recovered fragments and the linearized vector were recombined and connected using a pEASY*-Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant connection product was transformed into the DH5α strain and spread on an LB plate containing ampicillin for overnight growth. Single clones were selected for sequencing. The cloned bacterial solution with correct sequencing was inoculated in LB liquid culture medium, cultured overnight, and the plasmid was extracted. After the sequencing was correct again, subsequent experiments were carried out.

[0268] Example 10 Functional verification of INESS-mul-TCR

[0269] The same as in Example 2. Figure 14-16 .

[0270] Example 11 Construction of INESS-mTCR

[0271] 1. TCR constant region sequence determination

[0272] The TCR constant region contained in INESS-mTCR is based on the original mouse TCR (mTCR) constant region sequence, and the amino acid residues at the mCYS1-9 sites of the constant region of the α chain and the β chain are mutated to cysteine. The gene sequences of the constant region (C region) of the α chain and the β chain of the mTCR and the variable region (V region) of the TCR targeting NY-ESO-1 are synthesized at Qingke Biotechnology. This embodiment includes two kinds of TCRs: TP-WTMC5 and TP-WTMC9. The TCR constant region contained in TP-WTMC5 is based on the mTCR constant region sequence, and the mCYS5 mutation is carried out; the TCR constant region contained in TP-WTMC5 is based on the mTCR constant region sequence, and the mCYS9 mutation is carried out. The control group is TP-16M: containing the original mouse TCR (mTCR) constant region sequence. The nucleotide sequence of TP-16M is SEQ ID NO: 81 (TRBC) and SEQ ID NO: 82 (TRAC).

[0273] 2. Construction of INESS-mTCR Plasmid

[0274] The fragments constituting each plasmid were obtained from the synthesized gene fragments by PCR cloning, and the corresponding site mutations were introduced by primers. All PCR primers carried 15-20 bp bases homologous to the previous and next sequence fragments or vectors. The pCDH vector was digested by XbaI and BamHI. The PCR product and the linearized vector were subjected to agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The recovered fragments and the linearized vector were recombinantly connected by pEASY*-Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant connection product was transformed into the DH5α strain and spread on an LB plate containing ampicillin for overnight growth. Single clones were selected for sequencing. The cloned bacterial solution with correct sequencing was inoculated in LB liquid culture medium, cultured overnight, and the plasmid was extracted. After the sequencing was correct again, subsequent experiments were carried out.

[0275] Example 12 Functional verification of INESS-mTCR

[0276] The same as in Example 2. Figure 17-Figure 19 .

[0277] Example 13 Construction of INESS-mutEC-mTCR

[0278] 1. TCR constant region sequence determination

[0279] INESS-mutEC-mTCR includes two types of TCR: TP-MC5 and TP-MC9; the control group is TP-17M. TP-17M is based on the TP-16M sequence in Example 11, and the 91st amino acid site (cysteine) of the constant region of the α chain is mutated to arginine, and the 127th amino acid site (cysteine) of the constant region of the β chain is mutated to arginine, respectively, to help remove the naturally formed disulfide bond between the α chain and the β chain. The nucleotide sequence of TP-17M is SEQ ID NO: 83 (TRBC) and SEQ ID NO: 84 (TRAC). Based on the TP-17M sequence, mCYS5 is mutated and named TP-MC5; based on the TP-17M sequence, mCYS9 is mutated and named TP-MC9.

[0280] 2. Construction of INESS-mutEC-mTCR Plasmid

[0281] The fragments constituting each plasmid were obtained from the TP-17M plasmid by PCR cloning, and the corresponding site mutations were introduced by primers. All PCR primers carried 15-20 bp bases homologous to the previous and next sequence fragments or vectors. The pCDH vector was digested by XbaI and BamHI. The PCR product and the linearized vector were subjected to agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The recovered fragments and the linearized vector were recombined and connected by pEASY*-Basic Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant connection product was transformed into the DH5α strain and spread on an LB plate containing ampicillin for overnight growth. Single clones were selected for sequencing. The cloned bacterial solution with correct sequencing was inoculated in LB liquid culture medium, cultured overnight, and the plasmid was extracted. After the sequencing was correct again, subsequent experiments were carried out.

[0282] Example 14 Functional verification of INESS-mutEC-mTCR

[0283] The same as in Example 2. Figure 20-22 .

Claims

1. A TCR constant region with an exogenous disulfide bond, characterized in that: include: (1) Point mutation 1 located in the α chain: The site 4 amino acids away from the amino acid site involved in endogenous disulfide bond formation in the C-terminal direction of the amino acid sequence is mutated to C; and (2) point mutation 2 located in the β strand: The site that is one amino acid away from the amino acid site involved in endogenous disulfide bond formation in the N-terminal direction of the amino acid sequence is mutated to C; The TCR constant region is selected from any one of the following: TCR constant region 1: hTRAC includes point mutation 1, and hTRBC includes point mutation 2; the amino acid sequence of hTRAC is numbered A0A5H1ZRS8 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in hTRAC is C95; the amino acid sequence of hTRBC is numbered A0A5H1ZRT1 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in hTRBC is C131; Or, TCR constant region 2: including point mutation 1 based on hTRAC, and including point mutation 2 based on hTRBC; the amino acid sequence of hTRAC is numbered A0A5H1ZRS8 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in hTRAC is C95; the amino acid sequence of hTRBC is numbered A0A5H1ZRR3 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in hTRBC is C131; Or, TCR constant region 3: including point mutation 1 based on hTRAC, and including point mutation 2 based on hTRBC; the amino acid sequence of hTRAC is numbered A0A5H1ZRS8 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in hTRAC is C95; the amino acid sequence of hTRBC is numbered A0A0G2JMB4 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in hTRBC is C131; Or, TCR constant region 4: mTRAC includes point mutation 1, and mTRBC includes point mutation 2; the amino acid sequence of mTRAC is numbered A0A991BQX6 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in hTRAC is C91; the amino acid sequence of mTRBC is numbered P01851 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in mTRBC is C127; Or, TCR constant region 5: mTRAC includes point mutation 1, and mTRBC includes point mutation 2; the amino acid sequence of mTRAC is numbered A0A991BQX6 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in hTRAC is C91; the amino acid sequence of mTRBC is numbered P01852 in UniProtKB, and the amino acid site involved in the formation of endogenous disulfide bonds in mTRBC is C127.

2. The TCR constant region according to claim 1, characterized in that It also includes mutations of endogenous disulfide bond sites, wherein the endogenous disulfide bond sites do not form endogenous disulfide bonds after mutation, and the mutations of the endogenous disulfide bond sites are selected from any of the following: (1) mTRAC includes a mutation at C91 to A, and mTRBC includes a mutation at C127 to A; (2) hTRAC includes a mutation at the C95 site to A, and hTRBC includes a mutation at the C131 site to A.

3. The TCR constant region according to any one of claims 1 to 2, characterized in that Also included are any of the following groups of mutations or combinations of three mutations: (1) T48C of hTRAC and S57C of hTRBC; (2) P91S, E92D, S93V, and S94P of hTRAC and E18R, S22A, F133I, V136A, and Q139H of hTRBC; (3) S115L, G119V and F120L of hTRAC.

4. Use of the TCR constant region according to any one of claims 1 to 3 in constructing TCR.

5. A product comprising the TCR constant region according to any one of claims 1 to 3, characterized in that The product is selected from: TCR or TCR chimeric receptor.

6. The product according to claim 5, characterized in that The TCR also includes a TCR variable region.

7. The product according to claim 6, characterized in that The antigen types targeted by the TCR variable region include any one or more of NY-ESO-1, MAGE-A1 / 3 / 4 / 6, MAGE-C2, MART-1, WT1, PRAME, gp100, MCPyV, hTG, TRAIL, HERV-E, HA-1, Tyrosinase, TGFβRII, HBV, HPV, CMV, EBV, HIV, AFP, HER2, MSLN, KK-LC-1, KRAS mutants, and P53 mutants.

8. The product according to claim 5, characterized in that The TCR chimeric receptor also includes an antigen recognition region, which comprises an antibody, a receptor or a ligand.

9. The product according to claim 8, characterized in that The antigen types targeted by the antigen recognition region include any one or more of CD19, CD20, CD22, BCMA, CD79A, TRBC1, TRBC2, GPRC5D, CD123, CD33, CLL1, Siglec6, CD70, GCC, GPC3, Claudin18.2, Claudin6, MSLN, GD2, IL13Rα2, CD7, Gucy2c, B7H3, EpCAM, CEA, CEACAM-1, EGFR, EGFRVIII, DLL3, and Nectin4.

10. A genetic engineering product, characterized in that: Select from any of the following: (1) a nucleic acid molecule encoding: a TCR constant region according to any one of claims 1 to 3 or a product according to any one of claims 5 to 9; (2) an expression vector comprising the nucleic acid molecule described in (1), or expressing the TCR constant region described in any one of claims 1 to 3 or the product described in any one of claims 5 to 9; (3) Cells: transferred with the expression vector described in (2), or expressing or secreting the TCR constant region described in any one of claims 1 to 3 or the product described in any one of claims 5 to 9.

11. The genetic engineering product according to claim 10, characterized in that The cells are selected from: T cells, NK cells, NKT cells, macrophages, dendritic cells, neutrophils; the cells are derived from the patient's own body, healthy volunteers or umbilical cord blood.

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