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WO2024260454A1 - 经修饰的tcr恒定区及其在tcr融合蛋白中的用途 - Google Patents

经修饰的tcr恒定区及其在tcr融合蛋白中的用途 Download PDF

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Publication number
WO2024260454A1
WO2024260454A1 PCT/CN2024/100701 CN2024100701W WO2024260454A1 WO 2024260454 A1 WO2024260454 A1 WO 2024260454A1 CN 2024100701 W CN2024100701 W CN 2024100701W WO 2024260454 A1 WO2024260454 A1 WO 2024260454A1
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Prior art keywords
constant region
amino acid
tcr
chain
chain constant
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PCT/CN2024/100701
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English (en)
French (fr)
Inventor
谢兴旺
蒋栋
毕晶磊
闫宝琪
付双莉
乔雪微
李贤�
李永红
王江华
王雪艳
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北京可瑞生物科技有限公司
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Priority claimed from PCT/CN2023/101881 external-priority patent/WO2023246911A1/zh
Application filed by 北京可瑞生物科技有限公司 filed Critical 北京可瑞生物科技有限公司
Publication of WO2024260454A1 publication Critical patent/WO2024260454A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present disclosure relates to modified T cell receptor (TCR) constant regions and their use in TCR-based multispecific antigen-binding molecules, in particular, the use of the polypeptide molecules in the treatment of cancer, infectious diseases, autoimmune diseases and/or inflammatory diseases.
  • TCR T cell receptor
  • the human immune system consists of "three major lines of defense", namely the first line of defense consisting of the skin and mucous membranes and their secretions; the second line of defense consisting of bactericidal substances (such as lysozyme) and phagocytes in body fluids; and the third line of defense consisting of immune organs and immune cells (T cells, B cells).
  • the first two are collectively referred to as the innate immune system, and the third is called the adaptive immune system.
  • the main function of innate immunity is to resist infection. It is the first line of defense for the body to defend against the invasion of pathogenic microorganisms. Its characteristics are congenital acquisition, immediate effect, non-specificity, and no memory.
  • the adaptive immune system is a targeted and more advanced immune function produced by humans after adapting to the living environment and contacting antigenic substances. It is characterized by acquired, delayed reaction, specificity, and memory.
  • a large number of biopharmaceutical products have been developed using the specificity and memory characteristics of adaptive immune responses for the treatment of viral infections, tumors, and autoimmune diseases, and huge clinical benefits have been achieved. These products mainly achieve therapeutic purposes by simulating the mechanism by which B cell immune response products (antibodies) exert immune functions in the body, such as monoclonal antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, CAT-T/CAR-NK and other products.
  • TCR recognizes major histocompatibility complex (MHC)-antigen peptide complex (pMHC), which is an important step in T cell-mediated immune response.
  • MHC major histocompatibility complex
  • pMHC antigen peptide complex
  • TCR can recognize antigen peptides that are processed and presented on the cell surface MHC molecules of intracellular and cell surface tumor-specific antigens, and has a wider recognition range.
  • TCR-T TCR-based T cell repositioning bispecific antibodies
  • TCR-T a product for the treatment of advanced and terminal tumors.
  • a large amount of in vitro and in vivo research data have proved the effectiveness of such products in tumor treatment.
  • Many products at home and abroad have been promoted to the clinical trial stage. Due to the process limitations of autologous TCR-T, factors such as high cost of TCR-T treatment, long product preparation cycle, low preparation success rate, and difficulty in reuse have greatly limited the widespread application of this type of product.
  • T cell repositioning molecule developed by fusing TCR with CD3 antibody in the form of a soluble molecule can not only give full play to the advantage of TCR's extensive recognition of a large number of targets, but also has the advantage of low marginal cost of antibody drugs. Its mechanism of action is shown in Figure 1.
  • the first clinical drug based on this structure has been approved for marketing. Clinical trials have shown that it is safe and effective, and the treatment cost is also much lower than that of adoptive therapy products (TCR-T). Since natural TCR is a transmembrane protein molecule, it does not have the same excellent soluble expression ability as antibodies, so the transformation of TCR soluble expression stability has become a primary problem that needs to be solved.
  • the inventors have successfully screened out a series of new TCR constant region mutations that can improve TCR soluble expression, especially the mutation combination CMM1, by combining computer prediction and wet experiment verification.
  • the mutation combination CMM1 can improve the stability and solubility of TCR fusion protein, increase the success rate and yield of soluble expression of TCR fusion protein, enhance the activity of TCR fusion protein, and improve the non-specific activation of PBMC cells under high concentration of TCR fusion protein, thereby improving the drug safety window.
  • This discovery breaks an important barrier to the development of TCR soluble molecular drugs, and is expected to significantly reduce the cost and cycle of TCR screening and TCR soluble expression in the drug development process.
  • the present disclosure provides a T cell receptor (TCR) ⁇ chain constant region, wherein the ⁇ chain constant region comprises amino acid substitutions at one or more of the following positions: Q6, S21, K83, S84.6 and I114; wherein the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) according to the TCR ⁇ chain constant region shown in SEQ ID NO:1.
  • TCR T cell receptor
  • IMGT International Immunogenetics Information System
  • amino acid substitution at position Q6 in the ⁇ chain constant region is selected from Q6R, Q6K, Q6D and Q6E, preferably selected from Q6R and Q6K;
  • amino acid substitution at position S21 in the ⁇ chain constant region is selected from S21I, S21L, S21V, S21G, S21A and S21P, preferably selected from S21I, S21L and S21V; and/or
  • amino acid substitution at position K83 in the ⁇ chain constant region is selected from K83P, K83G, K83A, K83I, K83L and K83V, preferably selected from K83P, K83G and K83A; and/or
  • amino acid substitution at position S84.6 in the ⁇ chain constant region is selected from S84.6D, S84.6E, S84.6N, S84.6Q, S84.6K and S84.6R, preferably selected from S84.6D, S84.6E, S84.6N and S84.6Q; and/or
  • the amino acid substitution at position I114 in the ⁇ chain constant region is selected from I114E, I114D, I114K and I114R, preferably selected from I114E and I114D.
  • amino acid at position Q6 in the ⁇ chain constant region is substituted with Q6R;
  • amino acid substitution at position S84.6 in the ⁇ chain constant region is selected from S84.6D, S84.6E and S84.6N; and/or
  • the ⁇ chain constant region comprises amino acid substitutions at S21, K83, and S84.6.
  • the ⁇ chain constant region comprises one or more of the following amino acid substitutions: S21I, K83P and S84.6D; preferably comprises at least two of the amino acid substitutions S21I, K83P and S84.6D; more preferably comprises amino acid substitutions S21I, K83P and S84.6D.
  • the ⁇ chain constant region comprises an amino acid substitution selected from any one of the following (1)-(4):
  • the ⁇ chain constant region further comprises one or more of the following amino acid mutations: N1.2K, T84C and FFPSP deletion mutations at positions 120-124, preferably comprising T84C and optionally N1.2K and/or FFPSP deletion mutations at positions 120-124; more preferably comprising N1.2K, T84C and FFPSP deletion mutations at positions 120-124.
  • the ⁇ chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO:3-13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:3-13.
  • the present disclosure provides a TCR ⁇ chain constant region, which comprises amino acid substitutions S21I, K83P and S84.6D, wherein the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) according to the TCR ⁇ chain constant region shown in SEQ ID NO:1; preferably, the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:13.
  • IMGT International Immunogenetics Information System
  • the TCR ⁇ chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:13.
  • the present disclosure provides a TCR ⁇ chain constant region, wherein the ⁇ chain constant region comprises amino acid substitutions at one or more of the following positions: S92, T94 and Q107; wherein the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) based on the TCR ⁇ constant region shown in SEQ ID NO:14.
  • IMGT International Immunogenetics Information System
  • amino acid substitution at position S92 in the ⁇ chain constant region is selected from S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V, S92Q, S92K and S92R, preferably selected from S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V and S92Q; and/or
  • the amino acid substitution at position Q107 in the ⁇ chain constant region is Q107L, Q107I, Q107V, Q107G, Q107A and Q107P, preferably selected from Q107L, Q107I and Q107V.
  • amino acid substitution at position S92 in the ⁇ chain constant region is selected from S92P, S92E, S92D, S92L and S92N; and/or
  • the beta chain constant region comprises amino acid substitutions at T94 and Q107.
  • the beta chain constant region comprises one or more of the following amino acid substitutions: T94D and Q107L; preferably comprises amino acid substitutions T94D and Q107L.
  • the beta chain constant region comprises an amino acid substitution selected from any one of the following (1)-(3):
  • the TCR ⁇ chain constant region further comprises one or more of the following amino acid mutations: S79C, C85.1A and N97D, preferably comprising S79C and optionally C85.1A and/or N97D; more preferably comprising S79C, C85.1A and N97D.
  • the TCR ⁇ chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO:16-23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:16-23.
  • the present disclosure provides a TCR ⁇ chain constant region comprising amino acid substitutions T94D and Q107L, wherein the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) according to the TCR ⁇ chain constant region shown in SEQ ID NO:14; preferably, the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:23.
  • IMGT International Immunogenetics Information System
  • the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:23.
  • the present disclosure provides a TCR constant region comprising a TCR ⁇ chain constant region and a TCR ⁇ chain constant region described herein.
  • the TCR constant region comprises an amino acid substitution selected from any one of the following (1)-(6):
  • the ⁇ chain constant region comprises amino acid substitutions S21I, K83P, and S84.6D, and the ⁇ chain constant region comprises amino acid substitutions T94D and Q107L.
  • the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:13; and the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:23.
  • the present disclosure provides a polypeptide molecule comprising a TCR ⁇ chain constant region, a TCR ⁇ chain constant region, or a TCR constant region described herein.
  • the polypeptide molecule further comprises an antigen binding region that binds at least one antigen.
  • the antigen binding region is selected from an antigen binding region derived from an antibody and an antigen binding region derived from a TCR.
  • the present disclosure provides a T cell receptor (TCR), the TCR comprising an ⁇ chain and a ⁇ chain, wherein the ⁇ chain comprises the TCR ⁇ chain constant region described herein, and/or the ⁇ chain comprises the TCR ⁇ chain constant region described herein; or the TCR comprises the TCR constant region described herein.
  • TCR T cell receptor
  • the TCR binds to an antigen selected from the group consisting of: melanoma-associated antigens (e.g., gp100, MAGEA1), HPV antigens (e.g., HPV E6 or E7), and KRAS antigens (e.g., KRAS G12V, KRAS G12D).
  • an antigen selected from the group consisting of: melanoma-associated antigens (e.g., gp100, MAGEA1), HPV antigens (e.g., HPV E6 or E7), and KRAS antigens (e.g., KRAS G12V, KRAS G12D).
  • the TCR comprises a variable region derived from a murine TCR or a human TCR.
  • the TCR is isolated or purified.
  • the present disclosure provides a T cell receptor (TCR), the TCR comprising an ⁇ chain and a ⁇ chain,
  • constant region of the ⁇ chain comprises an amino acid substitution at one or more of the following positions: Q6, S21, K83, S84.6 and I114; and/or
  • constant region of the beta chain comprises an amino acid substitution at one or more of the following positions: S92, T94, and Q107;
  • the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) based on the TCR ⁇ chain constant region shown in SEQ ID NO:1 and the TCR ⁇ constant region shown in SEQ ID NO:14.
  • IMGT International Immunogenetics Information System
  • amino acid substitution at position Q6 in the constant region of the ⁇ chain is selected from Q6R, Q6K, Q6D and Q6E, preferably selected from Q6R and Q6K;
  • amino acid substitution at position S21 in the constant region of the ⁇ chain is selected from S21I, S21L, S21V, S21G, S21A and S21P, preferably selected from S21I, S21L and S21V; and/or
  • the amino acid substitution at position K83 in the constant region of the ⁇ chain is selected from the group consisting of K83P, K83G, K83A, K83I, K83L and K83V, preferably selected from the group consisting of K83P, K83G and K83A; and/or
  • amino acid substitution at position S84.6 in the constant region of the ⁇ chain is selected from the group consisting of S84.6D, S84.6E, S84.6N, S84.6Q, S84.6K and S84.6R, preferably selected from the group consisting of S84.6D, S84.6E, S84.6N and S84.6Q; and/or
  • amino acid at position I114 in the constant region of the ⁇ chain is substituted with I114E, I114D, I114K and I114R, preferably selected from I114E and I114D; and/or
  • amino acid substitution at position S92 in the constant region of the ⁇ chain is selected from the group consisting of S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V, S92Q, S92K and S92R, preferably selected from the group consisting of S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V and S92Q; and/or
  • the amino acid at position T94 in the constant region of the ⁇ chain is substituted with T94D, T94E, T94K and T94R, preferably selected from T94D and T94E; and/or
  • the amino acid substitution at position Q107 in the constant region of the ⁇ chain is selected from Q107L, Q107I, Q107V, Q107G, Q107A and Q107P, preferably selected from Q107L, Q107I and Q107V.
  • amino acid at position Q6 in the constant region of the ⁇ chain is substituted with Q6R;
  • amino acid substitution at position S84.6 in the constant region of the ⁇ chain is selected from S84.6D, S84.6E and S84.6N; and/or
  • amino acid substitution at position S92 in the constant region of the ⁇ chain is selected from the group consisting of S92P, S92E, S92D, S92L and S92N; and/or
  • the constant region of the ⁇ chain comprises amino acid substitutions at S21, K83, and S84.6, and/or the constant region of the ⁇ chain comprises amino acid substitutions at T94 and Q107.
  • the constant region of the ⁇ chain comprises one or more of the following amino acid substitutions: S21I, K83P and S84.6D; preferably comprises at least two of the amino acid substitutions S21I, K83P and S84.6D; more preferably comprises amino acid substitutions S21I, K83P and S84.6D.
  • the constant region of the beta chain comprises one or more of the following amino acid substitutions: T94D and Q107L; preferably comprises amino acid substitutions T94D and Q107L.
  • the TCR comprises an amino acid substitution selected from any one of the following (1)-(6):
  • the constant region of the alpha chain comprises the amino acid substitutions S21I, K83P and S84.6D
  • the constant region of the beta chain comprises the amino acid substitutions T94D and Q107L.
  • the constant region of the ⁇ chain further comprises one or more of the following amino acid mutations: N1.2K, T84C and FFPSP deletion mutation at positions 120-124; and/or the constant region of the ⁇ chain further comprises one or more of the following amino acid mutations: S79C, C85.1A and N97D; preferably, the constant region of the ⁇ chain further comprises N1.2K, T84C and FFPSP deletion mutation at positions 120-124, and the constant region of the ⁇ chain further comprises S79C, C85.1A and N97D.
  • the TCR comprises an ⁇ chain constant region having an amino acid sequence shown in any one of SEQ ID NOs:3-13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs:3-13, and/or a ⁇ chain constant region having an amino acid sequence shown in any one of SEQ ID NOs:16-23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs:16-23.
  • the TCR binds to an antigen selected from the group consisting of: melanoma-associated antigens (e.g., gp100, MAGEA1), HPV antigens (e.g., HPV E6 or E7), and KRAS antigens (e.g., KRAS G12V, KRAS G12D).
  • an antigen selected from the group consisting of: melanoma-associated antigens (e.g., gp100, MAGEA1), HPV antigens (e.g., HPV E6 or E7), and KRAS antigens (e.g., KRAS G12V, KRAS G12D).
  • the TCR does not comprise a TRAV with an amino acid sequence as set forth in SEQ ID NO:24 and does not comprise a TRBV with an amino acid sequence as set forth in SEQ ID NO:25. In some embodiments, the TCR does not comprise a TRAV with a CDR1, CDR2, and CDR3 with amino acid sequences as set forth in SEQ ID NO:38-40, respectively, and does not comprise a TRBV with a CDR1, CDR2, and CDR3 with amino acid sequences as set forth in SEQ ID NO:41-43, respectively. In some embodiments, the TCR does not bind to the KRAS antigen epitope VVGAVGVGK or its complex with HLA-A*11. In some embodiments, the TCR does not bind to the KRAS antigen.
  • the TCR comprises a variable region derived from a murine TCR or a human TCR.
  • the TCR is isolated or purified.
  • the present disclosure provides a multispecific antigen-binding molecule comprising a TCR ⁇ chain constant region, a TCR ⁇ chain constant region, a TCR constant region, a polypeptide molecule, or a TCR as described herein.
  • the present disclosure provides a multispecific antigen-binding molecule comprising at least one antigen-binding region derived from a TCR and: (1) the TCR ⁇ chain constant region (TRAC) described herein and/or the TCR ⁇ chain constant region (TRBC) described herein; or (2) the TCR constant region described herein.
  • TCR TCR ⁇ chain constant region
  • TRBC TCR ⁇ chain constant region
  • the present disclosure provides a multispecific antigen-binding molecule comprising at least one antigen-binding region derived from a TCR and an alpha chain constant region (TRAC) and/or a beta chain constant region (TRBC) derived from a TCR;
  • a multispecific antigen-binding molecule comprising at least one antigen-binding region derived from a TCR and an alpha chain constant region (TRAC) and/or a beta chain constant region (TRBC) derived from a TCR;
  • ⁇ chain constant region comprises an amino acid substitution at one or more of the following positions: Q6, S21, K83, S84.6 and I114; and/or
  • beta chain constant region comprises an amino acid substitution at one or more of the following positions: S92, T94, and Q107;
  • the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) based on the TCR ⁇ chain constant region shown in SEQ ID NO:1 and the TCR ⁇ chain constant region shown in SEQ ID NO:14.
  • IMGT International Immunogenetics Information System
  • amino acid substitution at position Q6 in the ⁇ chain constant region is selected from Q6R, Q6K, Q6D and Q6E, preferably selected from Q6R and Q6K;
  • amino acid substitution at position S21 in the ⁇ chain constant region is selected from S21I, S21L, S21V, S21G, S21A and S21P, preferably selected from S21I, S21L and S21V; and/or
  • amino acid substitution at position K83 in the ⁇ chain constant region is selected from K83P, K83G, K83A, K83I, K83L and K83V, preferably selected from K83P, K83G and K83A; and/or
  • amino acid substitution at position S84.6 in the ⁇ chain constant region is selected from S84.6D, S84.6E, S84.6N, S84.6Q, S84.6K and S84.6R, preferably selected from S84.6D, S84.6E, S84.6N and S84.6Q; and/or
  • amino acid at position I114 in the ⁇ chain constant region is substituted with I114E, I114D, I114K and I114R, preferably selected from I114E and I114D; and/or
  • amino acid substitution at position S92 in the ⁇ chain constant region is selected from the group consisting of S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V, S92Q, S92K and S92R, preferably selected from the group consisting of S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V and S92Q; and/or
  • the amino acid at position T94 in the ⁇ chain constant region is substituted with T94D, T94E, T94K and T94R, preferably selected from T94D and T94E; and/or
  • the amino acid substitution at position Q107 in the ⁇ chain constant region is Q107L, Q107I, Q107V, Q107G, Q107A and Q107P, preferably selected from Q107L, Q107I and Q107V.
  • amino acid at position Q6 in the ⁇ chain constant region is substituted with Q6R;
  • amino acid substitution at position S84.6 in the ⁇ chain constant region is selected from S84.6D, S84.6E and S84.6N; and/or
  • amino acid substitution at position S92 in the ⁇ chain constant region is selected from the group consisting of S92P, S92E, S92D, S92L and S92N; and/or
  • the ⁇ chain constant region comprises amino acid substitutions at S21, K83, and S84.6, and/or the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises one or more of the following amino acid substitutions: S21I, K83P and S84.6D; preferably comprises at least two of the amino acid substitutions S21I, K83P and S84.6D; more preferably comprises amino acid substitutions S21I, K83P and S84.6D.
  • the beta chain constant region comprises one or more of the following amino acid substitutions: T94D and Q107L; preferably comprises amino acid substitutions T94D and Q107L.
  • the multispecific antigen-binding molecule comprises an amino acid substitution selected from any one of the following (1)-(6):
  • the ⁇ chain constant region comprises the amino acid substitutions S21I, K83P and S84.6D, and the ⁇ chain constant region comprises the amino acid substitutions T94D and Q107L.
  • the ⁇ chain constant region further comprises one or more of the following amino acid mutations: N1.2K, T84C and FFPSP deletion mutation at positions 120-124; and/or the ⁇ chain constant region further comprises one or more of the following amino acid mutations: S79C, C85.1A and N97D; preferably, the constant region of the ⁇ chain further comprises N1.2K, T84C and FFPSP deletion mutation at positions 120-124, and the constant region of the ⁇ chain further comprises S79C, C85.1A and N97D.
  • the ⁇ chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO:3-13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:3-13, and/or the ⁇ chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO:16-23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:16-23.
  • the multispecific antigen-binding molecule comprises two or more antigen-binding regions that bind two or more antigens, and the two or more antigens are each independently selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, an autoantigen, and an immune cell surface molecule;
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • viral antigen an autoantigen
  • autoantigen an immune cell surface molecule
  • one of the two or more antigens is selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens, autoantigens; and the other is an immune cell surface molecule;
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • viral antigens viral antigens
  • autoantigens autoantigens
  • the other is an immune cell surface molecule
  • the TAA is a melanoma-associated antigen (e.g., gp100, MAGEA1); and/or the TSA is a KRAS antigen (e.g., KRAS G12V, KRAS G12D); and/or the viral antigen is an HPV antigen (e.g., HPV E6 or E7); and/or the immune cell surface molecule is selected from CD3, CD28, 4-1BB (CD137), PD-1 and PD-L1.
  • a melanoma-associated antigen e.g., gp100, MAGEA1
  • the TSA is a KRAS antigen (e.g., KRAS G12V, KRAS G12D)
  • the viral antigen is an HPV antigen (e.g., HPV E6 or E7)
  • the immune cell surface molecule is selected from CD3, CD28, 4-1BB (CD137), PD-1 and PD-L1.
  • the multispecific antigen-binding molecule does not comprise TRAV with an amino acid sequence as set forth in SEQ ID NO:24 and does not comprise TRBV with an amino acid sequence as set forth in SEQ ID NO:25. In some embodiments, the multispecific antigen-binding molecule does not comprise TRAV with CDR1, CDR2, and CDR3 with amino acid sequences as set forth in SEQ ID NO:38-40, respectively, and does not comprise TRBV with CDR1, CDR2, and CDR3 with amino acid sequences as set forth in SEQ ID NO:41-43, respectively. In some embodiments, the multispecific antigen-binding molecule does not bind to the KRAS antigen epitope VVGAVGVGK or its complex with HLA-A*11. In some embodiments, the multispecific antigen-binding molecule does not bind to the KRAS antigen.
  • the multispecific antigen-binding molecule does not bind the gp100 antigen.
  • the multispecific antigen-binding molecule comprises a first binding region that binds a first antigen and a second binding region that binds a second antigen on at least two polypeptide chains.
  • the first binding region comprises an ⁇ chain variable region (TRAV) and a ⁇ chain variable region (TRBV) derived from a TCR that binds to the first antigen-MHC complex;
  • TRAV ⁇ chain variable region
  • TRBV ⁇ chain variable region
  • the second binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL) derived from an antibody that binds to the second antigen;
  • the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen and an autoantigen, and the second antigen is an immune cell surface molecule;
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • viral antigen a viral antigen and an autoantigen
  • the second antigen is an immune cell surface molecule
  • the multispecific antigen-binding molecule further comprises at least one of the following functional domains:
  • the TRAC and the TRBC are linked to the C-termini of the TRAV and the TRBV, respectively, via an optional linker; or the TRAC and the TRBC are linked to the C-termini of the TRBV and the TRAV, respectively, via an optional linker.
  • a TRAC or TRBC of the present disclosure is not linked to the C-terminus of the VH or VL, for example, with or without a linker.
  • the N-terminus of the antibody is linked to the C-terminus of the TCR.
  • the functional domain is derived from the hinge region of an antibody and/or the Fc domain of an antibody or its dimerization portion and/or the CH3 domain of an antibody and/or the constant region or its fragment derived from TCR; and any two polypeptide chains of the multispecific antigen-binding molecule are connected by covalent or non-covalent binding between the two chains of the functional domain;
  • the two chains of the functional domain are connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific antigen-binding molecule, preferably the C-terminus, respectively, through an optional linker; or
  • the first binding region and the second binding region are connected by covalent or non-covalent binding between the two chains of the functional domain;
  • the multispecific antigen-binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRBV-optional linker-TRBC-linker-VL-linker-VH-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: TRAV-optional linker-TRAC-optional linker-hinge region-CH2-CH3; or
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-optional linker-TRAC-linker-VL-linker-VH-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3; or
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRBV-optional linker-TRBC-linker-VH-linker-VL-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: TRAV-optional linker-TRAC-optional linker-hinge region-CH2-CH3; or
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-optional linker-TRBC-linker-VH-linker-VL-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3; or
  • the multispecific antigen-binding molecule comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first polypeptide chain comprises from N-terminus to C-terminus: TRBV-optional linker-TRBC-linker-VL, the second polypeptide chain comprises from N-terminus to C-terminus: VH-optional linker-hinge region-CH2-CH3, and the third polypeptide chain comprises from N-terminus to C-terminus: TRAV-optional linker-TRAC-optional linker-hinge region-CH2-CH3.
  • the multispecific antigen-binding molecule does not contain the amino acid sequences shown in SEQ ID NO:92 and 93.
  • the multispecific antigen-binding molecule does not comprise the following first and second polypeptide chains: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-TRBC.
  • the present disclosure provides a nucleic acid encoding a TCR alpha chain constant region, a TCR beta chain constant region, a TCR constant region, a polypeptide molecule, a TCR, or a multispecific antigen-binding molecule described herein.
  • the present disclosure provides a vector comprising a nucleic acid described herein.
  • the vector is selected from a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.
  • the present disclosure provides a host cell comprising a TCR alpha chain constant region, a TCR beta chain constant region, a TCR constant region, a polypeptide molecule, a TCR, a multispecific antigen-binding molecule, a nucleic acid, or a vector described herein.
  • the present disclosure provides a conjugate comprising a polypeptide molecule, a TCR or a multispecific antigen-binding molecule described herein, and a chemical moiety conjugated to the polypeptide molecule, the TCR or the multispecific antigen-binding molecule.
  • the chemical moiety is selected from the group consisting of a detectable label, an immunostimulatory molecule, and a therapeutic agent;
  • the detectable label is selected from biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent or chemiluminescent molecule;
  • the immunostimulatory molecules are selected from cytokines, chemokines, platelet factors and complement initiators;
  • the therapeutic agent is selected from the group consisting of an immunomodulator, a radioactive compound, an enzyme, a chemotherapeutic agent, and a toxin.
  • the present disclosure provides a composition comprising a polypeptide molecule, a TCR, a multispecific antigen-binding molecule, a nucleic acid, a vector, a cell, or a conjugate described herein; and a pharmaceutically acceptable carrier or excipient.
  • the composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
  • the present disclosure provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of a polypeptide molecule, TCR, multispecific antigen-binding molecule, nucleic acid, vector, host cell, conjugate, or composition described herein.
  • the disease is selected from cancer, infectious diseases, autoimmune diseases and inflammatory diseases.
  • the method further comprises administering a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.
  • the present disclosure provides a TCR, which comprises a TCR ⁇ chain variable region (TRAV) and a TCR ⁇ chain variable region (TRBV), wherein the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:56 (DSSMTY), SEQ ID NO:57 (IFAFESM) and SEQ ID NO:58 (AGSGGGTDK), respectively, or a functional variant formed by inserting, deleting or substituting one or several amino acids; and/or the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:59 (DFEATT), SEQ ID NO:60 (TDYGSKA) and SEQ ID NO:61 (SAREPGQGPWE), respectively, or a functional variant formed by inserting, deleting or substituting one or several amino acids.
  • TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:56 (DSSMTY), S
  • the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:56 (DSSMTY), SEQ ID NO:57 (IFAFESM) and SEQ ID NO:58 (AGSGGGTDK), respectively; and/or the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:59 (DFEATT), SEQ ID NO:60 (TDYGSKA) and SEQ ID NO:61 (SAREPGQGPWE), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:30, and/or the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:31.
  • the present disclosure provides a TCR, which comprises a TCR ⁇ chain variable region (TRAV) and a TCR ⁇ chain variable region (TRBV), wherein the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:62 (DSSMTY), SEQ ID NO:63 (IFFYQDM) and SEQ ID NO:64 (AGSGGGTDK), respectively, or a functional variant formed by inserting, deleting or substituting one or several amino acids; and/or the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:65 (DFEATT), SEQ ID NO:66 (TDYGSKA) and SEQ ID NO:67 (SAREPGQAWSD), respectively, or a functional variant formed by inserting, deleting or substituting one or several amino acids.
  • TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:62 (DSSMTY), S
  • the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:62 (DSSMTY), SEQ ID NO:63 (IFFYQDM) and SEQ ID NO:64 (AGSGGGTDK), respectively, and/or the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:65 (DFEATT), SEQ ID NO:66 (TDYGSKA) and SEQ ID NO:67 (SAREPGQAWSD), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:32, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:33.
  • Figure 1 shows the mechanism of action of TCR-CD3 antibody fusion protein.
  • Figure 2 shows the principle of protein stability design.
  • FIG3 shows the optimization process for soluble expression of TCR fusion proteins.
  • Figure 4 shows the three-dimensional structure of TCR predicted by AlphaFold2.
  • Figure 5 shows the three-dimensional structure of the TCR fusion protein gp100-B60 predicted by AlphaFold2.
  • FIG6 shows the protein-protein interaction sites (PPIs) of the TCR fusion protein gp100-B60A chain predicted by ISPRED4.
  • Figure 7 shows the ELISPOT results of TCR constant region single site mutants applied to format 62.
  • FIG8 shows a comparison between the activity gains predicted by the Stable single-item score, the Soluble single-item score, and the Stable/Soluble double-item score and the activity gains verified by the ELISPOT experiment.
  • FIG. 9 shows the expression of CR-KVA11-N03TCR on the surface of T cells detected by flow cytometry.
  • FIG10 shows the computer-predicted three-dimensional structural model of CR-KVA11-N03TCR and the mutation sites in the constant region.
  • FIG. 11 shows the SDS-PAGE results of CR-KVA11-N03-B62 molecules containing different mutations.
  • FIG. 12 shows the SDS-PAGE results and calculated protein yields of CMM1 mutations applied to different TCR sequences and different TCR fusion protein structures.
  • FIG13 shows the ELISPOT results of CMM1 mutations applied to different TCR sequences and different TCR fusion protein structures.
  • Figure 14 shows the ELISPOT results of TCR constant region single site mutants applied to format 22-2.
  • Figure 15 shows the SDS-PAGE results and calculated protein yields of the CMM1 mutation and its reverse mutations CMM2, CMM3, CMM4 and CMM5 applied to format 62.
  • TRAC comprises N1.2K, T84C and FFPSP deletion mutations at positions 120-124 relative to human TRAC*01 shown in SEQ ID No.1, and its amino acid sequence is shown in SEQ ID NO:2
  • TRBC comprises S79C, C85.1A, N97D relative to human TRBC2*01 shown in Seq ID No.14, and its amino acid sequence is shown in SEQ ID NO:15.
  • conservative substitutions of these mutated amino acids are also expected to improve the soluble expression of TCR fusion proteins, because the conservatively substituted amino acids have similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathicity.
  • conservative substitutions of amino acids can be made with reference to the following table, wherein it is preferred to select amino acids in the same block in the first column for conservative substitution, and it is more preferred to select amino acids in the same row in the second column for conservative substitution.
  • the modified TCR constant region of the invention may comprise one or more of the following amino acid substitutions: Q6R, Q6K, Q6D, Q6E; S21I, S21L, S21V, S21G, S21A, S21P; K83P, K83G, K83A, K83I, K83L, K83V; S84.6D, S84.6E, S84.6N, S84.6Q, S84.6K, S84.6R; I114E, I114D, I114N, I114Q, I114K, I114R; S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V, S92Q, S92K, S92R; T94D, T94E, T94N, T94Q, T94K, T94R; Q107L, Q107I, Q107V, Q107G, Q107A, Q107P.
  • the present disclosure provides a T cell receptor (TCR) ⁇ chain constant region, wherein the ⁇ chain constant region comprises amino acid substitutions at one or more of the following positions: Q6, S21, K83, S84.6 and I114; wherein the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) according to the TCR ⁇ chain constant region shown in SEQ ID NO:1.
  • TCR T cell receptor
  • IMGT International Immunogenetics Information System
  • the T cell receptor consists of two (separate) protein chains, which are produced by independent T cell receptor ⁇ and ⁇ (TCR ⁇ and TCR ⁇ ) genes and are called ⁇ chain and ⁇ chain.
  • Each chain of the 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.
  • TCR is a heterodimer of one ⁇ chain and one ⁇ chain that can bind to MHC molecules presenting antigenic peptides.
  • variable regions of the ⁇ and ⁇ chains are conferred by the variable regions of the ⁇ and ⁇ chains.
  • Both variable regions of the TCR ⁇ and ⁇ chains contain three hypervariable or complementary determining regions (CDR1 ⁇ / ⁇ , CDR2 ⁇ / ⁇ , and CDR3 ⁇ / ⁇ ) surrounded by framework (FR) regions.
  • CDR3 is the main determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with specific antigens), while CDR1 and CDR2 mainly interact with MHC molecules presenting antigenic peptides.
  • TCR recognizes antigenic peptides that are bound to major histocompatibility complex (MHC) molecules at the surface of antigen presenting cells ("presented/displayed on MHC molecules").
  • MHC major histocompatibility complex
  • Antigenic peptides presented on MHC molecules are also referred to herein as "complex of epitope with MHC molecule", “epitope-MHC complex” or "target antigen peptide-MHC complex”.
  • MHC I and MHC II which present peptides from different cell compartments.
  • MHC class I molecules are expressed on the surface of all nucleated cells in the human body and display peptides or protein fragments from intracellular compartments to cytotoxic T cells.
  • MHC is also known as human leukocyte antigen (HLA).
  • HLA human leukocyte antigen
  • MHC class I There are three main types of MHC class I: HLA-A, HLA-B and HLA-C.
  • the sequence of the TCR constant region can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant region sequence of the TCR molecule ⁇ chain is "TRAC*01", and the constant domain sequence of the TCR molecule ⁇ chain is "TRBC1*01" or "TRBC2*01".
  • IMGT International Immunogenetics Information System
  • the positions of the amino acid sequences of TCR in the present invention are numbered according to the naming rules of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the position numbering of the amino acid sequences of the TCR ⁇ chain constant region (TRAC) and the ⁇ chain constant region (TRBC) are numbered according to the positions listed in IMGT.
  • the position number listed in IMGT is 84.6, then it is described in the present invention as the 84.6th amino acid of TRAC;
  • the position number listed in IMGT is 92, then it is described in the present invention as the 92nd amino acid of TRBC; and so on.
  • special instructions for the sequence position numbering of other amino acids follow the special instructions.
  • the amino acid substitution at position Q6 in the ⁇ chain constant region is selected from Q6R, Q6K, Q6D and Q6E, preferably selected from Q6R and Q6K.
  • the amino acid substitution at position S21 in the ⁇ chain constant region is selected from S21I, S21L, S21V, S21G, S21A and S21P, preferably selected from S21I, S21L and S21V.
  • the amino acid substitution at position K83 in the ⁇ chain constant region is selected from K83P, K83G, K83A, K83I, K83L and K83V, preferably selected from K83P, K83G and K83A.
  • the amino acid substitution at position S84.6 in the ⁇ chain constant region is selected from S84.6D, S84.6E, S84.6N, S84.6Q, S84.6K and S84.6R, preferably selected from S84.6D, S84.6E, S84.6N and S84.6Q.
  • the amino acid substitution at position I114 in the ⁇ chain constant region is selected from I114E, I114D, I114K and I114R, preferably selected from I114E and I114D.
  • the amino acid at position Q6 in the ⁇ chain constant region is substituted with Q6R.
  • the amino acid at position S21 in the ⁇ chain constant region is substituted with S21I.
  • the amino acid substitution at position K83 in the ⁇ chain constant region is K83P.
  • the amino acid substitution at position S84.6 in the ⁇ chain constant region is selected from S84.6D, S84.6E and S84.6N.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of positions Q6, S21, K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any two positions of Q6, S21, K83, S84.6 and I114. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any three positions of Q6, S21, K83, S84.6 and I114. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any four positions of Q6, S21, K83, S84.6 and I114. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114.
  • the ⁇ chain constant region comprises one or more of the following amino acid substitutions: S21I, K83P and S84.6D; preferably comprises at least two of the amino acid substitutions S21I, K83P and S84.6D; more preferably comprises amino acid substitutions S21I, K83P and S84.6D.
  • the ⁇ chain constant region comprises an amino acid substitution selected from any one of the following (1)-(4):
  • the ⁇ chain constant region comprises S21I, K83P and S84.6D. In some embodiments, the ⁇ chain constant region comprises S21I and S84.6D.
  • the ⁇ chain constant region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:1.
  • the ⁇ chain constant region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2.
  • the ⁇ chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO:3-13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:3-13.
  • sequence identity refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions or substitutions may have a lower degree of identity.
  • sequence identity can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and ClustalW.
  • amino acid sequence of SEQ ID NO: 3 can be used as a "subject sequence” or “reference sequence”, while the amino acid sequence of the TCR constant region that is different therefrom can be used as a "query sequence”.
  • the ⁇ chain constant region comprises Q6R. In a preferred embodiment, the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:3.
  • the ⁇ chain constant region comprises S21I.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:4.
  • the ⁇ chain constant region comprises K83P. In a preferred embodiment, the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:5.
  • the ⁇ chain constant region comprises S84.6D. In a preferred embodiment, the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:6.
  • the ⁇ chain constant region comprises S84.6E. In a preferred embodiment, the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:7.
  • the ⁇ chain constant region comprises S84.6N. In a preferred embodiment, the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:8.
  • the ⁇ chain constant region comprises I114E.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:9.
  • the ⁇ chain constant region comprises K83P and S84.6D.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:10.
  • the ⁇ chain constant region comprises S21I and S84.6D. In a preferred embodiment, the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:11.
  • the ⁇ chain constant region comprises S21I and K83P.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:12.
  • the ⁇ chain constant region comprises S21I, K83P and S84.6D.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:13.
  • the present disclosure provides a TCR ⁇ chain constant region comprising amino acid substitutions S21I, K83P and S84.6D, wherein the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) based on the TCR ⁇ chain constant region shown in SEQ ID NO:1.
  • IMGT International Immunogenetics Information System
  • the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:13.
  • the TCR ⁇ chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13. Therefore, the present disclosure provides a TCR ⁇ chain constant region, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.
  • the present disclosure provides a TCR ⁇ chain constant region, wherein the ⁇ chain constant region comprises amino acid substitutions at one or more of the following positions: S92, T94 and Q107; wherein the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) based on the TCR ⁇ constant region shown in SEQ ID NO:14.
  • IMGT International Immunogenetics Information System
  • the amino acid substitution at position S92 in the beta chain constant region is selected from S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V, S92Q, S92K and S92R, preferably selected from S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V and S92Q.
  • the amino acid substitution at position T94 in the ⁇ chain constant region is T94D, T94E, T94K and T94R, preferably selected from T94D and T94E.
  • the amino acid substitution at position Q107 in the beta chain constant region is Q107L, Q107I, Q107V, Q107G, Q107A and Q107P, preferably selected from Q107L, Q107I and Q107V.
  • the amino acid substitution at position S92 in the beta chain constant region is selected from S92P, S92E, S92D, S92L and S92N.
  • the amino acid substitution at position T94 in the beta chain constant region is T94D.
  • amino acid at position Q107 in the beta chain constant region is substituted with Q107L.
  • the beta chain constant region comprises amino acid substitutions at S92, T94, and Q107. In some embodiments, the beta chain constant region comprises amino acid substitutions at T94 and Q107. In some embodiments, the beta chain constant region comprises amino acid substitutions at S92 and T94. In some embodiments, the beta chain constant region comprises amino acid substitutions at S92 and Q107. In some embodiments, the beta chain constant region comprises amino acid substitutions at S92. In some embodiments, the beta chain constant region comprises amino acid substitutions at T94. In some embodiments, the beta chain constant region comprises amino acid substitutions at Q107.
  • the beta chain constant region comprises one or more of the following amino acid substitutions: T94D and Q107L; preferably comprises amino acid substitutions T94D and Q107L.
  • the beta chain constant region comprises an amino acid substitution selected from any one of the following (1)-(3):
  • the beta chain constant region comprises T94D and Q107L. In some embodiments, the beta chain constant region comprises T94D. In some embodiments, the beta chain constant region comprises Q107L.
  • the TCR ⁇ chain constant region further comprises one or more of the following amino acid mutations: S79C, C85.1A and N97D.
  • the constant region of the ⁇ chain further comprises S79C and optionally C85.1A and/or N97D.
  • the TCR ⁇ chain constant region further comprises S79C, C85.1A and N97D.
  • the beta chain constant region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:14.
  • the beta chain constant region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:15.
  • the TCR ⁇ chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO:16-23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:16-23.
  • the beta chain constant region comprises S92P.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:16.
  • the ⁇ chain constant region comprises S92E.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:17.
  • the ⁇ chain constant region comprises S92D.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:18.
  • the ⁇ chain constant region comprises S92L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:19.
  • the ⁇ chain constant region comprises S92N.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:20.
  • the ⁇ chain constant region comprises T94D.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:21.
  • the ⁇ chain constant region comprises Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:22.
  • the ⁇ chain constant region comprises T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO:23.
  • the present disclosure provides a TCR ⁇ chain constant region comprising amino acid substitutions T94D and Q107L, wherein the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) based on the TCR ⁇ chain constant region shown in SEQ ID NO:14.
  • IMGT International Immunogenetics Information System
  • the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:23.
  • the TCR ⁇ chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 23. Therefore, the present disclosure provides a TCR ⁇ chain constant region, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 23.
  • the present disclosure provides a TCR constant region comprising a TCR ⁇ chain constant region and a TCR ⁇ chain constant region described herein.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92 and T94.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92, T94 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises Q6 and S21, Q6 and K83, Q6 and S84.6, amino acid substitutions at Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the beta chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94, and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94, and Q107.
  • the constant region of the ⁇ chain comprises one or more of the following amino acid substitutions: S21I, K83P, and S84.6D; preferably comprises at least two of the amino acid substitutions S21I, K83P, and S84.6D; more preferably comprises amino acid substitutions S21I, K83P, and S84.6D.
  • the ⁇ chain constant region comprises S21I, K83P, and S84.6D. In some embodiments, the ⁇ chain constant region comprises S21I and S84.6D.
  • the constant region of the beta chain comprises one or more of the following amino acid substitutions: T94D and Q107L; preferably comprises amino acid substitutions T94D and Q107L.
  • the beta chain constant region comprises T94D and Q107L.
  • the beta chain constant region comprises T94D.
  • the beta chain constant region comprises Q107L.
  • the TCR comprises an amino acid substitution selected from any one of the following (1)-(21):
  • the constant region of the ⁇ chain further comprises one or more of the following amino acid mutations: N1.2K, T84C, and 120-124 FFPSP deletion mutations; the constant region of the ⁇ chain further comprises one or more of the following amino acid mutations: S79C, C85.1A, and N97D.
  • the constant region of the ⁇ chain further comprises T84C and optionally N1.2K and/or 120-124 FFPSP deletion mutations; the constant region of the ⁇ chain further comprises S79C and optionally C85.1A and/or N97D.
  • the constant region of the ⁇ chain further comprises N1.2K, T84C, and 120-124 FFPSP deletion mutations, and the constant region of the ⁇ chain further comprises S79C, C85.1A, and N97D.
  • the constant region of the ⁇ chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:1
  • the constant region of the ⁇ chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:14.
  • the constant region of the ⁇ chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:2, and the constant region of the ⁇ chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:15.
  • the ⁇ chain constant region comprises an amino acid sequence shown in any one of SEQ ID NO:3-13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:3-13, and the ⁇ chain constant region comprises an amino acid sequence shown in any one of SEQ ID NO:16-23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:16-23.
  • the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:13; and the TCR ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:23.
  • the constant region of the ⁇ chain comprises amino acid substitutions S21I, K83P and S84.6D, and the constant region of the ⁇ chain comprises amino acid substitutions T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 13
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the constant region of the ⁇ chain comprises K83P and S84.6D, and the constant region of the ⁇ chain comprises T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 10
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the constant region of the ⁇ chain comprises S21I and K83P, and the constant region of the ⁇ chain comprises T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 12
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the constant region of the ⁇ chain comprises S21I, K83P and S84.6D, and the constant region of the ⁇ chain comprises Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 13
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the constant region of the ⁇ chain comprises S21I, K83P and S84.6D, and the constant region of the ⁇ chain comprises T94D.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 13
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 21.
  • the present disclosure provides a polypeptide molecule comprising a TCR ⁇ chain constant region, a TCR ⁇ chain constant region, or a TCR constant region described herein.
  • the polypeptide molecule further comprises an antigen binding region that binds at least one antigen.
  • the antigen binding region is selected from an antigen binding region derived from an antibody and an antigen binding region derived from a TCR.
  • the polypeptide molecule is a TCR.
  • the polypeptide molecule is a chimeric molecule fused with a TCR constant region and an antibody variable region.
  • the polypeptide molecule is a CAR.
  • the present disclosure provides a T cell receptor (TCR), the TCR comprising an ⁇ chain and a ⁇ chain, wherein the ⁇ chain comprises the TCR ⁇ chain constant region described herein, and/or the ⁇ chain comprises the TCR ⁇ chain constant region described herein.
  • TCR T cell receptor
  • the TCR comprises an ⁇ chain and a ⁇ chain, and comprises a TCR constant region described herein.
  • the present disclosure provides a T cell receptor (TCR), the TCR comprising an ⁇ chain and a ⁇ chain,
  • constant region of the ⁇ chain comprises an amino acid substitution at one or more of the following positions: Q6, S21, K83, S84.6 and I114; and/or
  • constant region of the beta chain comprises an amino acid substitution at one or more of the following positions: S92, T94, and Q107;
  • the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) based on the TCR ⁇ chain constant region shown in SEQ ID NO:1 and the TCR ⁇ chain constant region shown in SEQ ID NO:14.
  • IMGT International Immunogenetics Information System
  • the TCR comprises a constant region of an alpha chain having an amino acid substitution at one or more of the following positions: Q6, S21, K83, S84.6, and I114. In some embodiments, the TCR comprises a constant region of a beta chain having an amino acid substitution at one or more of the following positions: S92, T94, and Q107. In some embodiments, the TCR comprises a constant region of an alpha chain having an amino acid substitution at one or more of the following positions: Q6, S21, K83, S84.6, and I114, and a constant region of a beta chain having an amino acid substitution at one or more of the following positions: S92, T94, and Q107.
  • the amino acid substitution at position Q6 in the constant region of the ⁇ chain is selected from Q6R, Q6K, Q6D and Q6E, preferably selected from Q6R and Q6K.
  • the amino acid substitution at position S21 in the constant region of the ⁇ chain is selected from S21I, S21L, S21V, S21G, S21A and S21P, preferably selected from S21I, S21L and S21V.
  • the amino acid substitution at position K83 in the constant region of the ⁇ chain is selected from K83P, K83G, K83A, K83I, K83L and K83V, preferably selected from K83P, K83G and K83A.
  • the amino acid substitution at position S84.6 in the constant region of the ⁇ chain is selected from S84.6D, S84.6E, S84.6N, S84.6Q, S84.6K and S84.6R, preferably selected from S84.6D, S84.6E, S84.6N and S84.6Q.
  • the amino acid substitution at position I114 in the constant region of the ⁇ chain is I114E, I114D, I114K and I114R, preferably selected from I114E and I114D.
  • the amino acid substitution at position S92 in the constant region of the ⁇ chain is selected from S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V, S92Q, S92K and S92R, preferably selected from S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V and S92Q.
  • the amino acid substitution at position T94 in the constant region of the ⁇ chain is T94D, T94E, T94K and T94R, preferably selected from T94D and T94E.
  • the amino acid substitution at position Q107 in the constant region of the beta chain is Q107L, Q107I, Q107V, Q107G, Q107A and Q107P, preferably selected from Q107L, Q107I and Q107V.
  • the amino acid at position Q6 in the constant region of the ⁇ chain is substituted with Q6R.
  • the amino acid at position S21 in the constant region of the ⁇ chain is substituted with S21I.
  • the amino acid substitution at position K83 in the constant region of the ⁇ chain is K83P.
  • the amino acid substitution at position S84.6 in the constant region of the ⁇ chain is selected from S84.6D, S84.6E, and S84.6N.
  • the amino acid substitution at position I114 in the constant region of the ⁇ chain is I114E.
  • amino acid substitution at position S92 in the constant region of the beta chain is selected from S92P, S92E, S92D, S92L, and S92N.
  • the amino acid substitution at position T94 in the constant region of the beta chain is T94D.
  • the amino acid at position Q107 in the constant region of the beta chain is substituted with Q107L.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of positions Q6, S21, K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any two positions of Q6, S21, K83, S84.6 and I114. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any three positions of Q6, S21, K83, S84.6 and I114. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any four positions of Q6, S21, K83, S84.6 and I114. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114.
  • the beta chain constant region comprises an amino acid substitution at any one of S92, T94, and Q107.
  • the beta chain constant region comprises an amino acid substitution at any two of S92, T94 and Q107. In some embodiments, the beta chain constant region comprises an amino acid substitution at S92 and T94. In some embodiments, the beta chain constant region comprises an amino acid substitution at T94 and Q107. In some embodiments, the beta chain constant region comprises an amino acid substitution at S92 and Q107.
  • the beta chain constant region comprises amino acid substitutions at S92, T94, and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92 and T94.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92, T94 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94, and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, amino acid substitutions at K83, S84.6 and I114; and the beta chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94, and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6 and I114, and/or the ⁇ chain constant region comprises amino acid substitutions at S92, T94 and Q107. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, and S84.6, and/or the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at S21, K83, S84.6 and I114, and/or the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the constant region of the ⁇ chain comprises amino acid substitutions at S21, K83 and S84.6, and/or the constant region of the ⁇ chain comprises amino acid substitutions at T94 and Q107. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at S21 and S84.6, and/or the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the constant region of the ⁇ chain comprises one or more of the following amino acid substitutions: S21I, K83P, and S84.6D; preferably comprises at least two of the amino acid substitutions S21I, K83P, and S84.6D; more preferably comprises amino acid substitutions S21I, K83P, and S84.6D.
  • the ⁇ chain constant region comprises S21I, K83P, and S84.6D. In some embodiments, the ⁇ chain constant region comprises S21I and S84.6D.
  • the constant region of the beta chain comprises one or more of the following amino acid substitutions: T94D and Q107L; preferably comprises amino acid substitutions T94D and Q107L.
  • the beta chain constant region comprises T94D and Q107L.
  • the beta chain constant region comprises T94D.
  • the beta chain constant region comprises Q107L.
  • the TCR comprises an amino acid substitution selected from any one of the following (1)-(21):
  • the TCR comprises an amino acid substitution selected from any one of (1)-(21) above.
  • the constant region of the ⁇ chain further comprises one or more of the following amino acid mutations: N1.2K, T84C, and 120-124 FFPSP deletion mutations; and/or the constant region of the ⁇ chain further comprises one or more of the following amino acid mutations: S79C, C85.1A, and N97D.
  • the constant region of the ⁇ chain further comprises T84C and optionally N1.2K and/or 120-124 FFPSP deletion mutations; and/or the constant region of the ⁇ chain further comprises S79C and optionally C85.1A and/or N97D.
  • the constant region of the ⁇ chain further comprises N1.2K, T84C, and 120-124 FFPSP deletion mutations, and the constant region of the ⁇ chain further comprises S79C, C85.1A, and N97D.
  • the constant region of the alpha chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1, and/or the constant region of the beta chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:14.
  • the constant region of the ⁇ chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:2, and/or the constant region of the ⁇ chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:15.
  • the TCR comprises an ⁇ chain constant region having an amino acid sequence shown in any one of SEQ ID NOs:3-13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs:3-13, and/or a ⁇ chain constant region having an amino acid sequence shown in any one of SEQ ID NOs:16-23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs:16-23.
  • the constant region of the ⁇ chain comprises amino acid substitutions S21I, K83P and S84.6D, and the constant region of the ⁇ chain comprises amino acid substitutions T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 13
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the constant region of the ⁇ chain comprises K83P and S84.6D, and the constant region of the ⁇ chain comprises T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 10
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the constant region of the ⁇ chain comprises S21I and S84.6D, and the constant region of the ⁇ chain comprises T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 11
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the constant region of the ⁇ chain comprises S21I and K83P, and the constant region of the ⁇ chain comprises T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 12
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the constant region of the ⁇ chain comprises S21I, K83P and S84.6D, and the constant region of the ⁇ chain comprises Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 13
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the constant region of the ⁇ chain comprises S21I, K83P and S84.6D, and the constant region of the ⁇ chain comprises T94D.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 13
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 21.
  • the TCR binds an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and a self-antigen.
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • viral antigen a self-antigen
  • the TCR binds to a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen.
  • melanoma-associated antigens e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, M
  • the TCR binds to a TSA selected from the following: KRAS (e.g., KRAS G12 mutant antigens, such as G12D, G12V, G12C, G12R, G12A; G13D; Q61H; G125), TP53 (e.g., R175H, R173H, R273C, R248W, R248Q, R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (e.g., E542K, E545K, H1047R), CTNNB1 (e.g., S45P, T41A), EGFR (e.g., L858R, T790M), BRAF (e.g., V600E), and GNAS (e.g., R201C, R201H).
  • KRAS KRAS G12 mutant antigens, such as G12D, G12V, G12C,
  • the TCR binds to a viral antigen selected from the group consisting of: HPV antigens (e.g., HPV E6 or E7 antigens), CMV antigens, HBV antigens, EBV antigens, herpes virus antigens, human immunodeficiency virus (HIV) antigens, influenza virus antigens, and coronavirus antigens.
  • HPV antigens e.g., HPV E6 or E7 antigens
  • CMV antigens e.g., HBV antigens
  • HBV antigens e.g., HBV antigens
  • EBV antigens e.g., herpes virus antigens
  • human immunodeficiency virus (HIV) antigens e.g., human immunodeficiency virus (HIV) antigens
  • influenza virus antigens e.g., influenza virus antigens, and coronavirus antigens.
  • coronavirus antigens e.g
  • the TCR binds to an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin, G AD65, IA2, Znt8, PL7, TARS, ARS, MI2, topoisomerase 1, EXOSC9, EXOSC107, POLR3A, POLR3K, PTRN, GAD2, SLC30A8, AchR, MUSK, LRP4, PLA2R, THSD7A, TSHR, IFN- ⁇ , CHRNA1, MUSK, LRP4, AQP4, MOG, GRIN1, COL4A3, PLA2R, GM-SCF, PR3, and
  • the TCR binds to an antigen selected from the following: melanoma-associated antigens (e.g., gp100, MAGEA1), HPV antigens (e.g., HPV E6 or E7), and KRAS antigens (e.g., KRAS G12V, KRAS G12D).
  • an antigen selected from the following: melanoma-associated antigens (e.g., gp100, MAGEA1), HPV antigens (e.g., HPV E6 or E7), and KRAS antigens (e.g., KRAS G12V, KRAS G12D).
  • the TCR binds KRAS G12V and comprises a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV) that binds KRAS G12V.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 38 (NSASQS), SEQ ID NO: 39 (VYSSGN), and SEQ ID NO: 40 (VVPGGTGGGNKLT), respectively
  • the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 41 (LGHDT), SEQ ID NO: 42 (YNNKEL), and SEQ ID NO: 43 (ASSHWGAQETQY), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 24, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 25.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 44 (NSASDY), SEQ ID NO: 45 (IRSNMDK), and SEQ ID NO: 46 (AENSDGTSYGKLT), respectively, and the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 47 (SGHAT), SEQ ID NO: 48 (FQNNGV), and SEQ ID NO: 49 (ASSLVGSPDYEQY), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:26, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:27.
  • the TRAV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences shown as SEQ ID NO:50 (VSGNPY), SEQ ID NO:51 (YITGDNLV), and SEQ ID NO:52 (AVRDIEGAGNNRKLI), respectively, and the TRBV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences shown as SEQ ID NO:53 (SGHNS), SEQ ID NO:54 (FNNNVP), and SEQ ID NO:55 (ASSEGFYTEAF), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:29.
  • the TCR binds KRAS G12D and comprises a TCR that binds KRAS TCR ⁇ chain variable region (TRAV) and TCR ⁇ chain variable region (TRBV) of G12D.
  • the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences shown in SEQ ID NO: 78 (NSASQS), SEQ ID NO: 79 (VYSSGN) and SEQ ID NO: 80 (VVTNARLM), respectively
  • the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences shown in SEQ ID NO: 81 (MNHEY), SEQ ID NO: 82 (SMNVEV) and SEQ ID NO: 83 (ASTSIVSSGRGEQF), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 34, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 35.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO: 84 (DRGSQS), SEQ ID NO: 85 (IYSNGD), and SEQ ID NO: 86 (AVNLGAAGNKLT), respectively, and the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO: 87 (SGHAT), SEQ ID NO: 88 (FQNNGV), and SEQ ID NO: 89 (ASSFSDSYNSPLH), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:90 (QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNLGAAGNKLTFGGGTRVLVKP), and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:91 (EAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSFSDSYNSPLHFGNGTRLTVT).
  • the TCR binds to MAGEA1 and comprises a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV) that bind to MAGEA1.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 56 (DSSMTY), SEQ ID NO: 57 (IFAFESM), and SEQ ID NO: 58 (AGSGGGTDK), respectively
  • the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 59 (DFEATT), SEQ ID NO: 60 (TDYGSKA), and SEQ ID NO: 61 (SAREPGQGPWE), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:30, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:31.
  • the TRAV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NO:62 (DSSMTY), SEQ ID NO:63 (IFFYQDM), and SEQ ID NO:64 (AGSGGGTDK), respectively, and the TRBV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NO:65 (DFEATT), SEQ ID NO:66 (TDYGSKA), and SEQ ID NO:67 (SAREPGQAWSD), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:32, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:33.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:68 (GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGTGTRLQVFPN), and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:69 (GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSDYQTCVTYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPFEQYFGPGTRLTVTE).
  • the TCR binds to HPV E7 and comprises a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV) that binds to HPV E7.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 70 (NSASQS), SEQ ID NO: 71 (VYSSGN), and SEQ ID NO: 72 (AVISAGTALI), respectively
  • the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 73 (SGHDT), SEQ ID NO: 74 (YYEEEE), and SEQ ID NO: 75 (ASSLGWRGGLYTEAF), respectively.
  • the TRAV comprises a sequence that is at least 80%, at least 85%, at least 90%, or more similar to SEQ ID NO: 76 (RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCAVISAGTALIFGKGTTLSVSS).
  • TRBV comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:77 (DAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGWRGGLYTEAFFGQGTRLTVV).
  • the TCR binds gp100 and comprises a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV) that bind gp100.
  • TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:94
  • TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:95.
  • the TCR does not comprise a TRAV having an amino acid sequence as set forth in SEQ ID NO:24 and does not comprise a TRBV having an amino acid sequence as set forth in SEQ ID NO:25. In some embodiments, the TCR does not comprise a TRAV having a CDR1, CDR2, and CDR3 having amino acid sequences as set forth in SEQ ID NO:38-40, respectively, and does not comprise a TRBV having a CDR1, CDR2, and CDR3 having amino acid sequences as set forth in SEQ ID NO:41-43, respectively. In some embodiments, the TCR does not bind to an antigenic epitope of KRAS comprising VVGAVGVGK or a complex thereof with HLA-A*11. In some embodiments, the TCR does not bind to a KRAS antigen.
  • the TCR does not comprise the amino acid sequence TRAV as shown in SEQ ID NO: 94, and does not comprise the amino acid sequence TRB V as shown in SEQ ID NO: 95. In some embodiments, the TCR does not bind to gp100.
  • the TCR comprises a variable region derived from a murine TCR or a human TCR.
  • the TCR is isolated or purified.
  • an "isolated or purified” TCR means that it has been identified, separated and/or recovered from the components of its production environment, such that the "isolated or purified” TCR is free or substantially free of other contaminant components from its production environment that may interfere with its therapeutic or diagnostic use.
  • Contaminant components may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • an "isolated or purified" TCR can be prepared by at least one purification step that removes or substantially removes these contaminant components.
  • the present disclosure provides a multispecific antigen-binding molecule comprising a TCR ⁇ chain constant region, a TCR ⁇ chain constant region, a TCR constant region, a polypeptide molecule, or a TCR as described herein.
  • the present disclosure provides a multispecific antigen-binding molecule comprising at least one antigen-binding region derived from a TCR and any one of the following:
  • TCR ⁇ chain constant region (TRAC) described herein and/or the TCR ⁇ chain constant region (TRBC) described herein;
  • the multispecific antigen-binding molecule comprises a TCR described herein.
  • the present disclosure provides a multispecific antigen-binding molecule comprising at least one antigen-binding region derived from a TCR and an alpha chain constant region (TRAC) and/or a beta chain constant region (TRBC) derived from a TCR;
  • a multispecific antigen-binding molecule comprising at least one antigen-binding region derived from a TCR and an alpha chain constant region (TRAC) and/or a beta chain constant region (TRBC) derived from a TCR;
  • ⁇ chain constant region comprises an amino acid substitution at one or more of the following positions: Q6, S21, K83, S84.6 and I114; and/or
  • beta chain constant region comprises an amino acid substitution at one or more of the following positions: S92, T94, and Q107;
  • the amino acid positions are numbered according to the naming rules of the International Immunogenetics Information System (IMGT) based on the TCR ⁇ chain constant region shown in SEQ ID NO:1 and the TCR ⁇ chain constant region shown in SEQ ID NO:14.
  • IMGT International Immunogenetics Information System
  • the multispecific antigen-binding molecule comprises an alpha chain constant region having an amino acid substitution at one or more of the following positions: Q6, S21, K83, S84.6, and I114. In some embodiments, the multispecific antigen-binding molecule comprises a beta chain constant region having an amino acid substitution at one or more of the following positions: S92, T94, and Q107. In some embodiments, the multispecific antigen-binding molecule comprises an alpha chain constant region having an amino acid substitution at one or more of the following positions: Q6, S21, K83, S84.6, and I114, and a beta chain constant region having an amino acid substitution at one or more of the following positions: S92, T94, and Q107.
  • the amino acid substitution at position Q6 in the ⁇ chain constant region is selected from Q6R, Q6K, Q6D and Q6E, preferably selected from Q6R and Q6K.
  • the amino acid substitution at position S21 in the ⁇ chain constant region is selected from S21I, S21L, S21V, S21G, S21A and S21P, preferably selected from S21I, S21L and S21V.
  • the amino acid substitution at position K83 in the ⁇ chain constant region is selected from K83P, K83G, K83A, K83I, K83L and K83V, preferably selected from K83P, K83G and K83A.
  • the amino acid substitution at position S84.6 in the ⁇ chain constant region is selected from S84.6D, S84.6E, S84.6N, S84.6Q, S84.6K and S84.6R, preferably selected from S84.6D, S84.6E, S84.6N and S84.6Q.
  • the amino acid substitution at position I114 in the ⁇ chain constant region is I114E, I114D, I114K and I114R, preferably selected from I114E and I114D.
  • the amino acid substitution at position S92 in the beta chain constant region is selected from S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V, S92Q, S92K and S92R, preferably selected from S92P, S92E, S92D, S92L, S92N, S92G, S92A, S92I, S92V and S92Q.
  • the amino acid substitution at position T94 in the ⁇ chain constant region is T94D, T94E, T94K and T94R, preferably selected from T94D and T94E.
  • the amino acid substitution at position Q107 in the beta chain constant region is Q107L, Q107I, Q107V, Q107G, Q107A and Q107P, preferably selected from Q107L, Q107I and Q107V.
  • the amino acid at position Q6 in the ⁇ chain constant region is substituted with Q6R.
  • the amino acid at position S21 in the ⁇ chain constant region is substituted with S21I.
  • the amino acid substitution at position K83 in the ⁇ chain constant region is K83P.
  • the amino acid substitution at position S84.6 in the ⁇ chain constant region is selected from S84.6D, S84.6E and S84.6N.
  • the amino acid substitution at position I114 in the ⁇ chain constant region is I114E.
  • the amino acid substitution at position S92 in the beta chain constant region is selected from S92P, S92E, S92D, S92L and S92N.
  • the amino acid substitution at position T94 in the beta chain constant region is T94D.
  • the amino acid substitution at position Q107 in the beta chain constant region is Q107L.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of positions Q6, S21, K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any two positions of Q6, S21, K83, S84.6 and I114. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any three positions of Q6, S21, K83, S84.6 and I114.
  • the ⁇ chain constant region comprises Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Amino acid substitutions at Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at any four positions of Q6, S21, K83, S84.6 and I114. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114.
  • the beta chain constant region comprises an amino acid substitution at any one of S92, T94, and Q107.
  • the beta chain constant region comprises an amino acid substitution at any two of S92, T94 and Q107. In some embodiments, the beta chain constant region comprises an amino acid substitution at S92 and T94. In some embodiments, the beta chain constant region comprises an amino acid substitution at T94 and Q107. In some embodiments, the beta chain constant region comprises an amino acid substitution at S92 and Q107.
  • the beta chain constant region comprises amino acid substitutions at S92, T94, and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92 and T94.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at any one of Q6, S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S92, T94 and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6 and S21, Q6 and K83, Q6 and S84.6, Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises Q6 and S21, Q6 and K83, Q6 and S84.6, amino acid substitutions at Q6 and I114, S21 and K83, S21 and S84.6, S21 and I114, K83 and S84.6, K83 and I114, or S84.6 and I114; and the beta chain constant region comprises amino acid substitutions at S92, T94, and Q107.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises an amino acid substitution at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21 and K83, Q6, S21 and S84.6, Q6, S21 and I114, Q6, K83 and S84.6, Q6, K83 and I114, Q6, S84.6 and I114, S21, K83 and S84.6, S21, K83 and I114, S21, S84.6 and I114, or K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83 and S84.6, Q6, S21, K83 and I114, Q6, S21, S84.6 and I114, Q6, K83, S84.6 and I114, or S21, K83, S84.6 and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at S9.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises an amino acid substitution at Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and T94.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6, and I114; and the ⁇ chain constant region comprises amino acid substitutions at S92, T94, and Q107.
  • the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, S84.6 and I114, and/or the ⁇ chain constant region comprises amino acid substitutions at S92, T94 and Q107. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at Q6, S21, K83, and S84.6, and/or the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at S21, K83, S84.6 and I114, and/or the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the constant region of the ⁇ chain comprises amino acid substitutions at S21, K83 and S84.6, and/or the constant region of the ⁇ chain comprises amino acid substitutions at T94 and Q107. In some embodiments, the ⁇ chain constant region comprises amino acid substitutions at S21 and S84.6, and/or the ⁇ chain constant region comprises amino acid substitutions at T94 and Q107.
  • the constant region of the ⁇ chain comprises one or more of the following amino acid substitutions: S21I, K83P, and S84.6D; preferably comprises at least two of the amino acid substitutions S21I, K83P, and S84.6D; more preferably comprises amino acid substitutions S21I, K83P, and S84.6D.
  • the ⁇ chain constant region comprises S21I, K83P, and S84.6D. In some embodiments, the ⁇ chain constant region comprises S21I and S84.6D.
  • the constant region of the beta chain comprises one or more of the following amino acid substitutions: T94D and Q107L; preferably comprises amino acid substitutions T94D and Q107L.
  • the beta chain constant region comprises T94D and Q107L.
  • the beta chain constant region comprises T94D.
  • the beta chain constant region comprises Q107L.
  • the multispecific antigen-binding molecule comprises an amino acid substitution selected from any one of the following (1)-(21):
  • the TCR comprises an amino acid substitution selected from any one of (1)-(21) above.
  • the ⁇ chain constant region further comprises one or more of the following amino acid mutations: N1.2K, T84C, and 120-124 FFPSP deletion mutations; and/or the ⁇ chain constant region further comprises one or more of the following amino acid mutations: S79C, C85.1A, and N97D.
  • the constant region of the ⁇ chain further comprises T84C and optionally N1.2K and/or 120-124 FFPSP deletion mutations; and/or the constant region of the ⁇ chain further comprises S79C and optionally C85.1A and/or N97D.
  • the constant region of the ⁇ chain further comprises N1.2K, T84C, and 120-124 FFPSP deletion mutations, and the constant region of the ⁇ chain further comprises S79C, C85.1A, and N97D.
  • the ⁇ chain constant region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:1, and/or the ⁇ chain constant region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:14.
  • the ⁇ chain constant region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:2, and/or the ⁇ chain constant region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:15.
  • the ⁇ chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO:3-13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:3-13, and/or the ⁇ chain constant region comprises the amino acid sequence shown in any one of SEQ ID NO:16-23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NO:16-23.
  • the ⁇ chain constant region comprises amino acid substitutions S21I, K83P and S84.6D, and the ⁇ chain constant region comprises amino acid substitutions T94D and Q107L.
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the ⁇ chain constant region comprises K83P and S84.6D, and the ⁇ chain constant region comprises T94D and Q107L.
  • the constant region of the ⁇ chain comprises the amino acid sequence shown in SEQ ID NO: 10
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the ⁇ chain constant region comprises S21I and S84.6D, and the ⁇ chain constant region comprises T94D and Q107L.
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the ⁇ chain constant region comprises S21I and K83P, and the ⁇ chain constant region comprises T94D and Q107L.
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 12
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the ⁇ chain constant region comprises S21I, K83P and S84.6D, and the ⁇ chain constant region comprises Q107L.
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the ⁇ chain constant region comprises S21I, K83P and S84.6D, and the ⁇ chain constant region comprises T94D.
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the ⁇ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 21.
  • the multispecific antigen-binding molecule comprises two or more antigen-binding regions that bind two or more antigens, and the two or more antigens are each independently selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, an autoantigen, and an immune cell surface molecule.
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • viral antigen an autoantigen
  • autoantigen an immune cell surface molecule
  • the TAA is selected from: melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and endogenous reverse transcriptase antigens.
  • melanoma-associated antigens e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC
  • the TSA is selected from: KRAS (e.g., KRAS G12 mutant antigens, such as G12D, G12V, G12C, G12R, G12A; G13D; Q61H; G125), TP53 (e.g., R175H, R173H, R273C, R248W, R248Q, R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (e.g., E542K, E545K, H1047R), CTNNB1 (e.g., S45P, T41A), EGFR (e.g., L858R, T790M), BRAF (e.g., V600E), and GNAS (e.g., R201C, R201H).
  • KRAS KRAS G12 mutant antigens, such as G12D, G12V, G12C, G12R, G12A; G
  • the viral antigen is selected from: HPV antigen (e.g., HPV E6 or E7 antigen), CMV antigen, HBV antigen, EBV antigen, herpes virus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen and coronavirus antigen.
  • HPV antigen e.g., HPV E6 or E7 antigen
  • CMV antigen e.g., HPV E6 or E7 antigen
  • HBV antigen e.g., HPV E6 or E7 antigen
  • HBV antigen e.g., HBV antigen
  • EBV antigen e.g., E6 or E7 antigen
  • herpes virus antigen e.g., human immunodeficiency virus (HIV) antigen
  • influenza virus antigen e.g., influenza virus antigen and coronavirus antigen.
  • the autoantigen is selected from the group consisting of: AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin, GAD65 , IA2, Znt8, PL7, TARS, ARS, MI2, topoisomerase 1, EXOSC9, EXOSC107, POLR3A, POLR3K, PTRN, GAD2, SLC30A8, AchR, MUSK, LRP4, PLA2R, THSD7A, TSHR, IFN- ⁇ , CHRNA1, MUSK, LRP4, AQP4, MOG, GRIN1, COL4A3, PLA2R, GM-SCF, PR3, and MPO.
  • the immune cell surface molecule is selected from the group consisting of: CD3 (e.g., CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ chain), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, Fc ⁇ R I.
  • CD3 e.g., CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ chain
  • one of the two or more antigens is selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens, and autoantigens; the other is an immune cell surface molecule.
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • the antigen bound by the antigen binding region derived from TCR is selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens, and autoantigens.
  • the TAA is a melanoma-associated antigen (e.g., gp100, MAGEA1).
  • the TSA is a KRAS antigen (e.g., KRAS G12V, KRAS G12D).
  • the viral antigen is an HPV antigen (e.g., HPV E6 or E7).
  • the immune cell surface molecule is selected from CD3, CD28, 4-1BB (CD137), PD-1, and PD-L1.
  • the immune cell surface molecule is CD3.
  • the immune cell surface molecule is CD28.
  • the multispecific antigen-binding molecule binds to KRAS G12V and comprises a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV) that binds to KRAS G12V.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively
  • the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:24
  • the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:25
  • the TRAV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, respectively
  • the TRBV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49, respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:26
  • the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:27
  • the TRAV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, respectively
  • the TRBV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:29.
  • the multispecific antigen-binding molecule binds KRAS G12D and comprises a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV) that bind KRAS G12D.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively
  • the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83, respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 34, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 35.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively, and the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:90, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:91.
  • the multispecific antigen-binding molecule binds to MAGEA1 and comprises a TCR ⁇ chain variable region (TRAV) and a TCR ⁇ chain variable region (TRBV) that bind to MAGEA1.
  • TRAV comprises an ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively
  • the TRBV comprises a ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:30
  • the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:31
  • the TRAV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64, respectively
  • the TRBV comprises ⁇ chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:32, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:33.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:68
  • the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:69.
  • the multispecific antigen-binding molecule binds to HPV E7 and comprises a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV) that binds to HPV E7.
  • the TRAV comprises an alpha chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively
  • the TRBV comprises a beta chain CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:76, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:77.
  • the multispecific antigen-binding molecule binds gp100 and comprises a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV) that bind gp100.
  • TRAV TCR alpha chain variable region
  • TRBV TCR beta chain variable region
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:94
  • the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:95.
  • the multispecific antigen-binding molecule does not comprise TRAV with an amino acid sequence as shown in SEQ ID NO:24 and does not comprise TRBV with an amino acid sequence as shown in SEQ ID NO:25. In some embodiments, the multispecific antigen-binding molecule does not comprise TRAV with CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NO:38-40, respectively, and does not comprise TRBV with CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NO:41-43, respectively. In some embodiments, the multispecific antigen-binding molecule does not bind to the KRAS antigen epitope VVGAVGVGK or its complex with HLA-A*11. In some embodiments, the multispecific antigen-binding molecule does not bind to the KRAS antigen.
  • the TCR does not comprise the amino acid sequence TRAV as shown in SEQ ID NO: 94, and does not comprise the amino acid sequence TRB V as shown in SEQ ID NO: 95. In some embodiments, the TCR does not bind to gp100.
  • the multispecific antigen-binding molecule comprises a first binding region that binds a first antigen and a second binding region that binds a second antigen on at least two polypeptide chains.
  • the first binding region comprises an ⁇ chain variable region (TRAV) and a ⁇ chain variable region (TRBV) derived from a TCR that binds to the first antigen-MHC complex;
  • TRAV ⁇ chain variable region
  • TRBV ⁇ chain variable region
  • the second binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL) derived from an antibody that binds to the second antigen;
  • the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen and an autoantigen, and the second antigen is an immune cell surface molecule;
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • viral antigen a viral antigen and an autoantigen
  • the second antigen is an immune cell surface molecule
  • the multispecific antigen-binding molecule further comprises at least one of the following functional domains:
  • the functional domain comprises a constant region derived from a TCR or a fragment thereof.
  • the bispecific polypeptide molecule comprises two polypeptide chains, and the two polypeptide chains respectively comprise:
  • variable regions in the two polypeptide chains are connected by a linker; and the constant region or fragment thereof of the TCR is connected to the C-terminus of each polypeptide chain via an optional linker.
  • the constant region of the TCR or a fragment thereof is a constant region of a TCR or a fragment thereof described herein.
  • the TCR constant region comprises TRAC described herein and/or TRBC described herein.
  • one polypeptide chain of the bispecific polypeptide molecule comprises TRAC and the other polypeptide chain comprises TRBC.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VL-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VH-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VL-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC.
  • the multispecific antigen-binding molecule does not comprise the following first and second polypeptide chains: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-ANIGGGSGGGG (SEQ ID NO:36)-VH-S-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-GGGSGGGGD (SEQ ID NO:37)-TRBV-TRBC.
  • the multispecific antigen-binding molecule does not comprise the following first and second polypeptide chains: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.
  • the multispecific antigen-binding molecule does not comprise the following first polypeptide chain and second polypeptide chain: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.
  • the two polypeptide chains of the bispecific polypeptide molecule do not comprise TRAV-VH and VL-TRBV, respectively.
  • the multispecific antigen-binding molecule does not contain the amino acid sequences shown in SEQ ID NO:92 and 93.
  • the antigen binding region derived from the TCR comprises TRAV and TRBV, and TRAC connected to the C-terminus of TRAV via an optional linker, and TRBC connected to the C-terminus of TRBV via an optional linker; and wherein the TRAC is the TRAC described herein; and/or the TRBC is the TRBC described herein.
  • the TRAC described herein and the TRBC described herein are linked to the C-termini of the TRAV and the TRBV, respectively, via an optional linker; or the TRAC and the TRBC are linked to the C-termini of the TRBV and the TRAV, respectively, via an optional linker.
  • the bispecific polypeptide molecule comprises two polypeptide chains.
  • the multispecific antigen-binding molecule comprises a first binding region that binds a first antigen and a second binding region that binds a second antigen.
  • the first binding region comprises an alpha chain variable region (TRAV1) and a beta chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;
  • TRAV1 alpha chain variable region
  • TRBV1 beta chain variable region derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex
  • the second binding region comprises a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen or a single heavy chain variable region (VHH1) derived from a single domain antibody or nanobody that binds to the second antigen;
  • the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen and an autoantigen, and the second antigen is an immune cell surface molecule;
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • viral antigen a viral antigen and an autoantigen
  • the second antigen is an immune cell surface molecule
  • N-terminus of the antibody is connected to the C-terminus of the TCR
  • the multispecific antigen-binding molecule further comprises at least one of the following functional domains:
  • the N-terminus of the antibody is linked to the C-terminus of the TCR.
  • TRAV1 and TRBV1 are distributed on different polypeptide chains.
  • the VH1 and VL1 are distributed on one polypeptide chain (e.g., forming scFv1 or Fab1); and the TRAV1 and TRBV1 are distributed on different polypeptide chains.
  • the VH1 and VL1 are connected by a linker.
  • the multispecific antigen-binding molecule comprises a first binding region that binds a first antigen and a second binding region that binds a second antigen on two polypeptide chains;
  • TRAV1 and TRBV1 are distributed on different polypeptide chains, and the VHH1 or the VH1 and VL1 are distributed on one polypeptide chain (for example, forming scFv1 or Fab1) and connected to the C-terminus of the TCR; or TRAV1 and TRBV1 are distributed on different polypeptide chains, and the VH1 and VL1 are distributed on different polypeptide chains, and one of the VH1 and VL1 is located in a polypeptide chain connected to the C-terminus of the polypeptide chain where TRAV1 is located, and the other is located in a polypeptide chain connected to the C-terminus of the polypeptide chain where TRBV1 is located.
  • the multispecific antigen-binding molecule comprises a first binding region that binds a first antigen and a second binding region that binds a second antigen on three polypeptide chains.
  • TRAV1 and TRBV1 are distributed on different polypeptide chains
  • the VH1 and VL1 are distributed on different polypeptide chains
  • the polypeptide chain where VH1 or VL1 is located is connected to the C-terminus of the polypeptide chain where TRBV1 or TRAV1 is located.
  • the three polypeptide chains of the multispecific polypeptide molecule respectively comprise: TRBV1-VL1, VH1, and TRAV1; or TRBV1-VH1, VL1, and TRAV1; or TRAV1-VL1, VH1, and TRBV1; or TRAV1-VH1, VL1, and TRBV1.
  • polypeptide chains of the multispecific antigen-binding molecule each comprise:
  • TRBV1-scFv1 and TRAV1 TRBV1-scFv1 and TRAV1;
  • TRBV1-VHH1 and TRAV1 TRBV1-VHH1 and TRAV1;
  • TRBV1-VL1, VH1, and TRAV1 TRBV1-VL1, VH1, and TRAV1;
  • TRBV1-VH1, VL1, and TRAV1 TRBV1-VH1, VL1, and TRAV1;
  • the multispecific antigen-binding molecule further comprises a third binding region that binds to a third antigen, wherein the third binding region comprises an ⁇ chain variable region (TRAV2) and a ⁇ chain variable region (TRBV2) derived from a TCR that binds to the third antigen-MHC complex; wherein the third antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the third antigen is the same as or different from the first antigen.
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • viral antigen a viral antigen
  • autoantigen an autoantigen
  • the TRAV2 is identical to the TRAV1, and/or the TRBV2 is identical to the TRBV1.
  • the TRAV2 and the TRBV2 are connected on the same polypeptide chain via a linker, and are preferably connected between VH1 and VL1.
  • the multispecific antigen-binding molecule comprises two polypeptide chains, each of which comprises:
  • the multispecific antigen-binding molecule further comprises a fourth binding region that binds to a fourth antigen, wherein the fourth binding region comprises a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody that binds to the fourth antigen or a single heavy chain variable region (VHH2) derived from a single domain antibody or nanobody that binds to the fourth antigen; wherein the fourth antigen is an immune cell surface molecule, and the fourth antigen is the same as or different from the second antigen.
  • VHH2 single heavy chain variable region
  • the VH2 is identical to the VH1, and/or the VL2 is identical to the VL1, and/or the VHH2 is identical to the VHH1.
  • the VHH2 or the VH2 and VL2 are connected in tandem on a polypeptide chain (e.g., to form scFv2 or Fab2) and connected to the N-terminus of the TRAV1 or the TRBV1, or one of the VH2 and VL2 is connected to the N-terminus of the TRAV1 and the other is connected to the N-terminus of the TRBV1.
  • a polypeptide chain e.g., to form scFv2 or Fab2
  • one of the VH2 and VL2 is connected to the N-terminus of the TRAV1 and the other is connected to the N-terminus of the TRBV1.
  • the multispecific antigen-binding molecule comprises two polypeptide chains, each of which comprises:
  • VHH2-TRBV1-scFv1 and TRAV1 VHH2-TRBV1-scFv1 and TRAV1;
  • VHH2-TRBV1-VHH1 and TRAV1 VHH2-TRBV1-VHH1 and TRAV1;
  • TRBV1-scFv1 and VHH2-TRAV1 TRBV1-scFv1 and VHH2-TRAV1; or
  • the multispecific antigen-binding molecule comprises a first binding region, a second binding region, a third binding region, and a fourth binding region
  • the multispecific antigen-binding molecule comprises two polypeptide chains, the two polypeptide chains respectively comprising:
  • the functional domain is derived from albumin or its binding portion and optionally the hinge region of an antibody; and the functional domain is connected to the C-terminus or N-terminus, preferably the C-terminus, of any polypeptide chain of the multispecific antigen-binding molecule via an optional linker.
  • the albumin is serum albumin, preferably human serum albumin; and/or the hinge region of the antibody is human, for example, a hinge domain or a portion thereof derived from human IgG1, IgG2 or IgG4.
  • the functional domain is derived from the hinge region of an antibody and/or the Fc domain of an antibody or its dimerization portion and/or the CH3 domain of an antibody; and any two polypeptide chains of the multispecific antigen-binding molecule are connected by covalent or non-covalent binding between the two chains of the functional domain;
  • the two chains of the functional domain are connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific antigen-binding molecule, preferably the C-terminus, respectively, through an optional linker; or
  • the first binding region and the second binding region are connected by covalent or non-covalent binding between the two chains of the functional domain.
  • the hinge region of the antibody is of human origin, for example, a hinge domain or a portion thereof derived from human IgG1, IgG2 or IgG4; and/or
  • the Fc domain of the antibody or the dimerization part thereof is of human origin, for example, an Fc domain or a part thereof derived from human IgG1, IgG2 or IgG4.
  • the hinge region of the antibody comprises at least one amino acid mutation that can reduce or eliminate the binding of the multispecific molecule to the FcR receptor and/or C1q, for example, the mutation site is selected from positions 233, 234, 235 and 236; and/or
  • the hinge region of the antibody comprises at least one mutation that can reduce or eliminate the formation of a disulfide bond between the hinge region and the non-hinge region, for example, the mutation site is 220; and/or
  • the CH2 domain in the Fc domain comprises at least one amino acid mutation that can reduce or eliminate the binding of the multispecific molecule to the FcR receptor and/or C1q; for example, the mutation site is selected from positions 237, 250, 252, 254, 256, 270, 297, 322, 327, 330 and 331; and/or
  • the CH3 domain in the Fc domain comprises at least one amino acid mutation that can reduce or eliminate the binding of the multispecific molecule to the FcR receptor and/or C1q; for example, the mutation site is selected from positions 428 and 434;
  • the CH3 in the Fc domain comprises at least one of the following mutations that can promote the polypeptide molecule to form a heterodimer:
  • a) comprises at least two cysteine residues mutated to form an interchain disulfide bond, such as 354C and 349C or 242C and 334C;
  • each of the CH3 independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407, preferably, one of the CH3 comprises a 366W mutation and the other comprises 366S, 368A and 407V mutations;
  • the mutation sites of the two CH3 chains are each independently selected from at least one of Y391, K392, T393, T394, P395, P396, V397, L398, D399, S400, Q347, V348, Y349, T350, S354, R355, E356, E357, F405, Y407 and K409.
  • the CH3 domain of the antibody is of human origin, for example, a CH3 domain derived from human IgG1, IgG2 or IgG4; and/or
  • the CH3 domain comprises at least one of the following mutations that can promote the polypeptide molecule to form a heterodimer:
  • a) comprises at least two cysteine residues mutated to form an interchain disulfide bond, such as 354C and 349C or 242C and 334C;
  • each of the CH3 independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407, preferably, one of the CH3 comprises a 366W mutation and the other comprises 366S, 368A and 407V mutations;
  • the mutation sites of the two CH3 chains are each independently selected from at least one of Y391, K392, T393, T394, P395, P396, V397, L398, D399, S400, Q347, V348, Y349, T350, S354, R355, D356, E357, K370, F405, Y407, K409 and K439.
  • the functional domain is derived from the constant region of a TCR or a fragment thereof and optionally the hinge region of an antibody; and any two polypeptide chains of the multispecific antigen-binding molecule are connected by covalent or non-covalent binding between the two chains of the functional domain;
  • the two chains of the functional domain are connected to the C-terminus or N-terminus of any two polypeptide chains of the multispecific antigen-binding molecule, preferably the C-terminus, respectively, through an optional linker; or
  • the first binding region and the second binding region are connected by covalent or non-covalent binding between the two chains of the functional domain.
  • the hinge region of the antibody is of human origin, for example, a hinge domain or a portion thereof derived from human IgG1, IgG2 or IgG4; and/or
  • the constant region of the TCR or a fragment thereof is of human or murine origin and is selected from a TCR ⁇ chain constant region (TRAC) and a TCR ⁇ chain constant region (TRBC); preferably, the TRAC and/or TRBC contain at least one cysteine mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, more preferably, the cysteine mutation is at the following positions: position 48 in the wild-type TCR ⁇ chain constant region and position 57 in the wild-type TCR ⁇ chain constant region.
  • TRAC and/or TRBC contain at least one cysteine mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, more preferably, the cysteine mutation is at the following positions: position 48 in the wild-type TCR ⁇ chain constant region and position 57 in the wild-type TCR ⁇ chain constant region.
  • the multispecific antigen-binding molecule further comprises a constant domain selected from the following linked to the C-terminus of TRAV1 and/or TRBV1 via an optional linker:
  • TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR ⁇ chain constant region (TRAC) and/or a ⁇ chain constant region (TRBC);
  • TCR ⁇ chain constant region TCR ⁇ chain constant region
  • TRBC ⁇ chain constant region
  • the TRAC and/or TRBC comprises at least one cysteine mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, more preferably, the cysteine mutation is at the following positions: position 48 of the wild-type TCR ⁇ chain constant region and position 57 of the wild-type TCR ⁇ chain constant region;
  • CH3 wherein the CH3 is derived from the Fc domain of an antibody, and preferably the CH3 in any one or both polypeptide chains contains at least one mutation that can promote the formation of heterodimers by the polypeptide molecule;
  • each of the CH3s independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407 and the two CH3s do not comprise the same mutation, more preferably, one of the CH3s comprises a T366W mutation and the other comprises T366S, L368A and Y407V mutations;
  • the multispecific antigen-binding molecule comprises TRAC of the invention and TRBC of the invention linked to the C-terminus of TRAV1 and TRBV1, respectively, via an optional linker.
  • the multispecific antigen-binding molecule further comprises CH1 and CL derived from an antibody connected to the VH1 and VL1, respectively, wherein the VH1, VL1, CH1 and CL form a single-chain Fab and are connected to the C-terminus of the TRAV1 or the TRBV1 via a linker.
  • polypeptide chains of the multispecific antigen-binding molecule comprise, from N-terminus to C-terminus:
  • first polypeptide chain TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3
  • second polypeptide chain TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3;
  • first polypeptide chain TRAV1-optional linker-TRAC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3
  • second polypeptide chain TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3;
  • first polypeptide chain TRBV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-hinge region-CH2-CH3
  • second polypeptide chain TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3;
  • first polypeptide chain TRAV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-hinge region-CH2-CH3
  • second polypeptide chain TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3;
  • first polypeptide chain TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-linker-CH2-CH3
  • second polypeptide chain TRAV1-optional linker-TRAC-linker-CH2-CH3
  • first polypeptide chain TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH3
  • second polypeptide chain TRAV1-optional linker-TRAC-optional linker-hinge region-CH3;
  • first polypeptide chain TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-TRBC
  • second polypeptide chain TRAV1-optional linker-TRAC-optional linker-hinge region-TRAC
  • first polypeptide chain TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region
  • second polypeptide chain TRAV1-optional linker-TRAC-optional linker-hinge region-Alb; or
  • first polypeptide chain TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-linker-Alb
  • second polypeptide chain TRAV1-optional linker-TRAC
  • first polypeptide chain TRAV1-optional linker-CH3-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3
  • second polypeptide chain TRBV1-optional linker-CH3-optional linker-optional hinge region-CH2-CH3;
  • first polypeptide chain TRAV1-optional linker-CH1-linker-VL1-linker-VH1-optional linker-optional hinge region-CH2-CH3
  • second polypeptide chain TRBV1-optional linker-CL-optional linker-optional hinge region-CH2-CH3;
  • first polypeptide chain TRBV1-optional linker-TRBC-linker-VHH1-optional linker-hinge region-CH2-CH3
  • second polypeptide chain TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3;
  • the antibody is an antibody Fab, and wherein the first polypeptide chain: TRBV1-optional linker-TRBC-linker-VL1-CL-linker-VH1-CH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3;
  • the first polypeptide chain TRBV1-optional linker-TRBC-linker-VL1
  • the second polypeptide chain VH1-optional linker-hinge region-CH2-CH3
  • the third polypeptide chain TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3;
  • the first polypeptide chain TRBV1-optional linker-TRBC-linker-VH1
  • the second polypeptide chain VL1-optional linker-hinge region-CH2-CH3
  • the third polypeptide chain TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3;
  • the first polypeptide chain TRAV1-optional linker-TRAC-linker-VL1
  • the second polypeptide chain VH1-optional linker-hinge region-CH2-CH3
  • the third polypeptide chain TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3.
  • the first polypeptide chain TRAV1-optional linker-TRAC-linker-VH1
  • the second polypeptide chain VL1-optional linker-hinge region-CH2-CH3
  • the third polypeptide chain TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3;
  • the first polypeptide chain TRBV1-optional linker-TRBC-linker-VL1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain VH1-optional linker-hinge region-CH2-CH3
  • the third polypeptide chain TRAV1-optional linker-TRAC
  • the first polypeptide chain TRBV1-optional linker-TRBC-linker-VH1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain VL1-optional linker-hinge region-CH2-CH3
  • the third polypeptide TRAV1-optional linker-TRAC
  • the first polypeptide chain TRAV1-optional linker-TRAC-linker-VL1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain VH1-optional linker-hinge region-CH2-CH3
  • the third polypeptide chain TRBV1-optional linker-TRBC;
  • First polypeptide chain TRAV1-optional linker-TRAC-linker-VH1-optional linker-hinge region-CH2-CH3
  • second polypeptide chain VL1-optional linker-hinge region-CH2-CH3
  • third polypeptide chain TRBV1-optional linker-TRBC.
  • the multispecific antigen-binding molecule comprises a first polypeptide chain and a second polypeptide chain; wherein:
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3.
  • the multispecific antigen-binding molecule comprises a first polypeptide chain and a second polypeptide chain; wherein:
  • the first polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; or
  • the first polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV1-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-hinge region-CH2-CH3; or
  • the first polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3.
  • the multispecific antigen-binding molecule comprises a first polypeptide chain and a second polypeptide chain; wherein:
  • the first polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-linker-TRBV1-optional linker-TRBC-linker-VL1-linker-TRAV2-linker-TRBV2-linker-VH1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3.
  • the multispecific antigen-binding molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; or
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3; or
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC-optional linker-hinge region-CH2-CH3; or
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRBC-linker-VH1-linker-VL1-optional linker-hinge region-CH2-CH3
  • the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-TRBC-optional linker-hinge region-CH2-CH3.
  • the present disclosure provides a nucleic acid encoding a TCR alpha chain constant region, a TCR beta chain constant region, a TCR constant region, a polypeptide molecule, a TCR, or a multispecific antigen-binding molecule described herein.
  • the present disclosure provides a vector comprising a nucleic acid described herein.
  • vector is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material into a host cell, in which the nucleic acid molecule as a vector can, for example, be replicated and/or expressed.
  • vector encompasses, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors and adenovirus-associated vectors (AAV)), bacteriophages, phagemids, cosmids and artificial chromosomes (including BACs and YACs).
  • the vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence as a vector "skeleton".
  • Engineered vectors are typically included in the starting point of autonomous replication in the host cell (if stable expression of polynucleotides is required), a selection marker and a restriction enzyme cleavage site (such as a multiple cloning site, MCS).
  • the vector may additionally include a promoter, a genetic marker, a reporter gene, a targeting sequence and/or a protein purification tag.
  • suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is incorporated herein by reference in its entirety.
  • the vector is selected from the group consisting of a lentiviral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.
  • the present disclosure provides a host cell comprising a TCR alpha chain constant region, a TCR beta chain constant region, a TCR constant region, a polypeptide molecule, a TCR, a multispecific antigen-binding molecule, a nucleic acid, or a vector described herein.
  • the term "host cell” as used herein refers to any type of cell capable of expressing the modified TCR of the present invention.
  • the cell can be a eukaryotic cell, for example, a plant (without the potential to develop into a plant), an animal, a fungus or an algae, or can be a prokaryotic cell, for example, a bacterium or a protozoan.
  • the cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism, such as a human.
  • the cell can be an adherent cell or a suspension cell, i.e., a cell grown in suspension.
  • Suitable host cells include, for example, DH5 ⁇ Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc.
  • the cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.
  • the present disclosure provides a conjugate comprising a polypeptide molecule, a TCR or a multispecific antigen-binding molecule described herein, and a chemical moiety conjugated to the polypeptide molecule, the TCR or the multispecific antigen-binding molecule.
  • the chemical moiety is selected from a detectable label, an immunostimulatory molecule and a therapeutic agent.
  • the detectable label is selected from biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent or chemiluminescent molecule.
  • the immunostimulatory molecule is selected from a cytokine (such as IL-2 and IFN- ⁇ ), a chemokine (such as IL-8), a platelet factor (such as platelet factor 4) and a complement initiator.
  • the therapeutic agent is selected from an immunomodulator, a radioactive compound, an enzyme, a chemotherapeutic agent and a toxin.
  • suitable therapeutic agents include small molecule cytotoxic agents, i.e. compounds having the ability to kill mammalian cells with a molecular weight of less than 700 Daltons. Such compounds may also contain toxic metals that can have cytotoxic effects.
  • these small molecule cytotoxic agents also include prodrugs, i.e. compounds that decay or are converted to release cytotoxic agents under physiological conditions.
  • agents include cisplatin, maytansine derivatives, rachlombycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, pyroximate sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronide, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells; for example, ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA e and RNase; radionuclides, i.e.
  • the present disclosure provides a composition comprising a polypeptide molecule, a TCR, a multispecific antigen-binding molecule, a nucleic acid, a vector, a cell, or a conjugate described herein; and a pharmaceutically acceptable carrier or excipient.
  • Excipients can include, for example, fillers, binders, disintegrants, coating agents, adsorbents, antiadhesives, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tension regulators.
  • suitable excipients are selected to prepare compositions of the present invention.
  • Exemplary carriers used in compositions of the present invention include saline, buffered saline, glucose and water.
  • the selection of suitable excipients depends especially on the desired dosage form of the activating agent used, the disease to be treated and the compositions.
  • compositions of the present disclosure may be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration.
  • the processes known to the invention for producing drugs are shown in the 22nd edition of Remington’s Pharmaceutical Sciences (Ed.
  • compositions comprising, for example, host cells or soluble TCRs as described herein are typically provided in liquid form and preferably contain a pharmaceutically acceptable buffer.
  • the composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
  • Preferred examples of the second therapeutic agent include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E (auristatin E), vincristine and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNA enzymes and RNA enzymes; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs
  • the present disclosure provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of a polypeptide molecule, TCR, multispecific antigen-binding molecule, nucleic acid, vector, cell, conjugate, or composition described herein.
  • the disease is selected from cancer, infectious disease, autoimmune disease, and inflammatory disease.
  • the cancer can be any cancer, such as cancers of the hematological system, cancers of the central and peripheral nervous systems, cancers of the lymphoid lineage, cancers of the myeloid lineage, cancers of mesenchymal origin, solid tumors, and the like.
  • cancer examples include, but are not limited to, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ova
  • Infectious diseases are diseases caused by infection with pathogens, including infectious diseases and non-infectious diseases.
  • Pathogens that cause infectious diseases include viruses, bacteria, mycoplasmas, chlamydia, rickettsia, prions, fungi, spirochetes, and parasites.
  • viruses include, but are not limited to, HPV, CMV, HBV, EBV, herpes viruses, human immunodeficiency virus (HIV), influenza viruses, and coronaviruses.
  • autoimmune and inflammatory diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, autoimmune destruction of the pancreatic islets leading to diabetes, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' thyroiditis, toxic diffuse goiter, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), psoriasis, glomerulonephritis, nephrotic syndrome, anti-glomerular basement membrane Disease, membranous nephropathy, systemic sclerosis, polymyositis, myasthenia gravis, psoriasis, pemphigus, vitiligo, autoimmune diseases of the central nervous system, celiac disease, autoimmune gastritis, primary biliary
  • the dosage applied to the subject may vary with the embodiment, the drug used, the method of administration, and the position and subject being treated. However, the dosage should be sufficient to provide a therapeutic response.
  • the clinician can determine the effective amount given to a person or other subject to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as the activity of the active agent and the route of administration.
  • the dosage of the polypeptide molecule, TCR, multispecific antigen-binding molecule, cell, conjugate, or composition of the present invention can be administered to a mammal once or in a series of sub-doses within an appropriate time period, for example, once a day, half a week, weekly, biweekly, half a month, bimonthly, half a year, or annually as needed.
  • a dosage unit containing an effective amount of a polypeptide molecule, TCR, multispecific antigen-binding molecule, cell, conjugate, or composition can be administered in a single daily dose, or the total daily dose can be administered in two, three, four, or more divided doses administered daily as needed.
  • Suitable modes of administration can be selected by a doctor.
  • the route of administration can be parenteral administration, such as by injection, nasal administration, pulmonary administration or transdermal administration.
  • Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.
  • the polypeptide molecule, conjugate or pharmaceutical composition is selected for parenteral delivery, inhalation or delivery through the digestive tract, such as oral administration.
  • the dosage and method of administration can vary according to the weight, age, condition, etc. of the subject, and can be appropriately selected.
  • the method further comprises administering a second therapeutic agent.
  • the polypeptide molecule, TCR, multispecific antigen binding molecule, cell, conjugate, or composition of the present invention is administered before, substantially simultaneously with, or after the administration of the second therapeutic agent.
  • the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. Preferred examples of the second therapeutic agent are as described above.
  • the present disclosure provides a TCR, which comprises a TCR ⁇ chain variable region (TRAV) and a TCR ⁇ chain variable region (TRBV), wherein the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:56 (DSSMTY), SEQ ID NO:57 (IFAFESM) and SEQ ID NO:58 (AGSGGGTDK), respectively, or a functional variant formed by inserting, deleting or substituting one or several amino acids; and/or the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:59 (DFEATT), SEQ ID NO:60 (TDYGSKA) and SEQ ID NO:61 (SAREPGQGPWE), respectively, or a functional variant formed by inserting, deleting or substituting one or several amino acids.
  • TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:56 (DSSMTY), S
  • the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:56 (DSSMTY), SEQ ID NO:57 (IFAFESM) and SEQ ID NO:58 (AGSGGGTDK), respectively; and/or the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:59 (DFEATT), SEQ ID NO:60 (TDYGSKA) and SEQ ID NO:61 (SAREPGQGPWE), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:30, and/or the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:31.
  • the present disclosure provides a TCR, which comprises a TCR ⁇ chain variable region (TRAV) and a TCR ⁇ chain variable region (TRBV), wherein the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:62 (DSSMTY), SEQ ID NO:63 (IFFYQDM) and SEQ ID NO:64 (AGSGGGTDK), respectively, or a functional variant formed by inserting, deleting or substituting one or several amino acids; and/or the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:65 (DFEATT), SEQ ID NO:66 (TDYGSKA) and SEQ ID NO:67 (SAREPGQAWSD), respectively, or a functional variant formed by inserting, deleting or substituting one or several amino acids.
  • TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:62 (DSSMTY), S
  • the TRAV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:62 (DSSMTY), SEQ ID NO:63 (IFFYQDM) and SEQ ID NO:64 (AGSGGGTDK), respectively, and/or the TRBV comprises ⁇ chain CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NO:65 (DFEATT), SEQ ID NO:66 (TDYGSKA) and SEQ ID NO:67 (SAREPGQAWSD), respectively.
  • the TRAV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:32, and the TRBV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:33.
  • the present disclosure provides the use of the TCR ⁇ chain constant region, TCR ⁇ chain constant region, TCR constant region, polypeptide molecule, TCR, multispecific antigen-binding molecule, nucleic acid, vector, cell, conjugate, or composition described herein in the preparation of a medicament for treating or preventing a disease in a subject.
  • the present disclosure provides a TCR ⁇ chain constant region, a TCR ⁇ chain constant region, a TCR constant region, a polypeptide molecule, a TCR, a multispecific antigen-binding molecule, a nucleic acid, a vector, a cell, a conjugate, or a composition described herein for use in treating or preventing a disease in a subject.
  • the present disclosure provides a method for detecting a disease and/or condition caused by a positive antigen in a subject, wherein the method comprises: (i) contacting a sample obtained from the subject with a polypeptide molecule, TCR, multispecific antigen-binding molecule, nucleic acid, vector, cell, conjugate, or composition described herein; and (ii) detecting the presence of a positive antigen in the sample, wherein the presence of the positive antigen indicates a disease and/or condition caused by the positive antigen.
  • the present disclosure provides a method for detecting a disease and/or condition caused by a positive antigen in a subject, wherein the method comprises: administering to the subject an effective amount of a polypeptide molecule, TCR, multispecific antigen-binding molecule, nucleic acid, vector, cell, conjugate, or composition described herein coupled to a detectable marker (e.g., a radionuclide).
  • a detectable marker e.g., a radionuclide
  • the present disclosure provides the use of the polypeptide molecules, TCRs, multispecific antigen-binding molecules, nucleic acids, vectors, cells, conjugates, or compositions of the present disclosure in the preparation of products for detecting (e.g., diagnosing) the presence of a positive antigen in a test sample, e.g., a kit.
  • the present disclosure provides a polypeptide molecule, TCR, multispecific antigen-binding molecule, nucleic acid, vector, cell, conjugate, or composition as described herein, which detects (e.g., diagnoses) a product for the presence of a positive antigen in a test sample.
  • the presence of the positive antigen may indicate a disease and/or condition caused by the positive antigen.
  • the present disclosure provides a kit comprising a polypeptide molecule, TCR, multispecific antigen-binding molecule, nucleic acid, vector, cell, conjugate, or composition described herein.
  • the kit is used to detect (e.g., diagnose) a disease and/or condition caused by a positive antigen in a subject, and comprises a polypeptide molecule, TCR, multispecific antigen-binding molecule, nucleic acid, vector, cell, conjugate, or composition described herein.
  • the disease or disorder is selected from cancer, infectious diseases, autoimmune diseases and inflammatory diseases.
  • the cancer, infectious diseases, autoimmune diseases and inflammatory diseases are as defined above.
  • the positive antigen is an HPV positive antigen, more preferably an HPV E7 positive antigen.
  • the HPV positive condition or condition is selected from HPV infection, HPV precancer and cancer.
  • the cancer is preferably selected from cervical cancer, head and neck cancer, oropharyngeal cancer, esophageal adenocarcinoma, anal cancer, anal canal cancer, rectal cancer, vaginal cancer, vulvar cancer, penile cancer.
  • the positive antigen is a KRAS mutation positive antigen, for example, a G12V mutation or a G12D mutation.
  • the disease and/or condition caused by the KRAS mutation positive antigen is selected from tumors, precancers and cancers caused by KRAS mutations.
  • the cancer is selected from solid tumors and hematological tumors. More preferably, the tumor includes at least one of: pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, lung cancer, melanoma, prostate cancer, bile duct cancer, cervical cancer, bladder cancer, liver cancer and breast cancer.
  • the positive antigen is a MAGE positive antigen, preferably a MAGE-A1 positive antigen.
  • the MAGE-A1 positive cancer is selected from lung cancer, liver cancer (e.g., hepatocellular carcinoma), gastric cancer, melanoma, esophageal cancer, colorectal cancer, cervical cancer, breast cancer, bladder cancer (e.g., bladder urothelial carcinoma), ovarian cancer (e.g., ovarian serous cystadenocarcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), uterine cancer, endometrial cancer, bile duct cancer, prostate cancer, adrenocortical carcinoma, mesothelioma, sarcoma, glioblastoma multiforme, pheochromocytoma, neuroblastoma, retinoblastoma, paraganglioma and
  • the inventors used computer prediction, Seq ID No. 2 and Seq ID No. 15 as parental sequences, introduced single amino acid site mutations in the TCR constant region, combined with wet experiment verification, and finally screened out beneficial mutations that can significantly improve the soluble expression properties of TCR.
  • the amino acid sequences of the wild-type TCR constant region, the parental TCR constant region, and the TCR constant region screened by the present invention are as follows (the mutation sites are numbered according to the IMGT naming rules):
  • Seq ID No.2 parent TRAC, containing N1.2>K, T84>C and FFPSP deletion mutations at positions 120-124 relative to Seq ID No.1
  • Seq ID No.3 Single-site mutation Q6>R based on Seq ID No.2
  • Seq ID No.4 Single-site mutation S21>I based on Seq ID No.2
  • Seq ID No.5 Single-site mutation K83>P based on Seq ID No.2
  • Seq ID No.6 Single site mutation S84.6>D based on Seq ID No.2
  • Seq ID No.7 Single site mutation S84.6>E based on Seq ID No.2
  • Seq ID No.8 Single site mutation S84.6>N based on Seq ID No.2
  • Seq ID No.9 Single site mutation I114>E based on Seq ID No.2
  • Seq ID No.10 Multiple mutations K83>P, S84.6>D based on Seq ID No.2
  • Seq ID No.11 Multiple mutations S21>I, S84.6>D based on Seq ID No.2
  • Seq ID No.12 Multiple mutations S21>I, K83>P based on Seq ID No.2
  • Seq ID No.13 Multiple mutations S21>I, K83>P, S84.6>D based on Seq ID No.2
  • Seq ID No.15 parent TRBC, relative to Seq ID No.14, contains S79>C, C85.1>A, N97>D
  • Seq ID No.16 Single-site mutation S92>P based on Seq ID No.15
  • Seq ID No.17 Single-site mutation S92>E based on Seq ID No.15
  • Seq ID No.18 Single-site mutation S92>D based on Seq ID No.15
  • Seq ID No.19 Single-site mutation S92>L based on Seq ID No.15
  • Seq ID No.20 Single-site mutation S92>N based on Seq ID No.15
  • Seq ID No.21 Single site mutation T94>D based on Seq ID No.15
  • Seq ID No.22 Single site mutation Q107>L based on Seq ID No.15
  • Seq ID No.23 Multiple mutations T94>D, Q107>L based on Seq ID No.15
  • the native state stability and expressibility of a protein are related in principle, although the former is a thermodynamic property and the latter is primarily governed by the folding trajectory. This relationship is due to the fact that the stability of a protein is determined by the free energy difference between the folded state and the misfolded or unfolded state.
  • the misfolded state can be thought of as a trap that prevents the nascent chain from folding to the native state, thereby blocking the folding trajectory and reducing the yield of the native folded protein; misfolded states can also lead to terminal aggregation. Therefore, the free energy difference between the folded and misfolded states is also one of the determinants of expressibility.
  • the energy landscape of a marginally stable target protein includes many misfolded states, which are close in energy to the native state, so there may be a large number of misfolded states.
  • the protein homeostasis network blocks the occurrence and accumulation of these misfolded states, but misfolded states may limit the protein's ability to be expressed in heterologous hosts and limit the protein's lifespan in vitro.
  • the goal of stability design is to increase the energy gap between the intrinsic folded state and the misfolded or unfolded state.
  • the energy gap can be increased by lowering the energy of the correctly folded state, which is also beneficial for eliminating as many misfolded states as possible, keeping the remaining misfolded states at a high energy position relative to the new correctly folded state, thereby improving the expressibility of the protein.
  • Protein as an organic macromolecular compound, exists in a dispersed state (colloidal state) in water. Therefore, there is no strict solubility of protein in water.
  • the solubility of protein is just the dispersion amount or dispersion level of protein in water.
  • the solubility of protein is affected by some conditions such as pH value, ionic strength, temperature, solvent type, etc., but it is ultimately determined by the amino acid sequence of the protein. Optimizing the amino acid sequence, reducing the aggregation of protein molecules, and increasing the dispersibility of protein molecules in the solvent system is another important means to increase protein production.
  • the inventor loaded the gp100-TCR sequence onto the TCR fusion protein structure independently developed by the inventor (format 62, B62#) for mutation prediction, protein expression of the mutated sequence, and protein activity verification.
  • the overall experimental process is shown in Figure 3.
  • Second polypeptide chain TRAV-TRAC-hinge region-CH2-CH3Hole
  • amino acid sequences of each component are as follows:
  • TRBC SEQ ID NO: 15
  • AlphaFold2 has shown strong applicability in the prediction of the three-dimensional structure of various proteins, especially in the prediction of antibody structure, and the computer-predicted structure is highly consistent with the wet experimental structure.
  • the inventors successfully realized the three-dimensional structure prediction of TCR and TCR fusion protein through this algorithm, and the predicted three-dimensional structure diagrams are shown in Figures 4 and 5.
  • ISPRED4 is a web server that predicts protein-protein interaction sites (PPI) from protein structure. After machine deep learning training, ISPRED4 can extract several features from protein sequence and structure, predict the interaction state of each residue on the protein surface, and predict whether each amino acid is located on the surface of the protein molecule, at the interface, or buried in the protein core.
  • PPI prediction results of the A chain of the exemplary TCR fusion protein gp100-B62 are shown in Figure 6.
  • TCR fusion protein gp100-B62 double chain Based on the prediction results of the TCR fusion protein gp100-B62 double chain, a total of 130 amino acid sites were screened out. These sites are either located in the PPI region or on the surface of the protein molecule but are less hydrophilic. We believe that mutating these sites is more likely to improve the expression properties of the TCR fusion protein.
  • the protein was transiently expressed in a 12-well plate of CHO-K1 cells.
  • the supernatant collected on the 7th day of expression was gradiently diluted and the activity of the protein in the supernatant was detected by the ELISPOT method.
  • protein activity was used as an indicator to evaluate the comprehensive gain effect of amino acid mutations on the activity and expression of TCR fusion proteins. The specific operation is as follows.
  • CHO-K1 cells were plated in a 12-well plate and placed in a carbon dioxide constant temperature incubator and cultured at 37°C and 6% CO2.
  • target cells T2 with a live cell density of 1x10 6 cells/mL were suspended in a centrifuge tube, and the corresponding concentration of epitope peptides was added. The cells were incubated at 37°C for 2 hours, and the mixture was mixed by flicking every 20 minutes. The effector cells PBMC were resuspended in 10% FBS 1640 medium and the cell density was adjusted to 1 ⁇ 10 6 cells/mL. According to the experimental design, the corresponding number and type of effector cells, target cells and protein drugs were added to the ELISPOT wells and cultured in a 37°C, 5% CO2 incubator for 16-20 hours. The medium and cells in the wells were discarded and the plate was washed with PBS.
  • the plate was incubated at 37°C for 2 hours with biotin-labeled antibodies and then washed. TMB colorimetric solution was added to the wells for color development. When obvious spots were observed, the color development was stopped and the ELISPOT spot reading was performed.
  • TCR constant region single-site mutants that can significantly improve the in vitro activity of TCR fusion proteins were screened. These 14 significantly beneficial mutants are evenly distributed in TRAC and TRBC, including 7 amino acid mutations located at Q6R, S21I, K83P, S84.6D, S84.6E, S84.6N, and I114E in TRAC and 7 amino acid mutations located at S92P, S92E, S92D, S92L, S92N, T94D, and Q107L sites in TRBC. Their ELISPOT results are shown in Figure 7.
  • the ELISPOT results in Figure 7 show that the supernatant of the mutant experimental group exhibited significantly stronger PBMC activation activity than that of the wild-type control group under 20-fold dilution conditions. This increase in activity may be due to gains in protein production and activity.
  • TCR Engager structure B62#
  • KRAS G12V-specific TCR CR-KVA11-N03
  • the nucleotide sequence encoding the above CR-KVA11-N03-B62 molecule was cloned into an expression vector, The obtained vector clones were confirmed by sequencing and then amplified and cultured, and plasmids were extracted. The plasmids were then used to transfect CHO cells, cultured for 6-10 days, and then the culture supernatant was collected. The collected culture supernatant was used for Protein A affinity purification to obtain purified protein. The purified protein was subjected to SDS-PAGE gel electrophoresis after denaturation (R) or non-denaturation (N) treatment to evaluate the protein expression purity and aggregation degree.
  • R denaturation
  • N non-denaturation
  • the SDS-PAGE results are shown in Figure 11.
  • the results show that CR-KVA11-N03-B62 (WT) without mutations cannot be expressed and assembled into effective molecules; although the V region mutant molecules scored higher in the algorithm prediction, the V region mutation combinations VMM1, VMM2 and VMM3 did not significantly improve the expression of CR-KVA11-N03-B62 molecules; the introduction of the constant region mutation combination CMM1 successfully achieved the soluble expression of CR-KVA11-N03-B62 molecules, and the SDS-PAGE results showed that the molecular size was consistent with expectations.
  • amino acid sequences of the above five TCRs are as follows:
  • CR-KVA11-N03 contains TRAV shown in SEQ ID NO.24 and TRBV shown in SEQ ID NO.25
  • the B62-LJH2 structure has the following two polypeptide chains:
  • Second polypeptide chain TRAV-TRAC-hinge region-CH2-CH3.
  • Protein expression and SDS-PAGE were performed as described in Example 3.2.2, and the protein yield was calculated based on the SDS-PAGE results. The results are shown in FIG12 .
  • CMM1 mutations can broadly increase the soluble expression yields of a variety of different TCRs and can be applied to a variety of different TCR fusion proteins.
  • TCR constant region is far away from the CDR region and the TCR-pMHC interaction interface from a structural point of view, it cannot be ruled out that local conformational changes in the constant region will indirectly have a distal effect on the TCR-pMHC interaction interface.
  • the B62 structures (respectively named A1A2-M01-625_B62, KDA11-N02_B62, KVA11-N04_B62) loaded with TCRs targeting different antigen epitopes (A1A2-M01-625 (whose amino acid sequence is shown in Example 3.2.3), KDA11-N02, KVA11-N04 (whose amino acid sequence is shown in Example 3.2.3)) and the B62-BJH2 structure (respectively named A1A2-M01-648-LJH2_B62) loaded with the MAGE-A1-specific TCR A1A2-M01-648 (whose amino acid sequence is shown in Example 3.2.3) were used as vectors to verify whether the CMM1 mutation would have an adverse effect on the protein activity.
  • TCR-B62 protein molecules can stimulate PBMCs to produce specific activation responses in vitro in the presence of their unique pMHC epitopes; when pMHC epitopes are absent, PBMCs cannot be activated under appropriate protein concentration conditions, but PBMCs are easily activated non-specifically at higher protein concentrations (e.g., -7M). Therefore, CMM1 mutations were introduced into the molecular structures of these TCR-B62 proteins.
  • the parental TCR constant region was used as a wild-type control.
  • amino acid sequence of KDA11-N02 is as follows:

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Abstract

提供了经修饰的TCR恒定区及其在TCR融合蛋白中的用途，并具体涉及经修饰的TCRα链恒定区、TCRβ链恒定区、TCR恒定区，以及包含其的多肽分子、TCR和多特异性抗原结合分子。使用多肽分子、TCR或多特异性抗原结合分子治疗疾病的方法。

Description

经修饰的TCR恒定区及其在TCR融合蛋白中的用途
本申请要求于2023年6月21日提交的国际专利申请PCT/CN2023/101881、2023年6月25日提交的国际专利申请PCT/CN2023/102211以及2023年12月27日提交的中国专利申请202311826282.6的优先权，其全部内容通过引用并入本文。
技术领域
本公开涉及经修饰的T细胞受体(TCR)恒定区及其在基于TCR的多特异性抗原结合分子中的用途，特别是该多肽分子在治疗癌症、感染性疾病、自身免疫病和/或炎性疾病中的用途。
背景技术
众所周知，人体内的免疫系统由“三大防线”构成，分别是由皮肤和黏膜及其分泌物构成的第一道防线；体液中的杀菌物质(如溶菌酶)和吞噬细胞构成的第二道防线；由免疫器官和免疫细胞(T细胞、B细胞)构成的第三道防线。前两者统称为固有免疫系统，第三者称之为适应性免疫系统。固有免疫的主要功能是抗感染，是机体防御病原微生物入侵的第一道防线，它的特点是先天获得、即时起效、无特异性、无记忆性。而适应性免疫系统是人类适应生存环境、接触抗原物质后产生的具有针对性的、进化水平上更高级的免疫功能，它的特点是后天获得、反应滞后、特异性、记忆性。已经利用适用性免疫反应的特异性、记忆性特点，开发了大量生物医药产品用于病毒感染、肿瘤、自身免疫疾病的治疗，收获了巨大的临床获益。这些产品主要是通过模拟B细胞免疫反应产物(抗体)在体内发挥免疫功能的机制来实现治疗目的，例如单克隆抗体、双特异性抗体、多特异性抗体、抗体偶联药物、CAT-T/CAR-NK等产品。
然而抗体只能识别靶细胞膜表面展示的蛋白(约占总蛋白数量的10％)，这意味着仍有超过90％的疾病无法使用抗体及抗体衍生产品并从中获益，而从T细胞免疫衍生而来的TCR相关产品则可以完美地补缺。TCR识别主要组织相容性复合体(MHC)-抗原肽复合物(pMHC)，是T细胞介导免疫反应的重要步骤。TCR可识别细胞内和细胞表面肿瘤特异抗原经加工后呈递在细胞表面MHC分子的抗原肽，识别范围更加广泛。基于TCR的T细胞重定位双特异性抗体中的靶细胞识别区从传统抗体换成了TCR，这样就可以利用TCR的广泛识别特性来增加靶点的选择性。近年来大量研究把注意力聚焦在晚期、末期肿瘤治疗产品TCR-T的开发上，大量的体外、体内研究数据证明该类产品在肿瘤治疗方面的有效性，国内外均有多个产品已经推进到临床试验阶段。由于自体TCR-T的工艺限制，TCR-T治疗费用高昂、产品制备周期长、制备成功率低、难以复用等等因素极大地限制了该类产品的广泛应用。
通过将TCR以可溶分子的形式与CD3抗体融合表达而开发出的一款T细胞重定位分子(ImmTAC)，既能充分发挥TCR广泛识别大量靶点的优势，还兼具了抗体药物边际成本低的优势，其作用机制如图1所示。基于该种结构第一款临床药物已经获批上市，临床试验表现安全有效，治疗成本也极低于过继治疗产品(TCR-T)。由于天然TCR是跨膜蛋白分子，它并不具有像抗体一样优秀的可溶表达能力，于是TCR可溶表达稳定性的改造成为亟待解决的首要问题。
科学家们最初想通过ScFv的形式将TCR的Va-Vb进行单链融合表达，结果发现大部分的TCR都很难成功表达，推测是由于Va-Ca、Vb-Cb交界面疏水氨基酸残基暴露导致的。于是通过对这些氨基酸进行突变，筛选出可以实现可溶表达的突变ScTv分子，但是这些突变是TCR特异性的，对不同的TCR并不具有通用性。后来通过在截短的TCR恒定区内引入新的二硫键从而提高了TCR可溶表达成功率及产量，但对于基础表达水平比较差的TCR来说，仍然无法通过这样的策略成功实现可溶表达。
发明内容
本发明人通过结合计算机预测和湿实验验证，成功筛选出一系列新的能够改善TCR可溶性表达的TCR恒定区突变，特别是突变组合CMM1。该突变组合CMM1能够提高TCR融合蛋白的稳定性和可溶性，增加TCR融合蛋白的可溶表达成功率和产量，增强TCR融合蛋白的活性，改善高浓度TCR融合蛋白条件下对PBMC细胞的非特异性激活，从而改善用药安全窗口。这一发现打破了TCR可溶分子药物开发的一道重要屏障，有望大幅压缩药物开发过程中TCR筛选、TCR可溶表达的成本和周期。
相应地，在第一方面，本公开提供了T细胞受体(TCR)α链恒定区，其中所述α链恒定区在以下一个或多个位置处包含氨基酸取代：Q6、S21、K83、S84.6和I114；其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在一些实施方案中，(1)所述α链恒定区中Q6位置处的氨基酸取代选自Q6R、Q6K、Q6D和Q6E，优选选自Q6R和Q6K；和/或
(2)所述α链恒定区中S21位置处的氨基酸取代选自S21I、S21L、S21V、S21G、S21A和S21P，优选选自S21I、S21L和S21V；和/或
(3)所述α链恒定区中K83位置处的氨基酸取代选自K83P、K83G、K83A、K83I、K83L和K83V，优选选自K83P、K83G和K83A；和/或
(4)所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E、S84.6N、S84.6Q、S84.6K和S84.6R，优选选自S84.6D、S84.6E、S84.6N和S84.6Q；和/或
(5)所述α链恒定区中I114位置处的氨基酸取代选自I114E、I114D、I114K和I114R，优选选自I114E和I114D。
在一些实施方案中，(1)所述α链恒定区中Q6位置处的氨基酸取代为Q6R；和/或
(2)所述α链恒定区中S21位置处的氨基酸取代为S21I；和/或
(3)所述α链恒定区中K83位置处的氨基酸取代为K83P；和/或
(4)所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E和S84.6N；和/或
(5)所述α链恒定区中I114位置处的氨基酸取代为I114E。
在一些实施方案中，所述α链恒定区包含S21、K83和S84.6处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含以下氨基酸取代中的一种或多种：S21I、K83P和S84.6D；优选包含氨基酸取代S21I、K83P和S84.6D中的至少两种；更优选包含氨基酸取代S21I、K83P和S84.6D。
在一些实施方案中，所述α链恒定区包含选自以下(1)-(4)中任一项的氨基酸取代：
(1)S21I、K83P和S84.6D；
(2)K83P和S84.6D；
(3)S21I和S84.6D；
(4)S21I和K83P。
在一些实施方案中，所述α链恒定区还包含以下氨基酸突变中的一种或多种：N1.2K、T84C和120-124位FFPSP缺失突变，优选包含T84C以及可选的N1.2K和/或120-124位FFPSP缺失突变；更优选包含N1.2K、T84C和120-124位FFPSP缺失突变。
在一些实施方案中，所述α链恒定区包含SEQ ID NO:3-13中任一项所示的氨基酸序列或与SEQ ID NO:3-13中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在第二方面，本公开提供了TCRα链恒定区，其包含氨基酸取代S21I、K83P和S84.6D，其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号；优选地，所述TCRα链恒定区包含SEQ ID NO:13所示的氨基酸序列或与SEQ ID NO:13具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在优选的实施方案中，所述TCRα链恒定区包含或组成为SEQ ID NO:13所示的氨基酸序列。
在第三方面，本公开提供了TCRβ链恒定区，其中所述β链恒定区在以下一个或多个位置处包含氨基酸取代：S92、T94和Q107；其中氨基酸位置根据SEQ ID NO:14所示的TCRβ恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在一些实施方案中，(1)所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V、S92Q、S92K和S92R，优选选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V和S92Q；和/或
(2)所述β链恒定区中T94位置处的氨基酸取代为T94D、T94E、T94K和T94R，优选选自T94D和T94E；和/或
(3)所述β链恒定区中Q107位置处的氨基酸取代为Q107L、Q107I、Q107V、Q107G、Q107A和Q107P，优选选自Q107L、Q107I和Q107V。
在一些实施方案中，(1)所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L和S92N；和/或
(2)所述β链恒定区中T94位置处的氨基酸取代为T94D；和/或
(3)所述β链恒定区中Q107位置处的氨基酸取代为Q107L。
在一些实施方案中，所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述β链恒定区包含以下氨基酸取代中的一种或多种：T94D和Q107L；优选包含氨基酸取代T94D和Q107L。
在一些实施方案中，所述β链恒定区包含选自以下(1)-(3)中任一项的氨基酸取代：
(1)T94D和Q107L；
(2)Q107L；
(3)T94D。
在一些实施方案中，所述TCRβ链恒定区还包含以下氨基酸突变中的一种或多种：S79C、C85.1A和N97D，优选包含S79C以及可选的C85.1A和/或N97D；更优选包含S79C、C85.1A和N97D。
在一些实施方案中，所述TCRβ链恒定区包含SEQ ID NO:16-23中任一项所示的氨基酸序列或与SEQ ID NO:16-23中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在第四方面，本公开提供了TCRβ链恒定区，其包含氨基酸取代T94D和Q107L，其中氨基酸位置根据SEQ ID NO:14所示的TCRβ链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号；优选地，所述TCRβ链恒定区包含SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在优选的实施方案中，所述TCRβ链恒定区包含SEQ ID NO:23所示的氨基酸序列。
在第五方面，本公开提供了TCR恒定区，其包含本文所述的TCRα链恒定区和TCRβ链恒定区。
在一些实施方案中，所述TCR恒定区包含选自以下(1)-(6)中任一项的氨基酸取代：
(1)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的T94D和Q107L；
(2)所述α链恒定区中的K83P和S84.6D以及所述β链恒定区中的T94D和Q107L；
(3)所述α链恒定区中的S21I和S84.6D以及所述β链恒定区中的T94D和Q107L；
(4)所述α链恒定区中的S21I和K83P以及所述β链恒定区中的T94D和Q107L；
(5)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的Q107L；
(6)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的T94D。
在一些实施方案中，所述α链恒定区包含氨基酸取代S21I、K83P和S84.6D，并且所述β链恒定区包含氨基酸取代T94D和Q107L。
在一些实施方案中，所述TCRα链恒定区包含SEQ ID NO:13所示的氨基酸序列或与SEQ ID NO:13具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列；和所述TCRβ链恒定区包含SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在第六方面，本公开提供了多肽分子，其包含本文所述的TCRα链恒定区、TCRβ链恒定区、或TCR恒定区。
在一些实施方案中，所述多肽分子还包含结合至少一个抗原的抗原结合区。
在一些实施方案中，所述抗原结合区选自来源于抗体的抗原结合区和来源于TCR的抗原结合区。
在第七方面，本公开提供了T细胞受体(TCR)，所述TCR包含α链和β链，其中所述α链包含本文所述的TCRα链恒定区，和/或所述β链包含本文所述的TCRβ链恒定区；或者所述TCR包含本文所述的TCR恒定区。
在一些实施方案中，所述TCR结合选自以下的抗原：黑色素瘤相关抗原(例如gp100、MAGEA1)、HPV抗原(例如HPV E6或E7)和KRAS抗原(例如KRAS G12V、KRAS G12D)。
在一些实施方案中，所述TCR包含来源于鼠TCR或人TCR的可变区。
在一些实施方案中，所述TCR是分离或纯化的。
在第八方面，本公开提供了T细胞受体(TCR)，所述TCR包含α链和β链，
其中所述α链的恒定区在以下一个或多个位置处包含氨基酸取代：Q6、S21、K83、S84.6和I114；和/或
其中所述β链的恒定区在以下一个或多个位置处包含氨基酸取代：S92、T94和Q107；
其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区和SEQ ID NO:14所示的TCRβ恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在一些实施方案中，(1)所述α链的恒定区中Q6位置处的氨基酸取代选自Q6R、Q6K、Q6D和Q6E，优选选自Q6R和Q6K；和/或
(2)所述α链的恒定区中S21位置处的氨基酸取代选自S21I、S21L、S21V、S21G、S21A和S21P，优选选自S21I、S21L和S21V；和/或
(3)所述α链的恒定区中K83位置处的氨基酸取代选自K83P、K83G、K83A、K83I、K83L和K83V，优选选自K83P、K83G和K83A；和/或
(4)所述α链的恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E、S84.6N、S84.6Q、S84.6K和S84.6R，优选选自S84.6D、S84.6E、S84.6N和S84.6Q；和/或
(5)所述α链的恒定区中I114位置处的氨基酸取代为I114E、I114D、I114K和I114R，优选选自I114E和I114D；和/或
(6)所述β链的恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V、S92Q、S92K和S92R，优选选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V和S92Q；和/或
(7)所述β链的恒定区中T94位置处的氨基酸取代为T94D、T94E、T94K和T94R，优选选自T94D和T94E；和/或
(8)所述β链的恒定区中Q107位置处的氨基酸取代为Q107L、Q107I、Q107V、Q107G、Q107A和Q107P，优选选自Q107L、Q107I和Q107V。
在一些实施方案中，(1)所述α链的恒定区中Q6位置处的氨基酸取代为Q6R；和/或
(2)所述α链的恒定区中S21位置处的氨基酸取代为S21I；和/或
(3)所述α链的恒定区中K83位置处的氨基酸取代为K83P；和/或
(4)所述α链的恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E和S84.6N；和/或
(5)所述α链的恒定区中I114位置处的氨基酸取代为I114E；和/或
(6)所述β链的恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L和S92N；和/或
(7)所述β链的恒定区中T94位置处的氨基酸取代为T94D；和/或
(8)所述β链的恒定区中Q107位置处的氨基酸取代为Q107L。
在一些实施方案中，所述α链的恒定区包含S21、K83和S84.6处的氨基酸取代，和/或所述β链的恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链的恒定区包含以下氨基酸取代中的一种或多种：S21I、K83P和S84.6D；优选包含氨基酸取代S21I、K83P和S84.6D中的至少两种；更优选包含氨基酸取代S21I、K83P和S84.6D。
在一些实施方案中，所述β链的恒定区包含以下氨基酸取代中的一种或多种：T94D和Q107L；优选包含氨基酸取代T94D和Q107L。
在一些实施方案中，所述TCR包含选自以下(1)-(6)中任一项的氨基酸取代：
(1)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的T94D和Q107L；和/或
(2)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的T94D和Q107L；和/或
(3)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的T94D和Q107L；和/或
(4)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的T94D和Q107L；和/或
(5)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的Q107L；和/或
(6)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的T94D；
优选地，所述α链的恒定区包含氨基酸取代S21I、K83P和S84.6D，并且所述β链的恒定区包含氨基酸取代T94D和Q107L。
在一些实施方案中，所述α链的恒定区还包含以下氨基酸突变中的一种或多种：N1.2K、T84C和120-124位FFPSP缺失突变；和/或所述β链的恒定区还包含以下氨基酸突变中的一种或多种：S79C、C85.1A和N97D；优选地，所述α链的恒定区还包含N1.2K、T84C和120-124位FFPSP缺失突变，并且所述β链的恒定区还包含S79C、C85.1A和N97D。
在一些实施方案中，所述TCR包含具有SEQ ID NO:3-13中任一项所示的氨基酸序列或与SEQ ID NO:3-13中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列的α链恒定区，和/或具有SEQ ID NO:16-23中任一项所示的氨基酸序列或与SEQ ID NO:16-23中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列的β链恒定区。
在一些实施方案中，所述TCR结合选自以下的抗原：黑色素瘤相关抗原(例如gp100、MAGEA1)、HPV抗原(例如HPV E6或E7)和KRAS抗原(例如KRAS G12V、KRAS G12D)。
在一些实施方案中，所述TCR不包含氨基酸序列如SEQ ID NO:24所示的TRAV并且不包含氨基酸序列如SEQ ID NO:25所示的TRBV。在一些实施方案中，所述TCR不包含具有氨基酸序列分别如SEQ ID NO:38-40所示的CDR1、CDR2和CDR3的TRAV并且不包含具有氨基酸序列分别如SEQ ID NO:41-43所示的CDR1、CDR2和CDR3的TRBV。在一些实施方案中，所述TCR不结合KRAS抗原表位VVGAVGVGK或其与HLA-A*11的复合物。在一些实施方案中，所述TCR不结合KRAS抗原。
在一些实施方案中，所述TCR包含来源于鼠TCR或人TCR的可变区。
在一些实施方案中，所述TCR是分离或纯化的。
在第九方面，本公开提供了多特异性抗原结合分子，其本文所述的TCRα链恒定区、TCRβ链恒定区、TCR恒定区、多肽分子、或TCR。
第十方面，本公开提供了多特异性抗原结合分子，其包含至少一个来源于TCR的抗原结合区以及：(1)本文所述的TCRα链恒定区(TRAC)和/或本文所述的TCRβ链恒定区(TRBC)；或者(2)本文所述的TCR恒定区。
在第十一方面，本公开提供了多特异性抗原结合分子，其包含至少一个来源于TCR的抗原结合区和来源于TCR的α链恒定区(TRAC)和/或β链恒定区(TRBC)；
其中所述α链恒定区在以下一个或多个位置处包含氨基酸取代：Q6、S21、K83、S84.6和I114；和/或
其中所述β链恒定区在以下一个或多个位置处包含氨基酸取代：S92、T94和Q107；
其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区和SEQ ID NO:14所示的TCRβ链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在一些实施方案中，(1)所述α链恒定区中Q6位置处的氨基酸取代选自Q6R、Q6K、Q6D和Q6E，优选选自Q6R和Q6K；和/或
(2)所述α链恒定区中S21位置处的氨基酸取代选自S21I、S21L、S21V、S21G、S21A和S21P，优选选自S21I、S21L和S21V；和/或
(3)所述α链恒定区中K83位置处的氨基酸取代选自K83P、K83G、K83A、K83I、K83L和K83V，优选选自K83P、K83G和K83A；和/或
(4)所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E、S84.6N、S84.6Q、S84.6K和S84.6R，优选选自S84.6D、S84.6E、S84.6N和S84.6Q；和/或
(5)所述α链恒定区中I114位置处的氨基酸取代为I114E、I114D、I114K和I114R，优选选自I114E和I114D；和/或
(6)所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V、S92Q、S92K和S92R，优选选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V和S92Q；和/或
(7)所述β链恒定区中T94位置处的氨基酸取代为T94D、T94E、T94K和T94R，优选选自T94D和T94E；和/或
(8)所述β链恒定区中Q107位置处的氨基酸取代为Q107L、Q107I、Q107V、Q107G、Q107A和Q107P，优选选自Q107L、Q107I和Q107V。
在一些实施方案中，(1)所述α链恒定区中Q6位置处的氨基酸取代为Q6R；和/或
(2)所述α链恒定区中S21位置处的氨基酸取代为S21I；和/或
(3)所述α链恒定区中K83位置处的氨基酸取代为K83P；和/或
(4)所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E和S84.6N；和/或
(5)所述α链恒定区中I114位置处的氨基酸取代为I114E；和/或
(6)所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L和S92N；和/或
(7)所述β链恒定区中T94位置处的氨基酸取代为T94D；和/或
(8)所述β链恒定区中Q107位置处的氨基酸取代为Q107L。
在一些实施方案中，所述α链恒定区包含S21、K83和S84.6处的氨基酸取代，和/或所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含以下氨基酸取代中的一种或多种：S21I、K83P和S84.6D；优选包含氨基酸取代S21I、K83P和S84.6D中的至少两种；更优选包含氨基酸取代S21I、K83P和S84.6D。
在一些实施方案中，所述β链恒定区包含以下氨基酸取代中的一种或多种：T94D和Q107L；优选包含氨基酸取代T94D和Q107L。
在一些实施方案中，所述多特异性抗原结合分子包含选自以下(1)-(6)中任一项的氨基酸取代：
(1)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的T94D和Q107L；
(2)所述α链恒定区中的K83P和S84.6D以及所述β链恒定区中的T94D和Q107L；
(3)所述α链恒定区中的S21I和S84.6D以及所述β链恒定区中的T94D和Q107L；
(4)所述α链恒定区中的S21I和K83P以及所述β链恒定区中的T94D和Q107L；
(5)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的Q107L；
(6)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的T94D；
优选地，所述α链恒定区包含氨基酸取代S21I、K83P和S84.6D，并且所述β链恒定区包含氨基酸取代T94D和Q107L。
在一些实施方案中，所述α链恒定区还包含以下氨基酸突变中的一种或多种：N1.2K、T84C和120-124位FFPSP缺失突变；和/或所述β链恒定区还包含以下氨基酸突变中的一种或多种：S79C、C85.1A和N97D；优选地，所述α链的恒定区还包含N1.2K、T84C和120-124位FFPSP缺失突变，并且所述β链的恒定区还包含S79C、C85.1A和N97D。
在一些实施方案中，所述α链恒定区包含SEQ ID NO:3-13中任一项所示的氨基酸序列或与SEQ ID NO:3-13中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列，和/或所述β链恒定区包含SEQ ID NO:16-23中任一项所示的氨基酸序列或与SEQ ID NO:16-23中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在一些实施方案中，所述多特异性抗原结合分子包含结合两个或更多个抗原的两个或更多个抗原结合区，并且所述两个或更多个抗原各自独立地选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原、自身抗原、和免疫细胞表面分子；
优选地，所述两个或更多个抗原中的一个选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原、自身抗原；另一个为免疫细胞表面分子；
更优选地，所述TAA为黑色素瘤相关抗原(例如gp100、MAGEA1)；和/或所述TSA为KRAS抗原(例如KRAS G12V、KRAS G12D)；和/或所述病毒抗原为HPV抗原(例如HPV E6或E7)；和/或所述免疫细胞表面分子选自CD3、CD28、4-1BB(CD137)、PD-1和PD-L1。
在一些实施方案中，所述多特异性抗原结合分子不包含氨基酸序列如SEQ ID NO:24所示的TRAV并且不包含氨基酸序列如SEQ ID NO:25所示的TRBV。在一些实施方案中，所述多特异性抗原结合分子不包含具有氨基酸序列分别如SEQ ID NO:38-40所示的CDR1、CDR2和CDR3的TRAV并且不包含具有氨基酸序列分别如SEQ ID NO:41-43所示的CDR1、CDR2和CDR3的TRBV。在一些实施方案中，所述多特异性抗原结合分子不结合KRAS抗原表位VVGAVGVGK或其与HLA-A*11的复合物。在一些实施方案中，所述多特异性抗原结合分子不结合KRAS抗原。
在一些实施方案中，所述多特异性抗原结合分子不结合gp100抗原。
在一些实施方案中，所述多特异性抗原结合分子在至少两条多肽链上包含结合第一抗原的第一结合区和结合第二抗原的第二结合区，
其中所述第一结合区包含来源于与所述第一抗原-MHC复合物结合的TCR的α链可变区(TRAV)和β链可变区(TRBV)；
其中所述第二结合区包含来源于与所述第二抗原结合的抗体的重链可变区(VH)和轻链可变区(VL)；
其中所述第一抗原选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原，且所述第二抗原为免疫细胞表面分子；
其中所述多特异性抗原结合分子还包含以下至少一种功能结构域：
1)源于抗体的铰链区，
2)源于抗体的Fc结构域或其二聚化部分；
3)源于抗体的CH3结构域；
4)白蛋白(Alb)或其结合部分；
5)源于TCR的恒定区或其片段。
在一些实施方案中，所述TRAC和所述TRBC分别通过任选的接头连接至所述TRAV和所述TRBV的C端；或所述TRAC和所述TRBC分别通过任选的接头连接至所述TRBV和所述TRAV的C端。
在一些实施方案中，本公开的TRAC或TRBC不与所述VH或VL的C端连接，例如，通过接头的形式或者不通过接头的形式。
在一些实施方案中，所述抗体的N端连接在所述TCR的C端。
在一些实施方案中，所述功能结构域来源于抗体的铰链区和/或抗体的Fc结构域或其二聚化部分和/或抗体的CH3结构域和/或源于TCR的恒定区或其片段；并且所述多特异性抗原结合分子的任意两条多肽链通过所述功能结构域两条链之间的共价结合或非共价结合而连接；
优选地，所述功能结构域的两条链通过任选的接头分别连接在所述多特异性抗原结合分子的任意两条多肽链的C末端或N末端，优选C末端；或
所述第一结合区和所述第二结合区通过所述功能结构域两条链之间的共价结合或非共价结合而连接；
更优选地，所述多特异性抗原结合分子包含第一多肽链和第二多肽链，其中
所述第一多肽链从N端到C端包含：TRBV-任选的接头-TRBC-接头-VL-接头-VH-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
所述第一多肽链从N端到C端包含：TRAV-任选的接头-TRAC-接头-VL-接头-VH-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRBV-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3；或
所述第一多肽链从N端到C端包含：TRBV-任选的接头-TRBC-接头-VH-接头-VL-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
所述第一多肽链从N端到C端包含：TRAV-任选的接头-TRBC-接头-VH-接头-VL-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRBV-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3；或
所述多特异性抗原结合分子包含第一多肽链、第二多肽链和第三多肽链，其中所述第一多肽链从N端到C端包含：TRBV-任选的接头-TRBC-接头-VL，所述第二多肽链从N端到C端包含：VH-任选的接头-铰链区-CH2-CH3，并且所述第三多肽链从N端到C端包含：TRAV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。
在一些实施方案中，所述多特异性抗原结合分子不包含如SEQ ID NO:92和93所示的氨基酸序列。
在一些实施方案中，所述多特异性抗原结合分子不包含以下第一多肽链和第二多肽链：所述第一多肽链从N端到C端包含：TRAV-接头-VH-接头-TRAC，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-TRBC。
在第十二方面，本公开提供了核酸，其编码本文所述的TCRα链恒定区、TCRβ链恒定区、TCR恒定区、多肽分子、TCR、或多特异性抗原结合分子。
在第十三方面，本公开提供了载体，其包含本文所述的核酸。
在一些实施方案中，所述载体选自慢病毒载体、逆转录病毒载体、质粒、DNA载体、mRNA载体、基于转座子的载体和人工染色体。
在第十四方面，本公开提供了宿主细胞，其包含本文所述的TCRα链恒定区、TCRβ链恒定区、TCR恒定区、多肽分子、TCR、多特异性抗原结合分子、核酸、或载体。
在第十五方面，本公开提供了缀合物，其包含本文所述的多肽分子、TCR或多特异性抗原结合分子，以及与所述多肽分子、所述TCR或所述多特异性抗原结合分子缀合的化学部分。
在一些实施方案中，所述化学部分选自可检测标记、免疫刺激分子和治疗剂；
优选地，所述可检测标记选自生物素、链霉抗生物素蛋白、酶或其催化活性片段、放射性核素、纳米颗粒、顺磁性金属离子、核酸探针、造影剂、和萤光、磷光或化学发光分子；
优选地，所述免疫刺激分子选自细胞因子、趋化因子、血小板因子和补体启动剂；
优选地，所述治疗剂选自免疫调节剂、放射性化合物、酶、化学治疗剂和毒素。
在第十六方面，本公开提供了组合物，其包含本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、或缀合物；以及药学上可接受的载体或赋形剂。
在一些实施方案中，所述组合物还包含第二治疗剂，优选地，所述第二治疗剂选自抗体、化疗剂和小分子药物。
在第十七方面，本公开提供了治疗受试者的疾病的方法，其包括向所述受试者施用有效量的本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、宿主细胞、缀合物、或组合物。
在一些实施方案中，所述疾病选自癌症、感染性疾病、自身免疫病和炎性疾病。在一些实施方案中，所述方法还包括施用第二治疗剂，优选地，所述第二治疗剂选自抗体、化疗剂和小分子药物。
在第十八方面，本公开提供了TCR，其包含TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)，其中所述TRAV包含分别具有如SEQ ID NO:56(DSSMTY)、SEQ ID NO:57(IFAFESM)和SEQ ID NO:58(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3，或者插入、缺失或取代一个或几个氨基酸所形成的功能变体；和/或所述TRBV包含分别具有如SEQ ID NO:59(DFEATT)、SEQ ID NO:60(TDYGSKA)和SEQ ID NO:61(SAREPGQGPWE)所示的氨基酸序列的β链CDR1、CDR2和CDR3，或者插入、缺失或取代一个或几个氨基酸所形成的功能变体。
在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:56(DSSMTY)、SEQ ID NO:57(IFAFESM)和SEQ ID NO:58(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3；和/或所述TRBV包含分别具有如SEQ ID NO:59(DFEATT)、SEQ ID NO:60(TDYGSKA)和SEQ ID NO:61(SAREPGQGPWE)所示的氨基酸序列的β链CDR1、CDR2和CDR3。
在一些实施方案中，所述TRAV包含与SEQ ID NO:30具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，和/或所述TRBV包含与SEQ ID NO:31具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在第十九方面，本公开提供了TCR，其包含TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)，其中所述TRAV包含分别具有如SEQ ID NO:62(DSSMTY)、SEQ ID NO:63(IFFYQDM)和SEQ ID NO:64(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3，或者插入、缺失或取代一个或几个氨基酸所形成的功能变体；和/或所述TRBV包含分别具有如SEQ ID NO:65(DFEATT)、SEQ ID NO:66(TDYGSKA)和SEQ ID NO:67(SAREPGQAWSD)所示的氨基酸序列的β链CDR1、CDR2和CDR3，或者插入、缺失或取代一个或几个氨基酸所形成的功能变体。
在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:62(DSSMTY)、SEQ ID NO:63(IFFYQDM)和SEQ ID NO:64(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3，和/或所述TRBV包含分别具有如SEQ ID NO:65(DFEATT)、SEQ ID NO:66(TDYGSKA)和SEQ ID NO:67(SAREPGQAWSD)所示的氨基酸序列的β链CDR1、CDR2和CDR3。
在一些实施方案中，所述TRAV包含与SEQ ID NO:32具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:33具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
附图说明
图1显示了TCR-CD3抗体融合蛋白作用机制。
图2显示了蛋白稳定性设计原理。
图3显示了TCR融合蛋白可溶表达优化流程。
图4显示了通过AlphaFold2预测的TCR三维结构图。
图5显示了通过AlphaFold2预测的TCR融合蛋白gp100-B60的三维结构图。
图6显示了通过ISPRED4预测的TCR融合蛋白gp100-B60A链的蛋白质-蛋白质相互作用位点(PPI)。
图7显示了TCR恒定区单位点突变体应用于format 62的ELISPOT结果。
图8显示了Stable单项打分、Soluble单项打分以及Stable/Soluble双项打分预测的活性增益与ELISPOT实验验证的活性增益的比较。
图9显示了通过流式细胞术检测的CR-KVA11-N03TCR在T细胞表面的表达。
图10显示了通过计算机预测的CR-KVA11-N03TCR的三维结构模型及恒定区中的突变位点。
图11显示了含有不同突变的CR-KVA11-N03-B62分子的SDS-PAGE结果。
图12显示了CMM1突变应用于不同TCR序列和不同TCR融合蛋白结构的SDS-PAGE结果和计算的蛋白产量。
图13显示了CMM1突变应用于不同TCR序列和不同TCR融合蛋白结构的ELISPOT结果。
图14显示了TCR恒定区单位点突变体应用于format 22-2的ELISPOT结果。
图15显示了CMM1突变及其反向突变CMM2、CMM3、CMM4和CMM5应用于format 62的SDS-PAGE结果和计算的蛋白产量。
具体实施方式
除非另有限定，否则本文所使用的全部技术和科学术语的含义与本领域普通技术人员通常所理解的含义相同。例如，本文所使用的术语如Janeway CA Jr，Travers P，Walport M等的《免疫生物学》(Immunobiology)，第五版，New York：GarlandScience(2001)和“A multilingual glossary of biotechnological terms:(IUPAC Recommendations)”,Leuenberger,H.G.W,Nagel,B.和H.编辑(1995),Helvetica Chimica Acta,CH-4010Basel,Switzerland中所述的定义。
应当注意，如本文中及所附权利要求书中使用的，单数形式“一个”、“一种”和“该/所述”包括复数提及物，除非上下文另有明确规定。因此，术语“一个”、“一种”、“一个/种或多个/种”和“至少一个/种”可以互换使用。类似地，术语“包含”、“包括”和“具有”可以互换使用。
在本文中及所附权利要求书中使用术语“包含”时，其不排除其它元素。为了本发明的目的，术语“由...组成”被认为是术语“包含”的优选实施方案。如果在下文中将组定义为包括或包含至少一定数量的实施方案，则还应被理解为公开了优选仅由这些实施方案组成的组。
本发明人通过计算机预测和湿实验验证发现，相对于亲本TCR恒定区在以下一个或多个位置处包含氨基酸取代的本发明的修饰的TCR恒定区能够改善TCR融合蛋白的可溶表达和体外活性：TRAC中的Q6、S21、K83、S84.6和I114；TRBC中的S92、T94和Q107，例如，TRAC中的Q6R、S21I、K83P、S84.6D、S84.6E、S84.6N、I114E，TRBC中的S92P、S92E、S92D、S92L、S92N、T94D、Q107L；其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区和SEQ ID NO:14所示的TCRβ恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。这些单位点突变及其任何组合均可改善TCR融合蛋白的可溶表达和体外活性。
术语“亲本TCR恒定区”、“亲本序列”、“野生型(WT)TCR恒定区”、“野生型(WT)序列”在本文中可互换使用，其中TRAC相对于SEQ ID No.1所示的人TRAC*01包含N1.2K、T84C和120-124位FFPSP缺失突变，其氨基酸序列如SEQ ID NO:2所示；TRBC相对于Seq ID No.14所示的人TRBC2*01包含S79C、C85.1A、N97D，其氨基酸序列如SEQ ID NO:15所示。
另外，这些突变后的氨基酸的保守性替换预期也能改善TCR融合蛋白的可溶表达，这是因为保守性替换的氨基酸在极性、电荷、溶解度、疏水性、亲水性和/或两亲性等性质方面具有相似性。例如，氨基酸的保守取代可参照下表进行，其中，优选选择第一列相同区块中的氨基酸进行保守取代，更优选选择第二列相同行中的氨基酸进行保守取代。
因此，本发明的修饰的TCR恒定区可包含以下氨基酸取代中的一种或多种：Q6R、Q6K、Q6D、Q6E；S21I、S21L、S21V、S21G、S21A、S21P；K83P、K83G、K83A、K83I、K83L、K83V；S84.6D、S84.6E、S84.6N、S84.6Q、S84.6K、S84.6R；I114E、I114D、I114N、I114Q、I114K、I114R；S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V、S92Q、S92K、S92R； T94D、T94E、T94N、T94Q、T94K、T94R；Q107L、Q107I、Q107V、Q107G、Q107A、Q107P。
相应地，在第一方面，本公开提供了T细胞受体(TCR)α链恒定区，其中所述α链恒定区在以下一个或多个位置处包含氨基酸取代：Q6、S21、K83、S84.6和I114；其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在TCR的天然形式中，其作为T细胞表面上的若干蛋白质的复合物存在。T细胞受体由两条(单独的)蛋白质链组成，这些蛋白质链由独立的T细胞受体α和β(TCRα和TCRβ)基因产生，并被称为α链和β链。TCR的每条链具有一个N末端免疫球蛋白样(Ig)-可变(V)区/结构域，一个Ig-恒定(C)区/结构域，将链锚定在质膜中的跨膜/细胞膜跨越区域，和C-末端的短细胞质尾。
通常，TCR是一条α链和一条β链的异二聚体，该异二聚体可以结合呈递抗原肽的MHC分子。
抗原特异性是由α和β链的可变区赋予的。TCRα链和β链的两个可变区(α链可变区(Vα)和β链可变区(Vβ))均包含由框架(FR)区包围的三个高变或互补决定区(CDR1α/β、CDR2α/β和CDR3α/β)。CDR3是抗原识别和特异性(即识别特异性抗原并与之相互作用的能力)的主要决定簇，而CDR1和CDR2主要与呈递抗原肽的MHC分子相互作用。
TCR识别抗原肽，该抗原肽与抗原呈递细胞表面处的主要组织相容性复合体(MHC)分子结合(“在MHC分子上呈递/展示”)。在MHC分子上呈递的抗原肽在本文中也称为“表位与MHC分子的复合物”、“表位-MHC复合物”或“靶抗原肽-MHC复合物”。存在两种不同类别的MHC分子：MHC I和MHC II，其呈递来自不同细胞区室的肽。MHC I类分子在人体所有有核细胞的表面上均有表达，并展示从细胞内区室到细胞毒性T细胞的肽或蛋白质片段。在人类中，MHC也称为人白细胞抗原(HLA)。MHC I类有三种主要类型：HLA-A、HLA-B和HLA-C。一旦TCR结合其特异性表位-MHC复合物，则T细胞被活化并发挥生物效应功能。
TCR恒定区的序列可以在国际免疫遗传学信息系统(IMGT)的公开数据库中找到，如TCR分子α链的恒定区序列为“TRAC*01”，TCR分子β链的恒定域序列为“TRBC1*01”或“TRBC2*01”。
本发明中TCR的氨基酸序列的位置按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。例如，TCRα链恒定区(TRAC)和β链恒定区(TRBC)的氨基酸序列的位置编号，按照IMGT中列出的位置进行编号。例如，TRAC中的某个氨基酸，IMGT中列出的位置编号为84.6，则本发明中将其描述为TRAC第84.6位氨基酸；TRBC中的某个氨基酸，IMGT中列出的位置编号为92，则本发明中将其描述为TRBC第92位氨基酸；其他以此类推。本文中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。
在一些实施方案中，所述α链恒定区中Q6位置处的氨基酸取代选自Q6R、Q6K、Q6D和Q6E，优选选自Q6R和Q6K。
在一些实施方案中，所述α链恒定区中S21位置处的氨基酸取代选自S21I、S21L、S21V、S21G、S21A和S21P，优选选自S21I、S21L和S21V。
在一些实施方案中，所述α链恒定区中K83位置处的氨基酸取代选自K83P、K83G、K83A、K83I、K83L和K83V，优选选自K83P、K83G和K83A。
在一些实施方案中，所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E、S84.6N、S84.6Q、S84.6K和S84.6R，优选选自S84.6D、S84.6E、S84.6N和S84.6Q。
在一些实施方案中，所述α链恒定区中I114位置处的氨基酸取代选自I114E、I114D、I114K和I114R，优选选自I114E和I114D。
在一些实施方案中，所述α链恒定区中Q6位置处的氨基酸取代为Q6R。
在一些实施方案中，所述α链恒定区中S21位置处的氨基酸取代为S21I。
在一些实施方案中，所述α链恒定区中K83位置处的氨基酸取代为K83P。
在一些实施方案中，所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E和S84.6N。
在一些实施方案中，所述α链恒定区中I114位置处的氨基酸取代为I114E。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意两个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意三个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意四个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含以下氨基酸取代中的一种或多种：S21I、K83P和S84.6D；优选包含氨基酸取代S21I、K83P和S84.6D中的至少两种；更优选包含氨基酸取代S21I、K83P和S84.6D。
在一些实施方案中，所述α链恒定区包含选自以下(1)-(4)中任一项的氨基酸取代：
(1)S21I、K83P和S84.6D；
(2)K83P和S84.6D；
(3)S21I和S84.6D；
(4)S21I和K83P。
在一些实施方案中，所述α链恒定区包含S21I、K83P和S84.6D。在一些实施方案中，所述α链恒定区包含S21I和S84.6D。
在一些实施方案中，所述α链恒定区还包含以下氨基酸突变中的一种或多种：N1.2K、T84C和120-124位FFPSP缺失突变。在一些优选的实施方案中，所述α链的恒定区还包含T84C以及可选的N1.2K和/或120-124位FFPSP缺失突变。在一些更优选的实施方案中，所述α链的恒定区还包含N1.2K、T84C和120-124位FFPSP缺失突变。
在一些实施方案中，所述α链恒定区与SEQ ID NO:1具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述α链恒定区与SEQ ID NO:2具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述α链恒定区包含SEQ ID NO:3-13中任一项所示的氨基酸序列或与SEQ ID NO:3-13中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
如本文所用的术语“序列同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性，但是高度保守性较低且具有缺失、添加或替换的序列可具有较低程度的同一性。本领域技术人员将认识到，一些算法可以用于使用标准参数来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197)和ClustalW。
因此，例如SEQ ID NO：3的氨基酸序列可以作为“主体序列”或“参考序列”，而与之不同的TCR恒定区氨基酸序列可作为“查询序列”。
在一些实施方案中，所述α链恒定区包含Q6R。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:3所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S21I。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:4所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含K83P。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:5所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S84.6D。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:6所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S84.6E。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:7所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S84.6N。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:8所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含I114E。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:9所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含K83P和S84.6D。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:10所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S21I和S84.6D。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:11所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S21I和K83P。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:12所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S21I、K83P和S84.6D。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:13所示的氨基酸序列。
在第二方面，本公开提供了TCRα链恒定区，其包含氨基酸取代S21I、K83P和S84.6D，其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在一些优选的实施方案中，所述TCRα链恒定区包含SEQ ID NO:13所示的氨基酸序列或与SEQ ID NO:13具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在进一步优选的实施方案中，所述TCRα链恒定区包含或组成为SEQ ID NO:13所示的氨基酸序列。因此，本公开提供了TCRα链恒定区，其包含或组成为SEQ ID NO:13所示的氨基酸序列。
在第三方面，本公开提供了TCRβ链恒定区，其中所述β链恒定区在以下一个或多个位置处包含氨基酸取代：S92、T94和Q107；其中氨基酸位置根据SEQ ID NO:14所示的TCRβ恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在一些实施方案中，所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V、S92Q、S92K和S92R，优选选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V和S92Q。
在一些实施方案中，所述β链恒定区中T94位置处的氨基酸取代为T94D、T94E、T94K和T94R，优选选自T94D和T94E。
在一些实施方案中，所述β链恒定区中Q107位置处的氨基酸取代为Q107L、Q107I、Q107V、Q107G、Q107A和Q107P，优选选自Q107L、Q107I和Q107V。
在一些实施方案中，所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L和S92N。
在一些实施方案中，所述β链恒定区中T94位置处的氨基酸取代为T94D。
在一些实施方案中，所述β链恒定区中Q107位置处的氨基酸取代为Q107L。
在一些实施方案中，所述β链恒定区包含S92、T94和Q107处的氨基酸取代。在一些实施方案中，所述β链恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述β链恒定区包含S92和T94处的氨基酸取代。在一些实施方案中，所述β链恒定区包含S92和Q107处的氨基酸取代。在一些实施方案中，所述β链恒定区包含S92处的氨基酸取代。在一些实施方案中，所述β链恒定区包含T94处的氨基酸取代。在一些实施方案中，所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述β链恒定区包含以下氨基酸取代中的一种或多种：T94D和Q107L；优选包含氨基酸取代T94D和Q107L。
在一些实施方案中，所述β链恒定区包含选自以下(1)-(3)中任一项的氨基酸取代：
(1)T94D和Q107L；
(2)Q107L；
(3)T94D。
在一些实施方案中，所述β链恒定区包含T94D和Q107L。在一些实施方案中，所述β链恒定区包含T94D。在一些实施方案中，所述β链恒定区包含Q107L。
在一些实施方案中，所述TCRβ链恒定区还包含以下氨基酸突变中的一种或多种：S79C、C85.1A和N97D。在一些优选的实施方案中，所述β链的恒定区还包含S79C以及可选的C85.1A和/或N97D。在一些更优选的实施方案中，所述TCRβ链恒定区还包含S79C、C85.1A和N97D。
在一些实施方案中，所述β链恒定区与SEQ ID NO:14具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述β链恒定区与SEQ ID NO:15具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述TCRβ链恒定区包含SEQ ID NO:16-23中任一项所示的氨基酸序列或与SEQ ID NO:16-23中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在一些实施方案中，所述β链恒定区包含S92P。在优选的实施方案中， 所述β链的恒定区包含SEQ ID NO:16所示的氨基酸序列。
在一些实施方案中，所述β链恒定区包含S92E。在优选的实施方案中，所述β链的恒定区包含SEQ ID NO:17所示的氨基酸序列。
在一些实施方案中，所述β链恒定区包含S92D。在优选的实施方案中，所述β链的恒定区包含SEQ ID NO:18所示的氨基酸序列。
在一些实施方案中，所述β链恒定区包含S92L。在优选的实施方案中，所述β链的恒定区包含SEQ ID NO:19所示的氨基酸序列。
在一些实施方案中，所述β链恒定区包含S92N。在优选的实施方案中，所述β链的恒定区包含SEQ ID NO:20所示的氨基酸序列。
在一些实施方案中，所述β链恒定区包含T94D。在优选的实施方案中，所述β链的恒定区包含SEQ ID NO:21所示的氨基酸序列。
在一些实施方案中，所述β链恒定区包含Q107L。在优选的实施方案中，所述β链的恒定区包含SEQ ID NO:22所示的氨基酸序列。
在一些实施方案中，所述β链恒定区包含T94D和Q107L。在优选的实施方案中，所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在第四方面，本公开提供了TCRβ链恒定区，其包含氨基酸取代T94D和Q107L，其中氨基酸位置根据SEQ ID NO:14所示的TCRβ链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在优选的实施方案中，所述TCRβ链恒定区包含SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在更为优选的实施方案中，所述TCRβ链恒定区包含或组成为SEQ ID NO:23所示的氨基酸序列。因此，本公开提供了TCRβ链恒定区，其包含或组成为SEQ ID NO:23所示的氨基酸序列。
在第五方面，本公开提供了TCR恒定区，其包含本文所述的TCRα链恒定区和TCRβ链恒定区。
能够理解的是，在了解本公开第一方面TCRα链恒定区和第二方面TCRβ链恒定区的基础上，本领域技术人员能够将第一方面公开的各种TCRα链恒定区和第二方面公开的各种TCRβ链恒定区进行组合。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸 取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、 Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链的恒定区包含以下氨基酸取代中的一种或多种：S21I、K83P和S84.6D；优选包含氨基酸取代S21I、K83P和S84.6D中的至少两种；更优选包含氨基酸取代S21I、K83P和S84.6D。在一些实施方案中，所述α链恒定区包含S21I、K83P和S84.6D。在一些实施方案中，所述α链恒定区包含S21I和S84.6D。
在一些实施方案中，所述β链的恒定区包含以下氨基酸取代中的一种或多种：T94D和Q107L；优选包含氨基酸取代T94D和Q107L。在一些实施方案中，所述β链恒定区包含T94D和Q107L。在一些实施方案中，所述β链恒定区包含T94D。在一些实施方案中，所述β链恒定区包含Q107L。
在一些实施方案中，所述TCR包含选自以下(1)-(21)中任一项的氨基酸取代：
(1)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的T94D和Q107L；
(2)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的T94D和Q107L；
(3)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的T94D和Q107L；
(4)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的T94D和Q107L；
(5)所述α链的恒定区中的S21I以及所述β链的恒定区中的T94D和Q107L；
(6)所述α链的恒定区中的K83P以及所述β链的恒定区中的T94D和Q107L；
(7)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的T94D和Q107L；
(8)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的Q107L；
(9)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的T94D；
(10)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的T94D；
(11)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的Q107L；
(12)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的T94D；
(13)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的Q107L；
(14)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的T94D；
(15)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的Q107L；
(16)所述α链的恒定区中的S21I以及所述β链的恒定区中的Q107L；
(17)所述α链的恒定区中的S21I以及所述β链的恒定区中的T94D；
(18)所述α链的恒定区中的K83P以及所述β链的恒定区中的T94D；
(19)所述α链的恒定区中的K83P以及所述β链的恒定区中的Q107L；
(20)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的T94D；
(21)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的Q107L。
在一些实施方案中，所述α链的恒定区还包含以下氨基酸突变中的一种或多种：N1.2K、T84C和120-124位FFPSP缺失突变；所述β链的恒定区还包含以下氨基酸突变中的一种或多种：S79C、C85.1A和N97D。在一些优选的实施方案中，所述α链的恒定区还包含T84C以及可选的N1.2K和/或120-124位FFPSP缺失突变；所述β链的恒定区还包含S79C以及可选的C85.1A和/或N97D。在一些更优选的实施方案中，所述α链的恒定区还包含N1.2K、T84C和120-124位FFPSP缺失突变，并且所述β链的恒定区还包含S79C、C85.1A和N97D。
在一些实施方案中，所述α链的恒定区与SEQ ID NO:1具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性，所述β链的恒定区与SEQ ID NO:14具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述α链的恒定区与SEQ ID NO:2具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性，所述β链的恒定区与SEQ ID NO:15具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述α链恒定区包含具有SEQ ID NO:3-13中任一项所示的氨基酸序列或与SEQ ID NO:3-13中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列，所述β链恒定区具有SEQ ID NO:16-23中任一项所示的氨基酸序列或与SEQ ID NO:16-23中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。在一些优选的实施方式中，所述TCRα链恒定区包含SEQ ID NO:13所示的氨基酸序列或与SEQ ID NO:13具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列；和所述TCRβ链恒定区包含SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含氨基酸取代S21I、K83P和S84.6D，并且所述β链的恒定区包含氨基酸取代T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含K83P和S84.6D，并且所述β链的恒定区包含T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:10所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含S21I和S84.6D，并且所述β链的恒定区包含T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:11所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含S21I和K83P，并且所述β链的恒定区包含T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:12所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含S21I、K83P和S84.6D，并且所述β链的恒定区包含Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:22所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含S21I、K83P和S84.6D，并且所述β链的恒定区包含T94D。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:21所示的氨基酸序列。
在第六方面，本公开提供了多肽分子，其包含本文所述的TCRα链恒定区、TCRβ链恒定区、或TCR恒定区。
在一些实施方案中，所述多肽分子还包含结合至少一个抗原的抗原结合区。
在一些实施方案中，所述抗原结合区选自来源于抗体的抗原结合区和来源于TCR的抗原结合区。在一些实施方案中，所述多肽分子为TCR。在一些实施方案中，所述多肽分子为TCR恒定区与抗体可变区融合的嵌合分子。在一些实施方案中，所述多肽分子为CAR。
在第七方面，本公开提供了T细胞受体(TCR)，所述TCR包含α链和β链，其中所述α链包含本文所述的TCRα链恒定区，和/或所述β链包含本文所述的TCRβ链恒定区。
在一些实施方案中，所述TCR包含α链和β链，并且包含本文所述的TCR恒定区。
在第八方面，本公开提供了T细胞受体(TCR)，所述TCR包含α链和β链，
其中所述α链的恒定区在以下一个或多个位置处包含氨基酸取代：Q6、S21、K83、S84.6和I114；和/或
其中所述β链的恒定区在以下一个或多个位置处包含氨基酸取代：S92、T94和Q107；
其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区和SEQ ID NO:14所示的TCRβ链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在一些实施方案中，所述TCR包含在以下一个或多个位置处具有氨基酸取代的α链的恒定区：Q6、S21、K83、S84.6和I114。在一些实施方案中，所述TCR包含在以下一个或多个位置处具有氨基酸取代的β链的恒定区：S92、T94和Q107。在一些实施方案中，所述TCR包含在以下一个或多个位置处具有氨基酸取代的α链的恒定区：Q6、S21、K83、S84.6和I114，和在以下一个或多个位置处具有氨基酸取代的β链的恒定区：S92、T94和Q107。
在一些实施方案中，所述α链的恒定区中Q6位置处的氨基酸取代选自Q6R、Q6K、Q6D和Q6E，优选选自Q6R和Q6K。
在一些实施方案中，所述α链的恒定区中S21位置处的氨基酸取代选自S21I、S21L、S21V、S21G、S21A和S21P，优选选自S21I、S21L和S21V。
在一些实施方案中，所述α链的恒定区中K83位置处的氨基酸取代选自K83P、K83G、K83A、K83I、K83L和K83V，优选选自K83P、K83G和K83A。
在一些实施方案中，所述α链的恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E、S84.6N、S84.6Q、S84.6K和S84.6R，优选选自S84.6D、S84.6E、S84.6N和S84.6Q。
在一些实施方案中，所述α链的恒定区中I114位置处的氨基酸取代为I114E、I114D、I114K和I114R，优选选自I114E和I114D。
在一些实施方案中，所述β链的恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V、S92Q、S92K和S92R，优选选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V和S92Q。
在一些实施方案中，所述β链的恒定区中T94位置处的氨基酸取代为T94D、T94E、T94K和T94R，优选选自T94D和T94E。
在一些实施方案中，所述β链的恒定区中Q107位置处的氨基酸取代为Q107L、Q107I、Q107V、Q107G、Q107A和Q107P，优选选自Q107L、Q107I和Q107V。
在一些实施方案中，所述α链的恒定区中Q6位置处的氨基酸取代为Q6R。
在一些实施方案中，所述α链的恒定区中S21位置处的氨基酸取代为S21I。
在一些实施方案中，所述α链的恒定区中K83位置处的氨基酸取代为K83P。
在一些实施方案中，所述α链的恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E和S84.6N。
在一些实施方案中，所述α链的恒定区中I114位置处的氨基酸取代为I114E。
在一些实施方案中，所述β链的恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L和S92N。
在一些实施方案中，所述β链的恒定区中T94位置处的氨基酸取代为T94D。
在一些实施方案中，所述β链的恒定区中Q107位置处的氨基酸取代为Q107L。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意两个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意三个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意四个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述β链恒定区包含S92、T94和Q107中的任意一个位置处的氨基酸取代。
在一些实施方案中，所述β链恒定区包含S92、T94和Q107中的任意两个位置处的氨基酸取代。在一些实施方案中，所述β链恒定区包含S92和T94处的氨基酸取代。在一些实施方案中，所述β链恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、 K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代，和/或所述β链恒定区包含S92、T94和Q107处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6、S21、K83、和S84.6处的氨基酸取代，和/或所述β链恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述α链恒定区包含S21、K83、S84.6和I114处的氨基酸取代，和/或所述β链恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述α链的恒定区包含S21、K83和S84.6处的氨基酸取代，和/或所述β链的恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述α链恒定区包含S21和S84.6处的氨基酸取代，和/或所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链的恒定区包含以下氨基酸取代中的一种或多种：S21I、K83P和S84.6D；优选包含氨基酸取代S21I、K83P和S84.6D中的至少两种；更优选包含氨基酸取代S21I、K83P和S84.6D。在一些实施方案中，所述α链恒定区包含S21I、K83P和S84.6D。在一些实施方案中，所述α链恒定区包含S21I和S84.6D。
在一些实施方案中，所述β链的恒定区包含以下氨基酸取代中的一种或多种：T94D和Q107L；优选包含氨基酸取代T94D和Q107L。在一些实施方案中，所述β链恒定区包含T94D和Q107L。在一些实施方案中，所述β链恒定区包含T94D。在一些实施方案中，所述β链恒定区包含Q107L。
在一些实施方案中，所述TCR包含选自以下(1)-(21)中任一项的氨基酸取代：
(1)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的T94D和Q107L；
(2)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的T94D和Q107L；
(3)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的T94D和Q107L；
(4)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的T94D和Q107L；
(5)所述α链的恒定区中的S21I以及所述β链的恒定区中的T94D和Q107L；
(6)所述α链的恒定区中的K83P以及所述β链的恒定区中的T94D和Q107L；
(7)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的T94D和Q107L；
(8)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的Q107L；
(9)所述α链的恒定区中的S21I、K83P和S84.6D以及所述β链的恒定区中的T94D；
(10)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的T94D；
(11)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的Q107L；
(12)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的T94D；
(13)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的Q107L；
(14)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的T94D；
(15)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的Q107L；
(16)所述α链的恒定区中的S21I以及所述β链的恒定区中的Q107L；
(17)所述α链的恒定区中的S21I以及所述β链的恒定区中的T94D；
(18)所述α链的恒定区中的K83P以及所述β链的恒定区中的T94D；
(19)所述α链的恒定区中的K83P以及所述β链的恒定区中的Q107L；
(20)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的T94D；
(21)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的Q107L。
在一些实施方案中，所述TCR包含选自上述(1)-(21)中任一项的氨基酸取代。
在一些实施方案中，所述α链的恒定区还包含以下氨基酸突变中的一种或多种：N1.2K、T84C和120-124位FFPSP缺失突变；和/或所述β链的恒定区还包含以下氨基酸突变中的一种或多种：S79C、C85.1A和N97D。在一些优选的实施方案中，所述α链的恒定区还包含T84C以及可选的N1.2K和/或120-124位FFPSP缺失突变；和/或所述β链的恒定区还包含S79C以及可选的C85.1A和/或N97D。在一些更优选的实施方案中，所述α链的恒定区还包含N1.2K、T84C和120-124位FFPSP缺失突变，并且所述β链的恒定区还包含S79C、C85.1A和N97D。
在一些实施方案中，所述α链的恒定区与SEQ ID NO:1具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性，和/或所述β链的恒定区与SEQ ID NO:14具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述α链的恒定区与SEQ ID NO:2具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性，和/或所述β链的恒定区与SEQ ID NO:15具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述TCR包含具有SEQ ID NO:3-13中任一项所示的氨基酸序列或与SEQ ID NO:3-13中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列的α链恒定区，和/或具有SEQ ID NO:16-23中任一项所示的氨基酸序列或与SEQ ID NO:16-23中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列的β链恒定区。
在一些实施方案中，所述α链的恒定区包含氨基酸取代S21I、K83P和S84.6D，并且所述β链的恒定区包含氨基酸取代T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含K83P和S84.6D，并且所述β链的恒定区包含T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:10所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含S21I和S84.6D，并且所述β链的恒定区包含T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:11所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含S21I和K83P，并且所述β链的恒定区包含T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:12所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含S21I、K83P和S84.6D，并且所述β链的恒定区包含Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:22所示的氨基酸序列。
在一些实施方案中，所述α链的恒定区包含S21I、K83P和S84.6D，并且所述β链的恒定区包含T94D。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链的恒定区包含SEQ ID NO:21所示的氨基酸序列。
在一些实施方案中，所述TCR结合选自以下的抗原：肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原。
在一些实施方案中，所述TCR结合选自以下的TAA：黑色素瘤相关抗原(例如gp100、MAGEA1、MAGEA3、MAGEA6、MAGEA4、MAGEA2、MAGEA12、MAGEA2B、MAGEA9B、MAGEA10、MAGEA11、MAGEB2、MAGEC1、MAGEC2)、IGF2BP1、GNGT1、PI4K2B、CCR8、NPSR1、COX7B2、ONECUT3、SMC1B、FOXI3、GAGE2A、FBXO43、BRDT、PAGE2、GAGE13、POU5F1B、CTAG1A和内源性逆转录酶抗原。
在一些实施方案中，所述TCR结合选自以下的TSA：KRAS(例如KRAS G12突变抗原，如G12D、G12V、G12C、G12R、G12A；G13D；Q61H；G125)、TP53(例如R175H、R173H、R273C、R248W、R248Q、R282W、Y220C、V157F、G245S、Y163C、R249S)、PIK3CA(例如E542K、E545K、H1047R)、CTNNB1(例如S45P、T41A)、EGFR(例如L858R、T790M)、BRAF(例如V600E)和GNAS(例如R201C、R201H)。
在一些实施方案中，所述TCR结合选自以下的病毒抗原：HPV抗原(例如HPV E6或E7抗原)、CMV抗原、HBV抗原、EBV抗原、疱疹病毒抗原、人类免疫缺陷病毒(HIV)抗原、流感病毒抗原和冠状病毒抗原。
在一些实施方案中，所述TCR结合选自以下的自身抗原：AFP、CEA、CD19、CD20、BCMA、CD22、CD30、SLAM、CLDN18.2、GD2、间皮素、CD38、Her2、GPC3、MUC1、Ro52、Ro60、La、Jo-1、SRP、IFIH1、CENPA、CENPB、SNRPA1、SNRNP70、SNR-PD3、RNAP3、TOPO1、Insulin、GAD65、IA2、Znt8、PL7、TARS、ARS、MI2、拓扑异构酶1、EXOSC9、EXOSC107、POLR3A、POLR3K、PTRN、GAD2、SLC30A8、AchR、MUSK、LRP4、PLA2R、THSD7A、TSHR、IFN-γ、CHRNA1、MUSK、LRP4、AQP4、MOG、GRIN1、COL4A3、PLA2R、GM-SCF、PR3和MPO。
在优选的实施方案中，所述TCR结合选自以下的抗原：黑色素瘤相关抗原(例如gp100、MAGEA1)、HPV抗原(例如HPV E6或E7)和KRAS抗原(例如KRAS G12V、KRAS G12D)。
在一些实施方案中，所述TCR结合KRAS G12V，并且包含结合KRAS G12V的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:38(NSASQS)、SEQ ID NO:39(VYSSGN)和SEQ ID NO:40(VVPGGTGGGNKLT)所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:41(LGHDT)、SEQ ID NO:42(YNNKEL)和SEQ ID NO:43(ASSHWGAQETQY)所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:24具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:25具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:44(NSASDY)、SEQ ID NO:45(IRSNMDK)和SEQ ID NO:46(AENSDGTSYGKLT)所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:47(SGHAT)、SEQ ID NO:48(FQNNGV)和SEQ ID NO:49(ASSLVGSPDYEQY)所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:26具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:27具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:50(VSGNPY)、SEQ ID NO:51(YITGDNLV)和SEQ ID NO:52(AVRDIEGAGNNRKLI)所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:53(SGHNS)、SEQ ID NO:54(FNNNVP)和SEQ ID NO:55(ASSEGFYTEAF)所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:28具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:29具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述TCR结合KRAS G12D，并且包含结合KRAS  G12D的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:78(NSASQS)、SEQ ID NO:79(VYSSGN)和SEQ ID NO:80(VVTNARLM)所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:81(MNHEY)、SEQ ID NO:82(SMNVEV)和SEQ ID NO:83(ASTSIVSSGRGEQF)所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:34具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:35具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:84(DRGSQS)、SEQ ID NO:85(IYSNGD)和SEQ ID NO:86(AVNLGAAGNKLT)所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:87(SGHAT)、SEQ ID NO:88(FQNNGV)和SEQ ID NO:89(ASSFSDSYNSPLH)所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:90(QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNLGAAGNKLTFGGGTRVLVKP)具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:91(EAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSFSDSYNSPLHFGNGTRLTVT)具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述TCR结合MAGEA1，并且包含结合MAGEA1的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:56(DSSMTY)、SEQ ID NO:57(IFAFESM)和SEQ ID NO:58(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:59(DFEATT)、SEQ ID NO:60(TDYGSKA)和SEQ ID NO:61(SAREPGQGPWE)所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:30具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:31具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:62(DSSMTY)、SEQ ID NO:63(IFFYQDM)和SEQ ID NO:64(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:65(DFEATT)、SEQ ID NO:66(TDYGSKA)和SEQ ID NO:67(SAREPGQAWSD)所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:32具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:33具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含与SEQ ID NO:68(GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGTGTRLQVFPN)具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:69(GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSDYQTCVTYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPFEQYFGPGTRLTVTE)具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述TCR结合HPV E7，并且包含结合HPV E7的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:70(NSASQS)、SEQ ID NO:71(VYSSGN)和SEQ ID NO:72(AVISAGTALI)所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:73(SGHDT)、SEQ ID NO:74(YYEEEE)和SEQ ID NO:75(ASSLGWRGGLYTEAF)所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRA V包含与SEQ ID NO:76(RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCAVISAGTALIFGKGTTLSVSS)具有至少80％、至少85％、至少90％、 至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:77(DAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGWRGGLYTEAFFGQGTRLTVV)具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述TCR结合gp100，并且包含结合gp100的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含与SEQ ID NO:94具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:95具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述TCR不包含氨基酸序列如SEQ ID NO:24所示的TRAV并且不包含氨基酸序列如SEQ ID NO:25所示的TRBV。在一些实施方案中，所述TCR不包含具有氨基酸序列分别如SEQ ID NO:38-40所示的CDR1、CDR2和CDR3的TRAV并且不包含具有氨基酸序列分别如SEQ ID NO:41-43所示的CDR1、CDR2和CDR3的TRBV。在一些实施方案中，所述TCR不结合KRAS包含VVGAVGVGK的抗原表位或其与HLA-A*11的复合物。在一些实施方案中，所述TCR不结合KRAS抗原。
在一些实施方案中，所述TCR不包含氨基酸序列如SEQ ID NO:94所示的氨基酸序列TRAV，并且不包含氨基酸序列如SEQ ID NO:95所示的TRB V。在一些实施方案中，所述TCR不结合gp100。
在一些实施方案中，所述TCR包含来源于鼠TCR或人TCR的可变区。
在一些实施方案中，所述TCR是分离或纯化的。
如本文所用的术语“分离或纯化的”是指已经从其产生环境的组分中鉴别、分离和/或回收，使得该“分离或纯化的”TCR不含或基本上不含来自其产生环境的、可能干扰其治疗或诊断用途的其它污染物组分。污染物组分可包括酶、激素和其它蛋白质或非蛋白质溶质。因此，可以通过至少一个移除或基本上移除这些污染物组分的纯化步骤制备“分离或纯化的”TCR。
在第九方面，本公开提供了多特异性抗原结合分子，其本文所述的TCRα链恒定区、TCRβ链恒定区、TCR恒定区、多肽分子、或TCR。
在第十方面，本公开提供了多特异性抗原结合分子，其包含至少一个来源于TCR的抗原结合区以及以下任意一项：
(1)本文所述的TCRα链恒定区(TRAC)和/或本文所述的TCRβ链恒定区(TRBC)；
(2)本文所述的TCR恒定区。
在一些实施方案中，所述多特异性抗原结合分子包含本文所述的TCR。
在第十一方面，本公开提供了多特异性抗原结合分子，其包含至少一个来源于TCR的抗原结合区和来源于TCR的α链恒定区(TRAC)和/或β链恒定区(TRBC)；
其中所述α链恒定区在以下一个或多个位置处包含氨基酸取代：Q6、S21、K83、S84.6和I114；和/或
其中所述β链恒定区在以下一个或多个位置处包含氨基酸取代：S92、T94和Q107；
其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区和SEQ ID NO:14所示的TCRβ链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
在一些实施方案中，所述多特异性抗原结合分子包含在以下一个或多个位置处具有氨基酸取代的α链恒定区：Q6、S21、K83、S84.6和I114。在一些实施方案中，所述多特异性抗原结合分子包含在以下一个或多个位置处具有氨基酸取代的β链恒定区：S92、T94和Q107。在一些实施方案中，所述多特异性抗原结合分子包含在以下一个或多个位置处具有氨基酸取代的α链恒定区：Q6、S21、K83、S84.6和I114，和在以下一个或多个位置处具有氨基酸取代的β链恒定区：S92、T94和Q107。
在一些实施方案中，所述α链恒定区中Q6位置处的氨基酸取代选自Q6R、Q6K、Q6D和Q6E，优选选自Q6R和Q6K。
在一些实施方案中，所述α链恒定区中S21位置处的氨基酸取代选自S21I、S21L、S21V、S21G、S21A和S21P，优选选自S21I、S21L和S21V。
在一些实施方案中，所述α链恒定区中K83位置处的氨基酸取代选自K83P、K83G、K83A、K83I、K83L和K83V，优选选自K83P、K83G和K83A。
在一些实施方案中，所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E、S84.6N、S84.6Q、S84.6K和S84.6R，优选选自S84.6D、S84.6E、S84.6N和S84.6Q。
在一些实施方案中，所述α链恒定区中I114位置处的氨基酸取代为I114E、I114D、I114K和I114R，优选选自I114E和I114D。
在一些实施方案中，所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V、S92Q、S92K和S92R，优选选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V和S92Q。
在一些实施方案中，所述β链恒定区中T94位置处的氨基酸取代为T94D、T94E、T94K和T94R，优选选自T94D和T94E。
在一些实施方案中，所述β链恒定区中Q107位置处的氨基酸取代为Q107L、Q107I、Q107V、Q107G、Q107A和Q107P，优选选自Q107L、Q107I和Q107V。
在一些实施方案中，所述α链恒定区中Q6位置处的氨基酸取代为Q6R。
在一些实施方案中，所述α链恒定区中S21位置处的氨基酸取代为S21I。
在一些实施方案中，所述α链恒定区中K83位置处的氨基酸取代为K83P。
在一些实施方案中，所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E和S84.6N。
在一些实施方案中，所述α链恒定区中I114位置处的氨基酸取代为I114E。
在一些实施方案中，所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L和S92N。
在一些实施方案中，所述β链恒定区中T94位置处的氨基酸取代为T94D。
在一些实施方案中，所述β链恒定区中Q107位置处的氨基酸取代为Q107L。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意两个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意三个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6， Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意四个位置处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代。
在一些实施方案中，所述β链恒定区包含S92、T94和Q107中的任意一个位置处的氨基酸取代。
在一些实施方案中，所述β链恒定区包含S92、T94和Q107中的任意两个位置处的氨基酸取代。在一些实施方案中，所述β链恒定区包含S92和T94处的氨基酸取代。在一些实施方案中，所述β链恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114中的任意一个位置处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6和S21、Q6和K83、Q6和S84.6、 Q6和I114、S21和K83、S21和S84.6、S21和I114、K83和S84.6、K83和I114或S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21和K83，Q6、S21和S84.6，Q6、S21和I114，Q6、K83和S84.6，Q6、K83和I114，Q6、S84.6和I114，S21、K83和S84.6，S21、K83和I114，S21、S84.6和I114，或K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83和S84.6，Q6、S21、K83和I114，Q6、S21、S84.6和I114，Q6、K83、S84.6和I114，或S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S9处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和T94处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代；并且所述β链恒定区包含S92、T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链恒定区包含Q6、S21、K83、S84.6和I114处的氨基酸取代，和/或所述β链恒定区包含S92、T94和Q107处的氨基酸取代。在一些实施方案中，所述α链恒定区包含Q6、S21、K83、和S84.6处的氨基酸取代，和/或所述β链恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述α链恒定区包含S21、K83、S84.6和I114处的氨基酸取代，和/或所述β链恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述α链的恒定区包含S21、K83和S84.6处的氨基酸取代，和/或所述β链的恒定区包含T94和Q107处的氨基酸取代。在一些实施方案中，所述α链恒定区包含S21和S84.6处的氨基酸取代，和/或所述β链恒定区包含T94和Q107处的氨基酸取代。
在一些实施方案中，所述α链的恒定区包含以下氨基酸取代中的一种或多种：S21I、K83P和S84.6D；优选包含氨基酸取代S21I、K83P和S84.6D中的至少两种；更优选包含氨基酸取代S21I、K83P和S84.6D。在一些实施方案中，所述α链恒定区包含S21I、K83P和S84.6D。在一些实施方案中，所述α链恒定区包含S21I和S84.6D。
在一些实施方案中，所述β链的恒定区包含以下氨基酸取代中的一种或多种：T94D和Q107L；优选包含氨基酸取代T94D和Q107L。在一些实施方案中，所述β链恒定区包含T94D和Q107L。在一些实施方案中，所述β链恒定区包含T94D。在一些实施方案中，所述β链恒定区包含Q107L。
在一些实施方案中，所述多特异性抗原结合分子包含选自以下(1)-(21)中任一项的氨基酸取代：
(1)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的T94D和Q107L；
(2)所述α链恒定区中的K83P和S84.6D以及所述β链恒定区中的T94D和Q107L；
(3)所述α链恒定区中的S21I和S84.6D以及所述β链恒定区中的T94D和Q107L；
(4)所述α链恒定区中的S21I和K83P以及所述β链恒定区中的T94D和Q107L；
(5)所述α链的恒定区中的S21I以及所述β链的恒定区中的T94D和Q107L；
(6)所述α链的恒定区中的K83P以及所述β链的恒定区中的T94D和Q107L；
(7)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的T94D和Q107L；
(8)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的Q107L；
(9)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的T94D；
(10)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的T94D；
(11)所述α链的恒定区中的K83P和S84.6D以及所述β链的恒定区中的Q107L；
(12)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的T94D；
(13)所述α链的恒定区中的S21I和S84.6D以及所述β链的恒定区中的Q107L；
(14)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的T94D；
(15)所述α链的恒定区中的S21I和K83P以及所述β链的恒定区中的Q107L。
(16)所述α链的恒定区中的S21I以及所述β链的恒定区中的Q107L；
(17)所述α链的恒定区中的S21I以及所述β链的恒定区中的T94D；
(18)所述α链的恒定区中的K83P以及所述β链的恒定区中的T94D；
(19)所述α链的恒定区中的K83P以及所述β链的恒定区中的Q107L；
(20)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的T94D；
(21)所述α链的恒定区中的S84.6D以及所述β链的恒定区中的Q107L。
在一些实施方案中，所述TCR包含选自上述(1)-(21)中任一项的氨基酸取代。
在一些实施方案中，所述α链恒定区还包含以下氨基酸突变中的一种或多种：N1.2K、T84C和120-124位FFPSP缺失突变；和/或所述β链恒定区还包含以下氨基酸突变中的一种或多种：S79C、C85.1A和N97D。在一些优选的实施方案中，所述α链的恒定区还包含T84C以及可选的N1.2K和/或120-124位FFPSP缺失突变；和/或所述β链的恒定区还包含S79C以及可选的C85.1A和/或N97D。在一些更优选的实施方案中，所述α链的恒定区还包含N1.2K、T84C和120-124位FFPSP缺失突变，并且所述β链的恒定区还包含S79C、C85.1A和N97D。
在一些实施方案中，所述α链恒定区与SEQ ID NO:1具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性，和/或所述β链恒定区与SEQ ID NO:14具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述α链恒定区与SEQ ID NO:2具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性，和/或所述β链恒定区与SEQ ID NO:15具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述α链恒定区包含SEQ ID NO:3-13中任一项所示的氨基酸序列或与SEQ ID NO:3-13中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列，和/或所述β链恒定区包含SEQ ID NO:16-23中任一项所示的氨基酸序列或与SEQ ID NO:16-23中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
在一些实施方案中，所述α链恒定区包含氨基酸取代S21I、K83P和S84.6D，并且所述β链恒定区包含氨基酸取代T94D和Q107L。在优选的实施方案中，所述α链恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含K83P和S84.6D，并且所述β链恒定区包含T94D和Q107L。在优选的实施方案中，所述α链的恒定区包含SEQ ID NO:10所示的氨基酸序列，并且所述β链恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S21I和S84.6D，并且所述β链恒定区包含T94D和Q107L。在优选的实施方案中，所述α链恒定区包含SEQ ID NO:11所示的氨基酸序列，并且所述β链恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S21I和K83P，并且所述β链恒定区包含T94D和Q107L。在优选的实施方案中，所述α链恒定区包含SEQ ID NO:12所示的氨基酸序列，并且所述β链恒定区包含SEQ ID NO:23所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S21I、K83P和S84.6D，并且所述β链恒定区包含Q107L。在优选的实施方案中，所述α链恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链恒定区包含SEQ ID NO:22所示的氨基酸序列。
在一些实施方案中，所述α链恒定区包含S21I、K83P和S84.6D，并且所述β链恒定区包含T94D。在优选的实施方案中，所述α链恒定区包含SEQ ID NO:13所示的氨基酸序列，并且所述β链恒定区包含SEQ ID NO:21所示的氨基酸序列。
在一些实施方案中，所述多特异性抗原结合分子包含结合两个或更多个抗原的两个或更多个抗原结合区，并且所述两个或更多个抗原各自独立地选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原、自身抗原、和免疫细胞表面分子。
在一些实施方案中，所述TAA选自：黑色素瘤相关抗原(例如gp100、MAGEA1、MAGEA3、MAGEA6、MAGEA4、MAGEA2、MAGEA12、MAGEA2B、MAGEA9B、MAGEA10、MAGEA11、MAGEB2、MAGEC1、MAGEC2)、IGF2BP1、GNGT1、PI4K2B、CCR8、NPSR1、COX7B2、ONECUT3、SMC1B、FOXI3、GAGE2A、FBXO43、BRDT、PAGE2、GAGE13、POU5F1B、CTAG1A和内源性逆转录酶抗原。
在一些实施方案中，所述TSA选自：KRAS(例如KRAS G12突变抗原，如G12D、G12V、G12C、G12R、G12A；G13D；Q61H；G125)、TP53(例如R175H、R173H、R273C、R248W、R248Q、R282W、Y220C、V157F、G245S、Y163C、R249S)、PIK3CA(例如E542K、E545K、H1047R)、CTNNB1(例如S45P、T41A)、EGFR(例如L858R、T790M)、BRAF(例如V600E)和GNAS(例如R201C、R201H)。
在一些实施方案中，所述病毒抗原选自：HPV抗原(例如HPV E6或E7抗原)、CMV抗原、HBV抗原、EBV抗原、疱疹病毒抗原、人类免疫缺陷病毒(HIV)抗原、流感病毒抗原和冠状病毒抗原。
在一些实施方案中，所述自身抗原选自：AFP、CEA、CD19、CD20、BCMA、CD22、CD30、SLAM、CLDN18.2、GD2、间皮素、CD38、Her2、GPC3、MUC1、Ro52、Ro60、La、Jo-1、SRP、IFIH1、CENPA、CENPB、SNRPA1、SNRNP70、SNR-PD3、RNAP3、TOPO1、Insulin、GAD65、IA2、Znt8、PL7、TARS、ARS、MI2、拓扑异构酶1、EXOSC9、EXOSC107、POLR3A、POLR3K、PTRN、GAD2、SLC30A8、AchR、MUSK、LRP4、PLA2R、THSD7A、TSHR、IFN-γ、CHRNA1、MUSK、LRP4、AQP4、MOG、GRIN1、COL4A3、PLA2R、GM-SCF、PR3和MPO。
在一些实施方案中，所述免疫细胞表面分子选自：CD3(例如CD3γ、CD3δ和CD3ε链)、CD4、CD8、CD10、CD11b、CD11c、CD14、CD16、CD18、CD25、CD32a、CD32b、CD41、CD41b、CD42a、CD42b、CD44、CD45RA、CD49、CD61、CD64、CD68、CD94、CD90、CD117、Nkp46、NKG2D、FcεRI、TCRα/β、TCRγ/δ、HLA-DR、CD28、4-1BB(CD137)、OX40(CD134)、ICOS(CD278)、2B4(CD244)、HVEM、LAG3、DAP10、DAP12、CD27、CD40、GITR、LFA-1、MyD88、CD2、CD7、LIGHT、B7-H3、CTLA-4、PD-1、PD-L1、CD80、BTLA、TIM3、TIGIT和LAG-3。
在一些实施方案中，所述两个或更多个抗原中的一个选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原、自身抗原；另一个为免疫细胞表面分子。在一些实施方案中，所述来源于TCR的抗原结合区结合的抗原选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原、自身抗原。
在一些实施方案中，所述TAA为黑色素瘤相关抗原(例如gp100、MAGEA1)。在一些实施方案中，所述TSA为KRAS抗原(例如KRAS G12V、KRAS G12D)。在一些实施方案中，所述病毒抗原为HPV抗原(例如HPV E6或E7)。在一些实施方案中，所述免疫细胞表面分子选自CD3、CD28、4-1BB(CD137)、PD-1和PD-L1。在一些实施方案中，所述免疫细胞表面分子为CD3。在一些实施方案中，所述免疫细胞表面分子为CD28。
在一些实施方案中，所述多特异性抗原结合分子结合KRAS G12V，并且包含结合KRAS G12V的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:41、SEQ ID NO:42和SEQ ID NO:43所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:24具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:25具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:26具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:27具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:28具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:29具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述多特异性抗原结合分子结合KRAS G12D，并且包含结合KRAS G12D的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:78、SE Q ID NO:79和SEQ ID NO:80所示的氨基酸序列的α链CDR1、CDR2和CDR 3，并且所述TRBV包含分别具有如SEQ ID NO:81、SEQ ID NO:82和SEQ ID NO:83所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:34具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:35具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:84、SEQ ID NO:85和SEQ ID NO:86所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:87、SEQ ID NO:88和SEQ ID NO:89所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:90具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SE Q ID NO:91具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述多特异性抗原结合分子结合MAGEA1，并且包含结合MAGEA1的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:59、SEQ ID NO:60和SEQ ID NO:61所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:30具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:31具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:62、SEQ ID NO:63和SEQ ID NO:64所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:32具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:33具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。在一些实施方案中，所述TRAV包含与SEQ ID NO:68具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:69具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述多特异性抗原结合分子结合HPV E7，并且包含结合HPV E7的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:70、SEQ ID NO:71和SEQ ID NO:72所示的氨基酸序列的α链CDR1、CDR2和CDR3，并且所述TRBV包含分别具有如SEQ ID NO:73、SEQ ID NO:74和SEQ ID NO:75所示的氨基酸序列的β链CDR1、CDR2和CDR3。在一些实施方案中，所述TRAV包含与SEQ ID NO:76具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:77具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述多特异性抗原结合分子结合gp100，并且包含结合gp100的TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)。在一些实施方案中，所述TRAV包含与SEQ ID NO:94具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:95具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在一些实施方案中，所述多特异性抗原结合分子不包含氨基酸序列如SEQ ID NO:24所示的TRAV并且不包含氨基酸序列如SEQ ID NO:25所示的TRBV。在一些实施方案中，所述多特异性抗原结合分子不包含具有氨基酸序列分别如SEQ ID NO:38-40所示的CDR1、CDR2和CDR3的TRAV并且不包含具有氨基酸序列分别如SEQ ID NO:41-43所示的CDR1、CDR2和CDR3的TRBV。在一些实施方案中，所述多特异性抗原结合分子不结合KRAS抗原表位VVGAVGVGK或其与HLA-A*11的复合物。在一些实施方案中，所述多特异性抗原结合分子不结合KRAS抗原。
在一些实施方案中，所述TCR不包含氨基酸序列如SEQ ID NO:94所示的氨基酸序列TRAV，并且不包含氨基酸序列如SEQ ID NO:95所示的TRB V。在一些实施方案中，所述TCR不结合gp100。
在一些实施方案中，所述多特异性抗原结合分子在至少两条多肽链上包含结合第一抗原的第一结合区和结合第二抗原的第二结合区，
其中所述第一结合区包含来源于与所述第一抗原-MHC复合物结合的TCR的α链可变区(TRAV)和β链可变区(TRBV)；
其中所述第二结合区包含来源于与所述第二抗原结合的抗体的重链可变区(VH)和轻链可变区(VL)；
其中所述第一抗原选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原，且所述第二抗原为免疫细胞表面分子；
其中所述多特异性抗原结合分子还包含以下至少一种功能结构域：
1)源于抗体的铰链区；
2)源于抗体的Fc结构域或其二聚化部分；
3)源于抗体的CH3结构域；
4)白蛋白(Alb)或其结合部分；
5)源于TCR的恒定区或其片段。
在一些实施方案中，所述功能结构域包含源于TCR的恒定区或其片段。
在一些实施方案中，所述双特异性多肽分子包含两条多肽链，并且所述两条多肽链分别包含：
TRAV-VH和VL-TRBV；
TRAV-VL和VH-TRBV；
TRBV-VH和VL-TRAV；或
TRBV-VL和VH-TRAV；
并且所述两条多肽链中相邻可变区之间通过接头连接；并且所述TCR的恒定区或其片段通过任选的接头连接在每条多肽链的C端。
在一些实施方案中，所述TCR的恒定区或其片段为本文所述的TCR的恒定区或其片段。在一些实施方案中，TCR恒定区包含本文所述的TRAC和/或本文所述的TRBC。在一些实施方案中，所述双特异性多肽分子的一条多肽链包含TRAC，另一条多肽链包含TRBC。
在一些实施方案中，所述双特异性多肽分子包含第一多肽链和第二多肽链，其中：所述第一多肽链从N端到C端包含：TRAV-接头-VH-任选的接头-TRAC，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-任选的接头-TRBC。
在一些实施方案中，所述双特异性多肽分子包含第一多肽链和第二多肽链，其中：所述第一多肽链从N端到C端包含：TRAV-接头-VH-任选的接头-TRBC，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-任选的接头-TRAC。
在一些实施方案中，所述双特异性多肽分子包含第一多肽链和第二多肽链，其中：所述第一多肽链从N端到C端包含：TRAV-接头-VL-任选的接头-TRAC，并且所述第二多肽链从N端到C端包含：VH-接头-TRBV-任选的接头-TRBC。
在一些实施方案中，所述双特异性多肽分子包含第一多肽链和第二多肽链，其中：所述第一多肽链从N端到C端包含：TRAV-接头-VL-任选的接头-TRBC，并且所述第二多肽链从N端到C端包含：VH-接头-TRBV-任选的接头-TRAC。
在一些实施方案中，所述双特异性多肽分子包含第一多肽链和第二多肽链，其中：所述第一多肽链从N端到C端包含：TRBV-接头-VH-任选的接头-TRAC，并且所述第二多肽链从N端到C端包含：VL-接头-TRAV-任选的接头-TRBC。
在一些实施方案中，所述双特异性多肽分子包含第一多肽链和第二多肽链，其中：所述第一多肽链从N端到C端包含：TRBV-接头-VH-任选的接头-TRBC，并且所述第二多肽链从N端到C端包含：VL-接头-TRAV-任选的接头-TRAC。
在一些实施方案中，所述双特异性多肽分子包含第一多肽链和第二多肽链，其中：所述第一多肽链从N端到C端包含：TRBV-接头-VL-任选的接头-TRAC，并且所述第二多肽链从N端到C端包含：VH-接头-TRAV-任选的接头-TRBC。
在一些实施方案中，所述双特异性多肽分子包含第一多肽链和第二多肽链，其中：所述第一多肽链从N端到C端包含：TRBV-接头-VL-任选的接头-TRBC，并且所述第二多肽链从N端到C端包含：VH-接头-TRAV-任选的接头-TRAC。
在一些实施方案中，所述多特异性抗原结合分子不包含以下第一多肽链和第二多肽链：所述第一多肽链从N端到C端包含：TRAV-ANIGGGSGGGG(SEQ ID NO:36)-VH-S-TRAC，并且所述第二多肽链从N端到C端包含：VL-GGGSGGGGD(SEQ ID NO:37)-TRBV-TRBC。在一些实施方案中，所述多特异性抗原结合分子不包含以下第一多肽链和第二多肽链：所述第一多肽链从N端到C端包含：TRAV-接头-VH-接头-TRAC，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-任选的接头-TRBC。在一些实施方案中，所述多特异性抗原结合分子不包含以下第一多肽链和第二多肽链：所述第一多肽链从N端到C端包含：TRAV-接头-VH-任选的接头-TRAC，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-任选的接头-TRBC。在一些实施方案中，所述双特异性多肽分子的两条多肽链分别不包含TRAV-VH和VL-TRBV。
在一些实施方案中，所述多特异性抗原结合分子不包含如SEQ ID NO:92和93所示的氨基酸序列。
SEQ ID NO:92
SEQ ID NO:93
在一些实施方案中，所述来源于TCR的抗原结合区包含TRAV和TRBV，以及通过任选的接头连接至所述TRAV的C端的TRAC，和通过任选的接头连接至所述TRBV的C端的TRBC；并且其中所述TRAC为本文所述的TRAC；和/或所述TRBC为本文所述的TRBC。
在一些实施方案中，本文所述的TRAC和本文所述TRBC分别通过任选的接头连接至所述TRAV和所述TRBV的C端；或所述TRAC和所述TRBC分别通过任选的接头连接至所述TRBV和所述TRAV的C端。
在一些实施方案中，所述双特异性多肽分子包含两条多肽链。
在一些实施方案中，所述多特异性抗原结合分子包含结合第一抗原的第一结合区和结合第二抗原的第二结合区，
其中所述第一结合区包含来源于与所述第一抗原-MHC复合物结合的T细胞受体(TCR)的α链可变区(TRAV1)和β链可变区(TRBV1)；
其中所述第二结合区包含来源于与所述第二抗原结合的抗体的重链可变区VH1和轻链可变区VL1或来源于与所述第二抗原结合的单域抗体或纳米抗体的单重链可变区(VHH1)；
其中所述第一抗原选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原，且所述第二抗原为免疫细胞表面分子；
其中所述抗体的N端连接在所述TCR的C端；
其中所述多特异性抗原结合分子还包含以下至少一种功能结构域：
1)源于抗体的铰链区；
2)源于抗体的Fc结构域或其二聚化部分；
3)源于抗体的CH3结构域；
4)白蛋白(Alb)或其结合部分；
5)源于TCR的恒定区或其片段。
在一些实施方案中，所述抗体的N末端连接在所述TCR的C末端。
在一些实施方案中，TRAV1和TRBV1分布在不同的多肽链上。在一些实施方案中，所述VH1和VL1分布在一条多肽链上(例如形成scFv1或Fab1)；且所述TRAV1和TRBV1分布在不同多肽链上。在一些实施方案中，在一些实施方案中，所述VH1和VL1通过接头连接。
在一些实施方案中，所述多特异性抗原结合分子在两条多肽链上包含结合第一抗原的第一结合区和结合第二抗原的第二结合区；
其中，TRAV1和TRBV1分布在不同多肽链上，所述VHH1或所述VH1和VL1分布在一条多肽链上(例如形成scFv1或Fab1)并连接在所述TCR的C端；或TRAV1和TRBV1分布在不同多肽链上，所述VH1和VL1分布在不同多肽链上，且所述VH1和VL1中的一个所在多肽链连接在所述TRAV1所在多肽链的C端，另一个所在多肽链连接在所述TRBV1所在多肽链的C端。
在一些实施方案中，所述多特异性抗原结合分子在三条多肽链上包含结合第一抗原的第一结合区和结合第二抗原的第二结合区。
在一些实施方案中，TRAV1和TRBV1分布在不同多肽链上，所述VH1和VL1分布在不同多肽链上，VH1或VL1所在多肽链分别连接在TRBV1或TRAV1所在多肽链的C端。在一些实施方案中，所述多特异性多肽分子的三条多肽链分别包含：TRBV1-VL1，VH1，和TRAV1；或TRBV1-VH1，VL1，和TRAV1；或TRAV1-VL1，VH1，和TRBV1；或TRAV1-VH1，VL1，和TRBV1。
在一些实施方案中，所述多特异性抗原结合分子的多肽链分别包含：
TRBV1-scFv1和TRAV1；或
TRAV1-scFv1和TRBV1；或
TRBV1-VHH1和TRAV1；或
TRAV1-VHH1和TRBV1；或
TRBV1-VL1和TRAV1-VH1；或
TRAV1-VL1和TRBV1-VH1；或
TRBV1-VL1-VH1和TRAV1；或
TRBV1-VH1-VL1和TRAV1；或
TRAV1-VL1-VH1和TRBV1；或
TRAV1-VH1-VL1和TRBV1；或
TRBV1-VL1，VH1，和TRAV1；或
TRBV1-VH1，VL1，和TRAV1；或
TRAV1-VL1，VH1，和TRBV1；或
TRAV1-VH1，VL1，和TRBV1。
在一些实施方案中，所述多特异性抗原结合分子还包含与第三抗原结合的第三结合区，所述第三结合区包含来源于与所述第三抗原-MHC复合物结合的TCR的α链可变区(TRAV2)和β链可变区(TRBV2)；其中所述第三抗原选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原，并且所述第三抗原与所述第一抗原相同或不同。
在一些实施方案中，所述TRAV2与所述TRAV1相同，和/或所述TRBV2与所述TRBV1相同。
在一些实施方案中，所述TRAV2和所述TRBV2通过接头连接在同一条多肽链上，并优选连接在VH1和VL1之间。
在一些实施方案中，所述多特异性抗原结合分子包含两条多肽链，所述两条多肽链分别包含：
TRBV1-VL1-TRAV2-TRBV2-VH1和TRAV1；或
TRBV1-VL1-TRBV2-TRAV2-VH1和TRAV1；或
TRBV1-VH1-TRBV2-TRAV2-VL1和TRAV1；或
TRBV1-VH1-TRAV2-TRBV2-VL1和TRAV1；或
TRAV1-VL1-TRBV2-TRAV2-VH1和TRBV1；或
TRAV1-VL1-TRAV2-TRBV2-VH1和TRBV1；或
TRAV1-VH1-TRAV2-TRBV2-VL1和TRBV1；或
TRAV1-VH1-TRBV2-TRAV2-VL1和TRBV1。
在一些实施方案中，所述多特异性抗原结合分子还包含与第四抗原结合的第四结合区，所述第四结合区包含来源于与所述第四抗原结合的抗体的重链可变区VH2和轻链可变区VL2或来源于与所述第四抗原结合的单域抗体或纳米抗体的单重链可变区(VHH2)；其中所述第四抗原为免疫细胞表面分子，并且所述第四抗原与所述第二抗原相同或不同。
在一些实施方案中，所述VH2与所述VH1相同，和/或所述VL2与所述VL1相同，和/或所述VHH2与所述VHH1相同。
在一些实施方案中，所述VHH2或所述VH2和VL2串联在一条多肽链上(例如，形成scFv2或Fab2)连接在所述TRAV1或所述TRBV1的N端，或者所述VH2和VL2中的一个连接在所述TRAV1的N端，另一个连接在所述TRBV1的N端。
在一些实施方案中，所述多特异性抗原结合分子包含两条多肽链，所述两条多肽链分别包含：
scFv2-TRBV1-scFv1和TRAV1；或
scFv2-TRAV1-scFv1和TRBV1；或
scFv2-TRBV1-VHH1和TRAV1；或
scFv2-TRAV1-VHH1和TRBV1；或
scFv2-TRBV1-VL1和TRAV1-VH1；或
scFv2-TRAV1-VL1和TRBV1-VH1；或
TRBV1-scFv1和scFv2-TRAV1；或
TRAV1-scFv1和scFv2-TRBV1；或
TRBV1-VHH1和scFv2-TRAV1；或
TRAV1-VHH1和scFv2-TRBV1；或
TRBV1-VL1和scFv2-TRAV1-VH1；或
TRAV1-VL1和scFv2-TRBV1-VH1；或
VHH2-TRBV1-scFv1和TRAV1；或
VHH2-TRAV1-scFv1和TRBV1；或
VHH2-TRBV1-VHH1和TRAV1；或
VHH2-TRAV1-VHH1和TRBV1；或
VHH2-TRBV1-VL1和TRAV1-VH1；或
VHH2-TRAV1-VL1和TRBV1-VH1；或
TRBV1-scFv1和VHH2-TRAV1；或
TRAV1-scFv1和VHH2-TRBV1；或
TRBV1-VHH1和VHH2-TRAV1；或
TRAV1-VHH1和VHH2-TRBV1；或
TRBV1-VL1和VHH2-TRAV1-VH1；或
TRAV1-VL1和VHH2-TRBV1-VH1；或
VL2-TRBV1-scFv1和VH2-TRAV1；或
VH2-TRBV1-scFv1和VL2-TRAV1；或
VL2-TRAV1-scFv1和VH2-TRBV1；或
VH2-TRAV1-scFv1和VL2-TRBV1；或
VL2-TRBV1-VHH1和VH2-TRAV1；或
VH2-TRBV1-VHH1和VL2-TRAV1；或
VL2-TRAV1-VHH1和VH2-TRBV1；或
VH2-TRAV1-VHH1和VL2-TRBV1；或
VL2-TRBV1-VL1和VH2-TRAV1-VH1；或
VH2-TRBV1-VL1和VL2-TRAV1-VH1；或
VL2-TRAV1-VL1和VH2-TRBV1-VH1；或
VH2-TRAV1-VL1和VL2-TRBV1-VH1。
在一些实施方案中，所述多特异性抗原结合分子包含第一结合区、第二结合区、第三结合区和第四结合区，并且所述多特异性抗原分子包含两条多肽链，所述两条多肽链分别包含：
scFv2-TRBV1-VL1-TRAV2-TRBV2-VH1和TRAV1；或
scFv2-TRBV1-VL1-TRBV2-TRAV2-VH1和TRAV1；或
scFv2-TRBV1-VH1-TRBV2-TRAV2-VL1和TRAV1；或
scFv2-TRBV1-VH1-TRAV2-TRBV2-VL1和TRAV1；或
scFv2-TRAV1-VL1-TRBV2-TRAV2-VH1和TRBV1；或
scFv2-TRAV1-VL1-TRAV2-TRBV2-VH1和TRBV1；或
scFv2-TRAV1-VH1-TRAV2-TRBV2-VL1和TRBV1；或
scFv2-TRAV1-VH1-TRBV2-TRAV2-VL1和TRBV1；或
TRBV1-VL1-TRAV2-TRBV2-VH1和scFv2-TRAV1；或
TRBV1-VL1-TRBV2-TRAV2-VH1和scFv2-TRAV1；或
TRBV1-VH1-TRBV2-TRAV2-VL1和scFv2-TRAV1；或
TRBV1-VH1-TRAV2-TRBV2-VL1和scFv2-TRAV1；或
TRAV1-VL1-TRBV2-TRAV2-VH1和scFv2-TRBV1；或
TRAV1-VL1-TRAV2-TRBV2-VH1和scFv2-TRBV1；或
TRAV1-VH1-TRAV2-TRBV2-VL1和scFv2-TRBV1；或
TRAV1-VH1-TRBV2-TRAV2-VL1和scFv2-TRBV1；或
VHH2-TRBV1-VL1-TRAV2-TRBV2-VH1和TRAV1；或
VHH2-TRBV1-VL1-TRBV2-TRAV2-VH1和TRAV1；或
VHH2-TRBV1-VH1-TRBV2-TRAV2-VL1和TRAV1；或
VHH2-TRBV1-VH1-TRAV2-TRBV2-VL1和TRAV1；或
VHH2-TRAV1-VL1-TRBV2-TRAV2-VH1和TRBV1；或
VHH2-TRAV1-VL1-TRAV2-TRBV2-VH1和TRBV1；或
VHH2-TRAV1-VH1-TRAV2-TRBV2-VL1和TRBV1；或
VHH2-TRAV1-VH1-TRBV2-TRAV2-VL1和TRBV1；或
TRBV1-VL1-TRAV2-TRBV2-VH1和VHH2-TRAV1；或
TRBV1-VL1-TRBV2-TRAV2-VH1和VHH2-TRAV1；或
TRBV1-VH1-TRBV2-TRAV2-VL1和VHH2-TRAV1；或
TRBV1-VH1-TRAV2-TRBV2-VL1和VHH2-TRAV1；或
TRAV1-VL1-TRBV2-TRAV2-VH1和VHH2-TRBV1；或
TRAV1-VL1-TRAV2-TRBV2-VH1和VHH2-TRBV1；或
TRAV1-VH1-TRAV2-TRBV2-VL1和VHH2-TRBV1；或
TRAV1-VH1-TRBV2-TRAV2-VL1和VHH2-TRBV1；或
VL2-TRBV1-VL1-TRAV2-TRBV2-VH1和VH2-TRAV1；或
VL2-TRBV1-VL1-TRBV2-TRAV2-VH1和VH2-TRAV1；或
VH2-TRBV1-VL1-TRAV2-TRBV2-VH1和VL2-TRAV1；或
VH2-TRBV1-VL1-TRBV2-TRAV2-VH1和VL2-TRAV1；或
VL2-TRBV1-VH1-TRBV2-TRAV2-VL1和VH2-TRAV1；或
VL2-TRBV1-VH1-TRAV2-TRBV2-VL1和VH2-TRAV1；或
VH2-TRBV1-VH1-TRBV2-TRAV2-VL1和VL2-TRAV1；或
VH2-TRBV1-VH1-TRAV2-TRBV2-VL1和VL2-TRAV1；或
VL2-TRAV1-VL1-TRBV2-TRAV2-VH1和VH2-TRBV1；或
VL2-TRAV1-VL1-TRAV2-TRBV2-VH1和VH2-TRBV1；或
VH2-TRAV1-VL1-TRBV2-TRAV2-VH1和VL2-TRBV1；或
VH2-TRAV1-VL1-TRAV2-TRBV2-VH1和VL2-TRBV1；或
VL2-TRAV1-VH1-TRAV2-TRBV2-VL1和VH2-TRBV1；或
VL2-TRAV1-VH1-TRBV2-TRAV2-VL1和VH2-TRBV1；或
VH2-TRAV1-VH1-TRAV2-TRBV2-VL1和VL2-TRBV1；或
VH2-TRAV1-VH1-TRBV2-TRAV2-VL1和VL2-TRBV1。
在一些实施方案中，所述功能结构域来源于白蛋白或其结合部分和任选的抗体的铰链区；并且所述功能结构域通过任选的接头连接在所述多特异性抗原结合分子的任一条多肽链的C末端或N末端，优选C末端。
在一些实施方案中，所述白蛋白为血清白蛋白，优选为人血清白蛋白；和/或所述抗体的铰链区为人源，例如，源自人IgG1、IgG2或IgG4的铰链结构域或其部分。
在一些实施方案中，所述功能结构域来源于抗体的铰链区和/或抗体的Fc结构域或其二聚化部分和/或抗体的CH3结构域；并且所述多特异性抗原结合分子的任意两条多肽链通过所述功能结构域两条链之间的共价结合或非共价结合而连接；
优选的，所述功能结构域的两条链通过任选的接头分别连接在所述多特异性抗原结合分子的任意两条多肽链的C末端或N末端，优选C末端；或
所述第一结合区和所述第二结合区通过所述功能结构域两条链之间的共价结合或非共价结合而连接。
在一些实施方案中，所述抗体的铰链区为人源，例如，源自人IgG1、IgG2或IgG4的铰链结构域或其部分；和/或
所述抗体的Fc结构域或其二聚化部分为人源，例如，源自人IgG1、IgG2或IgG4的Fc结构域或其部分。
在一些实施方案中，所述抗体的铰链区包含至少一个能够降低或消除所述多特异性分子与FcR受体和/或C1q结合的氨基酸突变，例如，所述突变位点选自233、234、235和236的位置；和/或
所述抗体的铰链区包含至少一个能够降低或消除铰链区和非铰链区之间形成二硫键的突变，例如，突变位点为220；和/或
所述Fc结构域中CH2结构域包含至少一个能够降低或消除所述多特异性分子与FcR受体和/或C1q结合的氨基酸突变；例如，所述突变位点选自237、250、252、254、256、270、297、322、327、330和331位置；和/或
所述Fc结构域中CH3结构域包含至少一个能够降低或消除所述多特异性分子与FcR受体和/或C1q结合的氨基酸突变；例如，所述突变位点选自428和434位置；
所述Fc结构域中的CH3包含以下至少一种能够促进所述多肽分子形成异二聚体的突变：
a)包含至少两个半胱氨酸残基突变以形成链间二硫键，例如354C和349C或242C和334C；
b)包含能够使两个CH3形成杵臼结构的突变，例如，所述CH3各自独立地包含位于选自氨基酸位置366、368、405和407的一个或更多个位置的突变，优选地，所述CH3中的一个包含366W突变且另一个包含366S、368A和407V突变；
c)包含能够改变CH3区域相互作用界面的氨基酸的电荷性质以促进异源二聚体形成的突变，例如，两条CH3链的突变位点各自独立的选自Y391、K392、T393、T394、P395、P396、V397、L398、D399、S400、Q347、V348、Y349、T350、S354、R355、E356、E357、F405、Y407和K409中的至少一个。
在一些实施方案中，所述抗体的CH3结构域为人源，例如，源自人IgG1、IgG2或IgG4的CH3结构域；和/或
所述CH3结构域包含以下至少一种能够促进所述多肽分子形成异二聚体的突变：
a)包含至少两个半胱氨酸残基突变以形成链间二硫键，例如354C和349C或242C和334C；
b)包含能够使两个CH3形成杵臼结构的突变，例如，所述CH3各自独立地包含位于选自氨基酸位置366、368、405和407的一个或更多个位置的突变，优选地，所述CH3中的一个包含366W突变且另一个包含366S、368A和407V突变；
c)包含能够改变CH3区域相互作用界面的氨基酸的电荷性质以促进异源二聚体形成的突变，例如，两条CH3链的突变位点各自独立的选自Y391、K392、T393、T394、P395、P396、V397、L398、D399、S400、Q347、V348、Y349、T350、S354、R355、D356、E357、K370、F405、Y407、K409和K439中的至少一个。
在一些实施方案中，所述功能结构域来源于TCR的恒定区或其片段和任选的抗体的铰链区；并且所述多特异性抗原结合分子的任意两条多肽链通过所述功能结构域两条链之间的共价结合或非共价结合而连接；
优选的，所述功能结构域的两条链通过任选的接头分别连接在所述多特异性抗原结合分子的任意两条多肽链的C末端或N末端，优选C末端；或
所述第一结合区和所述第二结合区通过所述功能结构域两条链之间的共价结合或非共价结合而连接。
在一些实施方案中，所述抗体的铰链区为人源，例如，源自人IgG1、IgG2或IgG4的铰链结构域或其部分；和/或
所述TCR的恒定区或其片段是人源或鼠源的，并且选自TCRα链恒定区(TRAC)和TCRβ链恒定区(TRBC)；优选地，所述TRAC和/或TRBC包含相对于野生型序列的至少一个半胱氨酸突变，以在TRAC和TRBC之间形成二硫键，更优选地，所述半胱氨酸突变在以下位置处：野生型TCRα链恒定区第48位和野生型TCRβ链恒定区第57位。
在一些实施方案中，所述多特异性抗原结合分子还包含通过任选的接头连接至TRAV1和/或TRBV1的C端的选自以下的恒定结构域：
(1)TCR恒定区或其片段，所述TCR恒定区例如包含TCRα链恒定区(TRAC)和/或β链恒定区(TRBC)；
优选地，所述TRAC和/或TRBC包含相对于野生型序列的至少一个半胱氨酸突变，以在TRAC和TRBC之间形成二硫键，更优选地，所述半胱氨酸突变在以下位置处：野生型TCRα链恒定区第48位和野生型TCRβ链恒定区第57位；
(2)CH3，所述CH3来源于抗体的Fc结构域，优选任何一条多肽链或两条多肽链中的CH3包含至少一个能够促进所述多肽分子形成异二聚体的突变；
优选地，所述CH3各自独立地包含位于选自氨基酸位置366、368、405和407的一个或更多个位置的突变且两个CH3不包含相同的突变，更优选地，所述CH3中的一个包含T366W突变且另一个包含T366S、L368A和Y407V突变；
(3)CL和CH1，所述CL来源于抗体轻链CL结构域，并且所述CH1来源于抗体重链CH1结构域。
在一些实施方案中，所述多特异性抗原结合分子包含通过任选的接头分别连接至TRAV1和TRBV1的C端的本发明的TRAC和本发明的TRBC。
在一些实施方案中，所述多特异性抗原结合分子还包含分别连接至所述VH1和VL1的来源于抗体的CH1和CL，其中所述VH1、VL1、CH1和CL形成单链Fab并通过接头连接在所述TRAV1或所述TRBV1的C端。
在一些实施方案中，所述多特异性抗原结合分子的多肽链从N端到C端分别包含：
第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-任选的接头-铰链区-CH2-CH3，和第二多肽链：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
第一多肽链：TRAV1-任选的接头-TRAC-接头-VL1-接头-VH1-任选的接头-铰链区-CH2-CH3，和第二多肽链：TRBV1-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3；或
第一多肽链：TRBV1-任选的接头-TRBC-接头-VH1-接头-VL1-任选的接头-铰链区-CH2-CH3，和第二多肽链：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
第一多肽链：TRAV1-任选的接头-TRBC-接头-VH1-接头-VL1-任选的接头-铰链区-CH2-CH3，和第二多肽链：TRBV1-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3；或
第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-接头-CH2-CH3，和第二多肽链：TRAV1-任选的接头-TRAC-接头-CH2-CH3；或
第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-任选的接头-铰链区-CH3，和第二多肽链：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH3；或
第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-任选的接头-铰链区-TRBC，和第二多肽链：TRAV1-任选的接头-TRAC-任选的接头-铰链区-TRAC；或
第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-任选的接头-铰链区，和第二多肽链：TRAV1-任选的接头-TRAC-任选的接头-铰链区-Alb；或
第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-接头-Alb，和第二多肽链：TRAV1-任选的接头-TRAC；或
第一多肽链：TRAV1-任选的接头-CH3-接头-VL1-接头-VH1-任选的接头-任选的铰链区-CH2-CH3，和第二多肽链：TRBV1-任选的接头-CH3-任选的接头-任选的铰链区-CH2-CH3；或
第一多肽链：TRAV1-任选的接头-CH1-接头-VL1-接头-VH1-任选的接头-任选的铰链区-CH2-CH3，和第二多肽链：TRBV1-任选的接头-CL-任选的接头-任选的铰链区-CH2-CH3；或
第一多肽链：TRBV1-任选的接头-TRBC-接头-VHH1-任选的接头-铰链区-CH2-CH3，和第二多肽链：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；
所述抗体是抗体Fab，并且其中第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1-CL-接头-VH1-CH1-任选的接头-铰链区-CH2-CH3，和第二多肽链： TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；
第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1，第二多肽链：VH1-任选的接头-铰链区-CH2-CH3，和第三多肽链：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；
第一多肽链：TRBV1-任选的接头-TRBC-接头-VH1，第二多肽链：VL1-任选的接头-铰链区-CH2-CH3，和第三多肽链：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；
第一多肽链：TRAV1-任选的接头-TRAC-接头-VL1，第二多肽链：VH1-任选的接头-铰链区-CH2-CH3，和第三多肽链：TRBV1-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3。
第一多肽链：TRAV1-任选的接头-TRAC-接头-VH1，第二多肽链：VL1-任选的接头-铰链区-CH2-CH3，和第三多肽链：TRBV1-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3；
第一多肽链：TRBV1-任选的接头-TRBC-接头-VL1-任选的接头-铰链区-CH2-CH3，第二多肽链：VH1-任选的接头-铰链区-CH2-CH3，和第三多肽链：TRAV1-任选的接头-TRAC；
第一多肽链：TRBV1-任选的接头-TRBC-接头-VH1-任选的接头-铰链区-CH2-CH3，第二多肽链：VL1-任选的接头-铰链区-CH2-CH3，和第三多肽：TRAV1-任选的接头-TRAC；
第一多肽链：TRAV1-任选的接头-TRAC-接头-VL1-任选的接头-铰链区-CH2-CH3，第二多肽链：VH1-任选的接头-铰链区-CH2-CH3，和第三多肽链：TRBV1-任选的接头-TRBC；
第一多肽链：TRAV1-任选的接头-TRAC-接头-VH1-任选的接头-铰链区-CH2-CH3，第二多肽链：VL1-任选的接头-铰链区-CH2-CH3，和第三多肽链：TRBV1-任选的接头-TRBC。
在一些实施方案中，所述多特异性抗原结合分子包含第一多肽链和第二多肽链；其中：
所述第一多肽链从N端到C端包含：TRBV1-任选的接头-TRBC-接头-VL1-接头-TRAV2-接头-TRBV2-接头-VH1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。
在一些实施方案中，所述多特异性抗原结合分子包含第一多肽链和第二多肽链；其中：
所述第一多肽链从N端到C端包含：VL2-接头-VH2-接头-TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
所述第一多肽链从N端到C端包含：VL2-接头-TRBV1-接头-VL1-接头-VH1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：VH2-接头-TRAV1-任选的接头-铰链区-CH2-CH3；或
所述第一多肽链从N端到C端包含：VL2-接头-TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：VH2-接头-TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。
在一些实施方案中，所述多特异性抗原结合分子包含第一多肽链和第二多肽链；其中：
所述第一多肽链从N端到C端包含：VL2-接头-VH2-接头-TRBV1-任选的接头-TRBC-接头-VL1-接头-TRAV2-接头-TRBV2-接头-VH1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。
在优选的实施方案中，所述多特异性抗原结合分子包含第一多肽链和第二多肽链，其中：
所述第一多肽链从N端到C端包含：TRBV1-任选的接头-TRBC-接头-VL1-接头-VH1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
所述第一多肽链从N端到C端包含：TRAV1-任选的接头-TRAC-接头-VL1-接头-VH1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRBV1-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3；或
所述第一多肽链从N端到C端包含：TRBV1-任选的接头-TRBC-接头-VH1-接头-VL1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV1-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
所述第一多肽链从N端到C端包含：TRAV1-任选的接头-TRBC-接头-VH1-接头-VL1-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRBV1-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3。
在第十二方面，本公开提供了核酸，其编码本文所述的TCRα链恒定区、TCRβ链恒定区、TCR恒定区、多肽分子、TCR、或多特异性抗原结合分子。
在第十三方面，本公开提供了载体，其包含本文所述的核酸。
如本文所用的术语“载体”是用作将(外源)遗传材料转移到宿主细胞中的媒介的核酸分子，在该宿主细胞中作为载体的所述核酸分子可以例如复制和/或表达。术语“载体”涵盖但不限于质粒、病毒载体(包括逆转录病毒载体、慢病毒载体、腺病毒载体、牛痘病毒载体、多瘤病毒载体和腺病毒相关载体(AAV))、噬菌体、噬菌粒、粘粒和人工染色体(包括BAC和YAC)。载体本身通常是核苷酸序列，通常是包含插入物(转基因)的DNA序列和作为载体“骨架”的较大序列。工程化载体通常包含在宿主细胞中自主复制的起点(如果需要多核苷酸的稳定表达)、选择标记和限制酶切割位点(如多克隆位点，MCS)。载体可另外包含启动子、遗传标记、报告基因、靶向序列和/或蛋白质纯化标签。如本领域技术人员已知的，大量合适的载体是本领域技术人员已知的，并且许多可商购获得。在J.Sambrook等，Molecular Cloning：A Laboratory Manual(第4版)，Cold Spring HarborLaboratory，Cold Spring Harbor Laboratory Press，New York(2012)中提供了合适载体的实例，其通过引用整体并入本文。
在一些实施方案中，所述载体选自慢病毒载体、逆转录病毒载体、质粒、DNA载体、mRNA载体、基于转座子的载体和人工染色体。
在第十四方面，本公开提供了宿主细胞，其包含本文所述的TCRα链恒定区、TCRβ链恒定区、TCR恒定区、多肽分子、TCR、多特异性抗原结合分子、核酸、或载体。
如本文所用的术语“宿主细胞”是指能够表达本发明的修饰的TCR的任何类型的细胞。细胞可以为真核细胞，例如，植物(不具有发育成植株的潜能)、动物、真菌或藻类，或可以为原核细胞，例如，细菌或原生动物。细胞可以为培养的细胞或原代细胞，即直接分离自生物体，例如人。细胞可以为贴壁细胞或悬浮细胞，即悬浮生长的细胞。合适的宿主细胞为本领域已知的并且包括，例如，DH5α大肠杆菌细胞、中国仓鼠卵巢细胞、猴VERO细胞、COS细胞、HEK293细胞等。为了产生本发明的TCR的目的，细胞优选为哺乳动物细胞。最优选地，宿主细胞为人细胞。
在第十五方面，本公开提供了缀合物，其包含本文所述的多肽分子、TCR或多特异性抗原结合分子，以及与所述多肽分子、所述TCR或所述多特异性抗原结合分子缀合的化学部分。
在一些实施方案中，所述化学部分选自可检测标记、免疫刺激分子和治疗剂。优选地，可检测标记选自生物素、链霉抗生物素蛋白、酶或其催化活性片段、放射性核素、纳米颗粒、顺磁性金属离子、核酸探针、造影剂、和萤光、磷光或化学发光分子。优选地，免疫刺激分子选自细胞因子(如IL-2和IFN-γ)、趋化因子(如IL-8)、血小板因子(如血小板因子4)和补体启动剂。优选地，治疗剂选自免疫调节剂、放射性化合物、酶、化学治疗剂和毒素。其他合适的治疗剂包括小分子细胞毒性剂，即具有杀死分子量小于700道尔顿的哺乳动物细胞能力的化合物。此类化合物还可含有能够具有细胞毒性作用的有毒金属。此外，应理解这些小分子细胞毒性剂还包括前体药物，即在生理条件下衰变或被转化以释放细胞毒性剂的化合物。此类药剂的实例包括顺铂、美登素衍生物、雷查霉素、卡奇霉素、多西他赛、依托泊苷、吉西他滨、异环磷醯胺、伊立替康、美法仑、米托蒽醌、吓吩姆钠光敏素II、替莫唑胺、拓扑替康、葡糖醛酸三甲曲沙、耳他汀E、长春新碱和多柔比星；肽细胞毒素，即，具有杀伤哺乳动物细胞能力的蛋白质或其片段；例如，蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、Dnase和RNase；放射性核素，即元素的不稳定同位素，其在衰减的同时发出一种或多种的α或β粒子或γ射线，例如，碘-131、铼-186、铟-111、钇-90、铋-210和-213、锕-225和砹-213；螯合剂，其可用于促进这些放射性核素与分子或其多聚体的结合；或异种蛋白结构域、同种异体蛋白结构域、病毒/细菌蛋白结构域、病毒/细菌肽。
在第十六方面，本公开提供了组合物，其包含本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、或缀合物；以及药学上可接受的载体或赋形剂。
赋形剂可以包括例如填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、防腐剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、粘度赋予剂、表面活性剂、稀释剂、润湿剂、载体、稀释剂、防腐剂、乳化剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋形剂以制备本发明的组合物。用于本发明的组合物中的示例性载体包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和组合物的期望剂型。
根据所采用的活性剂，可将本公开的组合物制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏剂、乳膏剂、透皮贴剂、凝胶剂、粉剂、片剂、溶液剂、气雾剂、颗粒剂、丸剂、混悬剂、乳剂、胶囊剂、糖浆剂、液体剂、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程在第22版的Remington’s Pharmaceutical Sciences(Ed.MaackPublishing Co，Easton，Pa.，2012)中显示，并可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的宿主细胞或可溶性TCR的组合物通常以液体形式提供，并且优选包含药学上可接受的缓冲剂。
在一些实施方案中，所述组合物还包含第二治疗剂，优选地，所述第二治疗剂选自抗体、化疗剂和小分子药物。
第二治疗剂的优选实例包括已知的抗癌药物，例如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；和肽细胞毒素，例如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，例如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，例如抗体定向的酶前药；免疫刺激剂，例如IL-2，趋化因子例如IL-8、血小板因子4；抗体或其片段，例如抗CD3抗体或其片段；补体活化剂；病毒/细菌蛋白结构域和病毒/细菌肽。
在第十七方面，本公开提供了治疗受试者的疾病的方法，其包括向所述受试者施用有效量的本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物。
在一些实施方案中，所述疾病选自癌症、感染性疾病、自身免疫病和炎性疾病。
癌症可为任何癌症，例如血液系统的癌症、中枢和外周神经系统的癌症、淋巴谱系的癌症、骨髓谱系的癌症、间充质来源的癌症、实体瘤等。癌症的实例包括但不限于急性淋巴细胞性癌症、急性骨髓性白血病、腺泡状横纹肌肉瘤、骨癌、脑癌、乳腺癌、肛门癌、肛管癌或直肠肛门癌、眼癌、肝内胆管癌、关节癌、颈癌、胆囊癌或胸膜癌、鼻癌、鼻腔癌或中耳癌、口腔癌、阴道癌、外阴癌、慢性淋巴细胞白血病、慢性骨髓性癌症、结肠癌、食管癌、宫颈癌、胃肠道类癌肿瘤、胶质瘤、霍奇金淋巴瘤、下咽癌、肾癌、喉癌、肝癌、肺癌、恶性间皮瘤、黑素瘤、多发性骨髓瘤、鼻咽癌、非霍奇金淋巴瘤、口咽癌、卵巢癌、阴茎癌、胰腺癌、腹膜癌、网膜癌和肠系膜癌、咽癌、前列腺癌、直肠癌、肾癌、皮肤癌、小肠癌、软组织癌、胃癌、睾丸癌、甲状腺癌、子宫癌、输尿管癌和膀胱癌。
感染性疾病是由病原体感染所导致的疾病，包括传染性疾病和非传染性疾病。引起感染性疾病的病原体包括病毒、细菌、支原体、衣原体、立克次体、朊粒、真菌、螺旋体和寄生虫等。病毒的实例包括但不限于HPV、CMV、HBV、EBV、疱疹病毒、人类免疫缺陷病毒(HIV)、流感病毒和冠状病毒。
常见的自身免疫病和炎性疾病包括但不限于类风湿性关节炎、系统性红斑狼疮、强直性脊柱炎、银屑病关节炎、自身免疫破坏胰岛导致的糖尿病、干燥综合征、桥本甲状腺炎、Graves’甲状腺炎、毒性弥漫性甲状腺肿、自身免疫性溶血性贫血、特发性血小板减少性紫癜、炎症肠病(例如溃疡性结肠炎和克罗恩病)、牛皮癣、肾小球肾炎、肾病综合征、抗肾小球基底膜病、膜性肾病、系统性硬化症、多发性肌炎、重症肌无力、银屑病、天疱疮、白癫风、中枢神经系统的自身免疫性疾病、乳糜泻、自身免疫性胃炎、原发性胆道胆管炎、自身免疫性肝炎、肺出血-肾炎综合征、自身免疫性卵巢炎、自身免疫性睾丸炎、特发性白细胞减少症、原发性肾上腺皮质萎缩、抗磷脂抗体综合征、感染后纤维化、心肌梗死后纤维化、肝纤维化、瘢痕增生。
在一些实施方案中，施用于受试者的剂量可随实施方案、所用药物、给药方法以及被治疗的部位和受试者而变化。然而，剂量应足以提供治疗反应。临床医生可以确定给予人或其他受试者以治疗医学病症的有效量。治疗有效所需的精确量可取决于许多因素，例如活性剂的活性和给药途径。
本发明的多肽分子、TCR、多特异性抗原结合分子、细胞、缀合物、或组合物的剂量可以在合适的时间段内一次性或以一系列亚剂量的形式施用给哺乳动物，例如根据需要，每天、每半周、每周、每两周、每半月、每两月、每半年或每年施用一次。包含有效量的多肽分子、TCR、多特异性抗原结合分子、细胞、缀合物、或组合物的剂量单位可以以单日剂量给药，或者总日剂量可以根据需要以每日给药的两个、三个、四个或更多个分剂量给药。
合适的给药方式可由医生选择。给药途径可以是肠胃外给药，例如通过注射给药、经鼻给药、经肺给药或经皮给药。可以通过静脉内注射、肌内注射、腹膜内注射、皮下注射进行全身或局部给药。在一些实施方案中，选择多肽分子、缀合物或药物组合物用于肠胃外递送、吸入或通过消化道递送，例如口服。给药剂量和方法可以根据受试者的重量、年龄、条件等而变化，并且可以适当地选择。
在一些实施方案中，所述方法还包括施用第二治疗剂。在某些实施方案中，在施用第二治疗剂之前、基本上同时或之后施用本发明的多肽分子、TCR、多特异性抗原结合分子、细胞、缀合物、或组合物。优选地，所述第二治疗剂选自抗体、化疗剂和小分子药物。第二治疗剂的优选实例如上文所述。
在第十八方面，本公开提供了TCR，其包含TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)，其中所述TRAV包含分别具有如SEQ ID NO:56(DSSMTY)、SEQ ID NO:57(IFAFESM)和SEQ ID NO:58(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3，或者插入、缺失或取代一个或几个氨基酸所形成的功能变体；和/或所述TRBV包含分别具有如SEQ ID NO:59(DFEATT)、SEQ ID NO:60(TDYGSKA)和SEQ ID NO:61(SAREPGQGPWE)所示的氨基酸序列的β链CDR1、CDR2和CDR3，或者插入、缺失或取代一个或几个氨基酸所形成的功能变体。
在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:56(DSSMTY)、SEQ ID NO:57(IFAFESM)和SEQ ID NO:58(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3；和/或所述TRBV包含分别具有如SEQ ID NO:59(DFEATT)、SEQ ID NO:60(TDYGSKA)和SEQ ID NO:61(SAREPGQGPWE)所示的氨基酸序列的β链CDR1、CDR2和CDR3。
在一些实施方案中，所述TRAV包含与SEQ ID NO:30具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，和/或所述TRBV包含与SEQ ID NO:31具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在第十九方面，本公开提供了TCR，其包含TCRα链可变区(TRAV)和TCRβ链可变区(TRBV)，其中所述TRAV包含分别具有如SEQ ID NO:62(DSSMTY)、SEQ ID NO:63(IFFYQDM)和SEQ ID NO:64(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3，或者插入、缺失或取代一个或几个氨基酸所形成的功能变体；和/或所述TRBV包含分别具有如SEQ ID NO:65(DFEATT)、SEQ ID NO:66(TDYGSKA)和SEQ ID NO:67(SAREPGQAWSD)所示的氨基酸序列的β链CDR1、CDR2和CDR3，或者插入、缺失或取代一个或几个氨基酸所形成的功能变体。
在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:62(DSSMTY)、SEQ ID NO:63(IFFYQDM)和SEQ ID NO:64(AGSGGGTDK)所示的氨基酸序列的α链CDR1、CDR2和CDR3，和/或所述TRBV包含分别具有如SEQ ID NO:65(DFEATT)、SEQ ID NO:66(TDYGSKA)和SEQ ID NO:67(SAREPGQAWSD)所示的氨基酸序列的β链CDR1、CDR2和CDR3。
在一些实施方案中，所述TRAV包含与SEQ ID NO:32具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列，并且所述TRBV包含与SEQ ID NO:33具有至少80％、至少85％、至少90％、至少95％或100％序列同一性的氨基酸序列。
在第二十方面，本公开提供了本文所述的TCRα链恒定区、TCRβ链恒定区、TCR恒定区、多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物在制备用于治疗或预防受试者中的疾病的药物中的用途。
在第二十一方面，本公开提供了本文所述的TCRα链恒定区、TCRβ链恒定区、TCR恒定区、多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物，其用于治疗或预防受试者中的疾病。
在二十二方面，本公开提供了一种检测受试者中由阳性抗原引起的疾病和/或病症的方法，其中所述方法包括：(i)使从所述受试者获得的样品与本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物接触；和(ii)检测所述样品中阳性抗原的存在，其中所述阳性抗原的存在指示由阳性抗原引起的疾病和/或病症。
在二十三方面，本公开提供了一种检测受试者中由阳性抗原引起的疾病和/或病症的方法，其中所述方法包括：所述受试者施用有效量的可检测标记(例如，放射性核素)偶联的本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物。
在二十四方面，本公开提供了本公开的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物在制备用于检测(例如诊断)待测样品中阳性抗原的存在的产品中的用途，例如，试剂盒。
在二十五方面，本公开提供了本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物，其检测(例如诊断)待测样品中阳性抗原存在的产品。所述阳性抗原的存在可以指示由阳性抗原引起的疾病和/或病症。
在二十六方面，本公开提供了种试剂盒，其包含本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物。
在一种具体的实施方案中，所述试剂盒用于检测(例如诊断)受试者中由阳性抗原引起的疾病和/或病症，其包含本文所述的多肽分子、TCR、多特异性抗原结合分子、核酸、载体、细胞、缀合物、或组合物。
在本发明的方法和用途的一些实施方案中，所述疾病或病症选自癌症、感染性疾病、自身免疫病和炎性疾病。所述癌症、感染性疾病、自身免疫病和炎性疾病如上文所定义。
在本发明的方法和用途的一些实施方案中，所述阳性抗原为HPV阳性抗原，更优选为HPV E7阳性抗原。在一些实施方案中，所述HPV阳性病症或病症选自HPV感染、HPV初癌和癌症。优选地，所述癌症优选选自宫颈癌、头颈癌、口咽癌、食道腺癌、肛门癌、肛管癌、直肠癌、阴道癌、外阴癌、阴茎癌。
在本发明的方法和用途的一些实施方案中，所述阳性抗原为KRAS突变阳性抗原，例如，G12V突变或G12D突变。在一些实施方案中，所述KRAS突变阳性抗原引起的疾病和/或病症选自KRAS突变引发的肿瘤、初癌和癌症。在一些实施方案中，所述癌症选自实体瘤和血液瘤。更优选的，所述肿瘤包括：胰腺癌、结肠癌、直肠癌、结直肠癌、子宫内膜癌、肺癌、黑色素瘤、前列腺癌、胆管癌、宫颈癌、膀胱癌、肝癌和乳腺癌中的至少一种。
在本发明的方法和用途的一些实施方案中，所述阳性抗原为MAGE阳性抗原，优选为MAGE-A1阳性抗原。在一些实施方案中，所述MAGE-A1阳性癌症选自肺癌、肝癌(例如肝细胞癌)、胃癌、黑色素瘤、食道癌、结直肠癌、宫颈癌、乳腺癌、膀胱癌(例如膀胱尿路上皮癌)、卵巢癌(例如卵巢浆液性囊腺癌)、头颈癌(例如头颈鳞状细胞癌)、子宫癌、子宫内膜癌、胆管癌、前列腺癌、肾上腺皮质癌、间皮瘤、肉瘤、多形胶质母细胞瘤、嗜铬细胞瘤、神经母细胞瘤、视网膜母细胞瘤、副神经节瘤和胸腺癌。
实施例
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。应当理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。以下实施例中未注明具体条件的实验方法，通过按照本领域的常规条件，例如，Sambrook和Russeii等人，分子克隆：实验室手册(第三版)(2001)，CSHL出版社中所述的条件，或按照制造商所建议的条件。除非另有说明，否则以下实施例中所用的实验材料和试剂均可商购获得。
实施例1.TCR恒定区突变序列信息
通过在如Seq ID No.2和Seq ID No.15所示的截短的人TCR恒定区内引入新的二硫键从而提高了TCR可溶表达成功率及产量，但对于基础表达水平比较差的TCR来说，仍然无法通过这样的策略成功实现可溶表达。
为了增加TCR的可溶性表达，本发明人通过计算机预测，以Seq ID No.2和Seq ID No.15作为亲本序列，在TCR恒定区引入单氨基酸位点突变，结合湿实验验证，最终筛选到可显著改善TCR可溶表达性质的有益突变。野生型TCR恒定区、亲本TCR恒定区和本发明筛选到的TCR恒定区的氨基酸序列如下(突变位点按照IMGT命名规则进行编号)：
Seq ID No.1:Homo sapiensTRAC*01
Seq ID No.2:亲本TRAC，相对于Seq ID No.1包含N1.2>K、T84>C和120-124位FFPSP缺失突变
Seq ID No.3:在Seq ID No.2的基础上单位点突变Q6>R
Seq ID No.4:在Seq ID No.2的基础上单位点突变S21>I

Seq ID No.5:在Seq ID No.2的基础上单位点突变K83>P
Seq ID No.6:在Seq ID No.2的基础上单位点突变S84.6>D
Seq ID No.7:在Seq ID No.2的基础上单位点突变S84.6>E
Seq ID No.8:在Seq ID No.2的基础上单位点突变S84.6>N
Seq ID No.9:在Seq ID No.2的基础上单位点突变I114>E
Seq ID No.10:在Seq ID No.2的基础上多位点突变K83>P、S84.6>D
Seq ID No.11:在Seq ID No.2的基础上多位点突变S21>I、S84.6>D
Seq ID No.12:在Seq ID No.2的基础上多位点突变S21>I、K83>P
Seq ID No.13:在Seq ID No.2的基础上多位点突变S21>I、K83>P、S84.6>D
Seq ID No.14:Homo sapiens TRBC2*01

Seq ID No.15:亲本TRBC，相对于Seq ID No.14包含S79>C、C85.1>A、N97>D
Seq ID No.16:在Seq ID No.15的基础上单位点突变S92>P
Seq ID No.17:在Seq ID No.15的基础上单位点突变S92>E
Seq ID No.18:在Seq ID No.15的基础上单位点突变S92>D
Seq ID No.19:在Seq ID No.15的基础上单位点突变S92>L
Seq ID No.20:在Seq ID No.15的基础上单位点突变S92>N
Seq ID No.21:在Seq ID No.15的基础上单位点突变T94>D
Seq ID No.22:在Seq ID No.15的基础上单位点突变Q107>L
Seq ID No.23:在Seq ID No.15的基础上多位点突变T94>D、Q107>L
实施例2.突变策略和方法选择
蛋白质的天然状态稳定性和可表达性在原则上是相关的，尽管前者是热力学性质，后者主要受折叠轨迹的支配。这种关系是由于蛋白质的稳定性是由折叠态和错误折叠态或未折叠态之间的自由能差决定的。错误折叠状态可以被认为是一种陷阱，使新生链无法折叠到天然状态，从而阻碍折叠轨迹并降低天然折叠蛋白的产量；错误折叠态也可能导致末端聚集。因此，折叠态和错误折叠态之间的自由能差也是可表达性的决定因素之一。我们可以使用图2中的示意图来考虑稳定性设计的目标：一个边缘稳定的目标蛋白的能量格局包括许多错误折叠状态，这些状态的能量与天然状态接近，因此可能会有大量的错误折叠状态。在原生宿主中，蛋白质稳态网络阻断了这些错误折叠状态的发生和积累，但错误折叠状态可能限制了蛋白质在异源宿主中的表达能力，并限制了蛋白质在体外的寿命。稳定性设计的目标是增加固有折叠态和错误折叠态或展开态之间的能量差距。可以通过降低正确折叠状态的能量来增加能隙，同时也有利于消除尽可能多的错误折叠态，使剩余的错误折叠态相对于新的正确折叠状态保持在能量高位，从而改善蛋白的可表达性。
蛋白质作为有机大分子分子化合物，在水中以分散态(胶体态)存在，因此，蛋白质在水中无严格意义上的溶解度，只是将蛋白质在水中的分散量或分散水平相应的称为蛋白质的溶解度。从表观上看，蛋白质溶解度的大小受到一些条件如pH值、离子强度、温度、溶剂类型等的影响，但归根究底都是由组成蛋白质的氨基酸序列决定的。通过氨基酸序列的优化，减少蛋白分子间的聚集，增加蛋白分子在溶剂体系中的分散性是增加蛋白产量的另一个重要手段。
综合以上信息，发明人明确了TCR融合蛋白可溶性表达性能优化的策略和方法：
a.通过增加蛋白稳定性和提高溶解度两个维度对TCR融合蛋白的可溶表达水平进行优化；
b.以TCR的恒定区为目标区域，通过生物学计算方法对该区域进行单氨基酸突变后的稳定性、溶解度进行预测；从计算结果中筛选增益效果显著的突变进行湿实验验证；
c.使用开源算法I mutant 2.0和SODA分别计算蛋白质稳定性和溶解度；
d.将经过湿实验验证的有益突变进行有限组合，验证不同空间位置上的有益氨基酸突变之间是否存在协同增益效果。
实施例3.算法预测和湿实验验证
为了便于TCR融合蛋白表达后进行功能验证，发明人将gp100-TCR序列装载到发明人自主研发的TCR融合蛋白结构(format 62，B62#)上进行突变预测、突变后序列的蛋白表达及蛋白活性验证。实验整体流程如图3所示。
Format 62具有以下两条多肽链：
第一多肽链：TRBV-TRBC-第一接头-VL-第二接头-VH-铰链区-CH2-CH3Knob
第二多肽链：TRAV-TRAC-铰链区-CH2-CH3Hole
其中，各组件的氨基酸序列如下所示：
TRAV
TRBV
TRAC：SEQ ID NO:2
TRBC：SEQ ID NO:15
VH
VL
第一接头
第二接头
铰链区-CH2-CH3Hole
铰链区-CH2-CH3Knob
3.1计算机预测
3.1.1三维结构预测
AlphaFold2在各类蛋白的三维结构预测中均表现出非常强的适用性，尤其在抗体的结构预测方面表现优异，计算机预测结构与湿实验结构高度吻合。发明人通过该算法成功实现了TCR和TCR融合蛋白的三维结构预测，预测的三维结构图如图4和图5所示。
3.1.2突变氨基酸位点预测及筛选
ISPRED4是从蛋白质结构开始预测蛋白质-蛋白质相互作用位点(PPI)的web服务器。ISPRED4经过机器深度学习训练，可以从蛋白质序列和结构中提取若干特征，预测蛋白质表面各残基的相互作用状态，预测每一个氨基酸是否位于蛋白分子表面、作用交接面或是被包埋在蛋白内核。示例性的TCR融合蛋白gp100-B62的A链PPI预测结果如图6所示。
根据TCR融合蛋白gp100-B62双链的预测结果，从中筛选出共计130个氨基酸位点，这些位点或位于PPI区域、或位于蛋白分子表面但亲水性较弱，我们认为对这些位点进行突变有更大的可能改善TCR融合蛋白的表达性质。
3.1.3突变稳定性、溶解度预测及筛选
然后，分别使用I Mutant和SODA算法对前面选定的130个氨基酸位点进行随机饱和突变(每个氨基酸位点突变为其余19种)，并且通过算法对突变后的蛋白稳定性(Stable)和溶解度(Soluble)进行打分并排序。最后从共计2470种突变序列中，挑选出150条突变序列进行蛋白表达，通过湿实验验证这些突变对TCR融合蛋白活性的影响。挑选原则如下：
a.同一氨基酸位点处有多种突变均产生增益，仅挑选打分最高的Top2进行验证；
b.分别考虑I Mutant和SODA得分，各选Top 100；
c.将I Mutant和SODA Top100突变序列并集，去重复后，最终筛选出双项打分均增加的突变(50克隆)、仅单项打分增加的突变(100克隆)。
3.2湿实验验证
3.2.1单位点突变TCR融合蛋白活性验证
共计150个克隆进入湿实验验证。为了便于快速表达蛋白分子并进行活性验证，通过CHO-K1细胞12孔板瞬转表达蛋白，将表达第7天时收取的上清液，经过梯度稀释后用ELISPOT方法检测上清液中蛋白的活性。在此以蛋白活性为指标，评价氨基酸突变对TCR融合蛋白的活性、表达量产生的综合增益效果。具体操作如下。
将CHO-K1细胞铺板在12孔板中，放入二氧化碳恒温培养箱，于37℃，6％CO2下培养。待细胞占比80％～95％时，使用Mirus转染试剂，按照质粒(μg)：转染试剂(μL)＝1:2.5的比例，将待筛选蛋白的质粒转染到细胞中，放入二氧化碳恒温培养箱，于37℃，6％的CO2下培养。培养第7天，用移液枪在每个孔板孔中吸取1mL培养上清，移至EP管中，在500g下离心5min，将上清液转移到新的EP管中，然后分别用5倍和20倍稀释的上清液进行ELISPOT检测。以亲本TCR恒定区(其TRAC和TRBC序列分别如SEQ ID NO.2和SEQ ID NO.15所示)作为野生型对照。
为进行ELISPOT检测，将活细胞密度为1x106个细胞/mL的靶细胞T2悬浮于离心管中，加入对应浓度表位多肽，置于37℃孵育2小时，期间每20分钟轻弹混匀一次；用10％FBS的1640培养基重悬效应细胞PBMC并将细胞密度调整至1×106个细胞/mL。根据实验方案设计，向ELISPOT孔中加入相应数量和种类的效应细胞、靶细胞以及蛋白药物，放在37℃,5％ CO2培养箱中培养16-20小时。弃掉孔中培养基与细胞，用PBS洗板。用生物素标记抗体将板在37℃下孵育2小时，然后洗板。将TMB显色液加入孔中进行显色，当观察到明显的斑点时，终止显色并进行ELISPOT斑点读板。
经过多轮蛋白表达-ELISPOT实验，筛选出14种可以显著改善TCR融合蛋白体外活性的TCR恒定区单位点突变体。这14种显著获益的突变体，平均分布于TRAC和TRBC，包括位于TRAC的Q6R、S21I、K83P、S84.6D、S84.6E、S84.6N、I114E的7种氨基酸突变和位于TRBC的S92P、S92E、S92D、S92L、S92N、T94D、Q107L位点的7种氨基酸突变。它们的ELISPOT结果如图7所示。
图7的ELISPOT结果显示，突变体实验组上清液在20倍稀释条件下表现出显著强于野生型对照组的PBMC激活活性，这样的活性增加可能来源于蛋白产量、活性多方面的增益。
另外，将ELISPOT验证的结果与实施例3.1.3中的Stable/Soluble双项打分预测结果进行比较，结果显示于图8。结果显示，Stable/Soluble双项评分均增加的实验分组中，20％的突变克隆均有显著的活性获益，而单项评分增加的实验分组中阳性率均显著降低(Stable:5.56％；Soluble:2.3％)，提示Stable/Soluble双项评分均增加的克隆有更大的可能显著改善蛋白活性。
3.2.2多位点突变TCR融合蛋白表达验证
发明人先前自主研发TCR Engager结构(B62#)，在早期研究中显示出优异的表达稳定性，可以支持不同种系来源的TCR实现可溶性表达，但也有少数特殊TCR(例如KRAS G12V特异性TCR，CR-KVA11-N03)在装载到该结构后无法成功表达。
CR-KVA11-N03 TCR的氨基酸如下所示：
TRAV
TRBV
深入研究发现，该TCR即便在T细胞膜表面也难以实现高峰度的表达(如图9所示)。这样的结果可能是由于特殊的V区序列导致的。那么具有此类特征的TCR是否注定无法应用于TCR融合蛋白中呢？考虑到TCR的稀缺性，本发明人尝试通过以上筛选到的TCR恒定区突变来提高该TCR的可溶表达性质。
结合计算机预测的CR-KVA11-N03TCR三维结构模型(图10)的指导，从以上筛选到的有效单位点突变中选择多个空间位置互不干扰的突变进行组合(将突变组合命名为CMM)，尝试在B62#结构上装载CR-KVA11-N03TCR(CR-KVA11-N03-B62)并引入CMM突变，观察是否能有效改善TCR融合蛋白的表达。
为了比较C区突变和V区突变对TCR可溶表达性质影响的权重，用与实施例3.1.3中相同的方法(I Mutant、SODA双项得分增加)在CR-KVA11-N03的可变区的FR区预测筛选了多个高分突变，并将之组合(命名为VMM1、VMM2、VMM3)后引入CR-KVA11-N03-B62结构中。在同批表达实验中，验证无突变野生型(WT，CR-KVA11-N03TCR可变区野生型+亲本恒定区)、C区组合突变(CMM1(TRAC中的S21I、K83P、S84.6D和TRBC中的T94D、Q107L的组合))、V区组合突变(VMM1、VMM2、VMM3)以及V区(VMM1)+C区(CMM1)组合突变对TCR可溶表达的影响。
将编码上述CR-KVA11-N03-B62分子的核苷酸序列克隆至表达载体中， 获得的载体克隆经测序确认后进行扩增培养、质粒提取。随后用质粒转染CHO细胞，培养6-10天，然后收集培养上清液。用收集的培养上清液进行Protein A亲和纯化，得到纯化蛋白。将纯化的蛋白经变性(R)或未变性(N)处理后进行SDS-PAGE凝胶电泳，以评估蛋白表达纯度及聚集程度。
SDS-PAGE结果如图11所示。结果显示，不包含突变的CR-KVA11-N03-B62(WT)无法表达组装成为有效分子；V区突变分子虽然在算法预测中得分较高，但V区突变组合VMM1、VMM2和VMM3均未显著改善CR-KVA11-N03-B62分子的表达；引入恒定区突变组合CMM1成功实现了CR-KVA11-N03-B62分子的可溶表达，SDS-PAGE结果显示分子大小与预期相符。同时，该表达结果仅仅经过一步Protein A亲和纯化，SDS-PAGE结果显示纯化后蛋白纯度较高，仅仅产生了少量的聚体。在未改善可溶表达的VMM1基础上引入CMM1组合突变(VCMM1)明显提高了CR-KVA11-N03-B62分子的可溶表达。
3.2.3 CMM1突变对TCR融合蛋白产量的影响
前述实验已经证实CMM1突变可以有效改善TCR的可溶表达，甚至可以帮助某些膜表达都十分困难的TCR实现可溶表达。接下来将进一步验证CMM1突变的这一属性是否可兼容不同的TCR。将5种不同种系来源、识别不同表位的TCR(CR-KVA11-N03、CR-KVA11-N04、CR-KVA11-N02、A1A2-M01-625、A1A2-M01-648)分别装载到B62结构中，并分别使用B62-WT(TCR恒定区为亲本TCR恒定区)和B62-CMM1(TCR恒定区包含CMM1突变)结构进行表达。同时还将A1A2-M01-648TCR转载到B62-LJH2结构中(称为A1A2-M01-648-LJH2)，并分别使用上述WT和CMM1结构进行表达。
上述5种TCR的氨基酸序列如下所示：
CR-KVA11-N03：包含SEQ ID NO.24所示TRAV和SEQ ID NO.25所示TRBV
CR-KVA11-N04
TRAV
TRBV
CR-KVA11-N02
TRAV
TRBV
A1A2-M01-625
TRAV
TRBV
A1A2-M01-648
TRAV
TRBV
B62-LJH2结构具有以下两条多肽链：
第一多肽链：TRBV-TRBC-第一接头-VH-第二接头-VL-铰链区-CH2-CH3；
第二多肽链：TRAV-TRAC-铰链区-CH2-CH3。
如实施例3.2.2中所述进行蛋白表达和SDS-PAGE，并根据SDS-PAGE结果计算蛋白产量。结果如图12所示。
结果证明，CMM1突变可以广泛地提高多种不同TCR的可溶表达产量，并且可应用于多种不同的TCR融合蛋白中。
3.2.4 CMM1突变对TCR融合蛋白活性的影响
众所周知，氨基酸突变导致的蛋白分子结构改变有可能直接影响蛋白活性。虽然从结构上看TCR恒定区远离CDR区以及TCR-pMHC交互作用界面，但无法排除恒定区局部的构象变化是否会间接对TCR-pMHC交互作用界面产生远端影响。
因此，使用分别装载有靶向不同抗原表位的TCR(A1A2-M01-625(其氨基酸序列如实施例3.2.3所示)、KDA11-N02、KVA11-N04(其氨基酸序列如实施例3.2.3所示))的B62结构(分别称为A1A2-M01-625_B62、KDA11-N02_B62、KVA11-N04_B62)以及装载有MAGE-A1特异性TCR A1A2-M01-648(其氨基酸序列如实施例3.2.3所示)的B62-BJH2结构(称为A1A2-M01-648-LJH2_B62)作为载体，验证CMM1突变是否会对蛋白活性产生不良影响。早期研究已明确，这些TCR-B62蛋白分子在体外可以在其独特的pMHC表位存在的情况下刺激PBMC产生特异性的激活反应；而在pMHC表位缺乏的时，适当的蛋白浓度条件下无法激活PBMC，但蛋白浓度较高时(例如-7M)则容易非特异性地激活PBMC。因此，将CMM1突变引入这些TCR-B62蛋白分子结构中。以亲本TCR恒定区作为野生型对照。
KDA11-N02的氨基酸序列如下：
TRAV
TRBV
使用与实施例3.2.1类似的实验方法进行蛋白表达和ELISPOT实验。ELISPOT结果如图13所示。
实验结果显示：
a)CMM1突变不影响蛋白对靶标的活性：引入CMM1突变，并未对A1A2-M01-625_B62、A1A2-M01-648-LJH2_B62、KDA11-N02_B62、KVA11-N04_B62蛋白的活性产生负面影响，在低浓度蛋白条件下，对照组(C-WT)和CMM1实验组激活效果相近，部分CMM1实验组还表现出更强的激活；
b)CMM1突变降低高浓度蛋白导致的非特异性激活：在高浓度蛋白(-7M)条件下，对照组的靶标阴性队列中的PBMC均表现为明确的非特异性激活，且非特异性激活强度与特异性激活强度呈现一定的相关；而CMM1实验组的靶标阴性队列中，PBMC的非特异性激活无一例外地显著降低；
c)CMM1突变降低非相关表位导致的非特异性激活：在高浓度蛋白(-7M)条件下，对照组的无关肽队列中的PBMC均表现为明确的非特异性激活，且非特异性激活强度与特异性激活强度呈现一定的相关；而CMM1实验组的靶标阴性队列中，PBMC的非特异性激活无一例外地显著降低。
以上结果表明，在B62以及B62-LJH2结构中引入CMM1突变，不会使蛋白对靶标的活性降低，同时还有效降低了高浓度蛋白条件下对PBMC的非特异性激活。
3.2.5 CMM1对不同TCR融合蛋白的兼容性
为验证上述筛选出来的CMM1是否可适用于不同的TCR融合蛋白结构，采用format 22-2融合蛋白进行蛋白表达和ELISPOT实验，其中，TCR为CR-KVA11-N03，同时以亲本TCR恒定区作为野生型对照。
Format 22-2具有以下两条多肽链：
第一多肽链：TRAV-接头-VH-接头-TRAC，氨基酸序列如SEQ ID NO:92所示；
第二多肽链：VL-接头-TRBV-TRBC，氨基酸序列如SEQ ID NO:93所示；
实验操作具体如下：
1.载体构建和质粒提取
将表达SEQ ID NO:92和SEQ ID NO:93的核苷酸序列直接克隆至表达载体pTT5中，获得的载体克隆经测序确认后进行扩增培养、质粒提取。
2.转染
将CHO细胞密度调整为1X106个细胞/ml，每瓶细胞液体积为40mL，然后旋紧瓶口放入摇床继续培养，在36.5℃，175rpm，5％CO2的条件下培养2-4小时后用质粒进行转染。配制转染液(1ml)：取约800μL的150mM灭菌的NaCl溶液稀释10μg DNA，混匀后在工作台放置5min；向DNA稀释液中加入约50μl的转染试剂混匀，最终转染液的总体积为1mL。在工作台放置10min后将转染液逐滴加入到细胞培养液中，摇匀后旋紧瓶口放回摇床(36.5℃，关闭5％CO2，175rpm)。转染20-24h后加入SMS293-I加料液(0.7mL/瓶)，以后隔天加料培养6-10天，然后收集培养上清液。
将收集的培养上清液进行梯度稀释(通常使用2倍稀释、5倍稀释)。然后将对应的靶肽和非相关肽分别加入DMSO中溶解，并用水稀释至使用浓度10-4M。用T2细胞分别负载10-6M的靶肽和非相关肽。向ELISPOT板中加入含10％FBS的1640完全培养基，在室温下封闭30min。弃去培养基，向板中加入5x105个细胞/mL的PBMC(100μL/孔)，5x105个细胞/mL的负载多肽的T2细胞(100μL/孔)，和不同浓度的format(2倍稀释、5倍稀释)。阴性对照加入不负载多肽的T2细胞和PBMC，候选多肽分子的浓度与实验组一致；阳性对照为PBMC加入10μL试剂盒自带的抗CD3-2的mAb作为阳性对照；非相关肽对照加入不相关多肽作为对照。加完所有样品后，盖好板盖，放在37℃、5％ CO2培养箱培养20-24小时后通过ELISPOT检测来确定IFN-γ的分泌，以评估本发明的format通过靶抗原肽-MHC复合物诱导的免疫细胞活化。
结果如图14所示。结果表明，CMM1实验组上清液在2倍和5倍稀释下均能激活免疫细胞，表现出明显强于野生型对照组的PBMC激活活性，这样的活性增加可能来源于蛋白产量、活性多方面的增益，表明CMM1突变可应用于多种不同的TCR融合蛋白中以提高融合蛋白的可溶表达。
3.2.6不同CMM组合效果验证
进一步，在CMM1突变组合的基础上对突变组合中的单个突变氨基酸位点逐个进行反向突变(突变为WT)，以期用尽量少的突变获得最好的增益效果，同时保留蛋白分子中更多的天然氨基酸序列。
有CMM1反向突变得到的组合突变包括CMM2(包含突变S21I、K83P、S84.6D、Q107L)、CMM3(包含突变S21I、K83P、S84.6D、T94D)、CMM4(包含突变K83P、S84.6D、T94D、Q107L)、CMM5(包含突变S21I、K83P、T94D、Q107L)。
使用装载有KRAS特异性TCR CR-KVA11-N03(其氨基酸序列如实施例3.2.4所示)的B62结构(称为CR-KVA11-N03_B62)作为载体，将上述CMM突变引入CR-KVA11-N03_B62分子中，如实施例3.2.2中所述进行蛋白表达和SDS-PAGE，并根据SDS-PAGE结果计算蛋白产量。结果如图15所示。
蛋白表达结果显示，TRAC突变序列S21>I、S84.6>D反向突变后，可溶蛋白分子表达产量显著降低；TRBC突变序列T94>D反向突变后，可溶蛋白分子表达产量没有发生显著变化；Q107>L反向突变后，蛋白产量反而更高。
以上结果表明，虽然各单点突变在三维构象上的空间距离都比较远，单位点突变组合后部分突变表现出积极的正向协同作用，比如TRAC中的S21>I和S84.6>D；也有部分突变并未积极参与贡献，比如TRBC中的T94>D；甚至TRBC中的Q107>L参与组合后反而会导致表达产量的降低。
本申请参考了各种发行的专利、公开的专利申请、期刊文章和其他出版物，将所有这些引入本申请作为参考。若任何引入的参考文献和本说明书有冲突，则以本说明书为准。此外，落入现有技术范围的本发明的任何具体实施方案可以明确地从任何一个或多个权利要求中排除。因为所述实施方案被认为是本领域技术人员已知的，它们可以被排除，即使所述排除没有在本申请中明确列出。本发明的任何具体实施方案可从任何权利要求中以任何理由排除，不管是否与现有技术的存在有关。
虽然已经参考其特定实施方案描述本发明，本领域的技术人员应当理解可以进行各种改变且可以替换等同物而不脱离本发明的真正的精神和范围。 另外，可作出许多修改以使特定的情况，材料，组合物，方法，方法步骤适于本发明的目的，精神和范围。所有这些修改都旨在权利要求的范围内。

Claims (43)

  1. T细胞受体(TCR)α链恒定区(TRAC)，其中所述α链恒定区(TRAC)在以下一个或多个位置处包含氨基酸取代：Q6、S21、K83、S84.6和I114；
    其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
  2. 根据权利要求1所述的TCRα链恒定区，其中：
    (1)所述α链恒定区中Q6位置处的氨基酸取代选自Q6R、Q6K、Q6D和Q6E，优选选自Q6R和Q6K；和/或
    (2)所述α链恒定区中S21位置处的氨基酸取代选自S21I、S21L、S21V、S21G、S21A和S21P，优选选自S21I、S21L和S21V；和/或
    (3)所述α链恒定区中K83位置处的氨基酸取代选自K83P、K83G、K83A、K83I、K83L和K83V，优选选自K83P、K83G和K83A；和/或
    (4)所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E、S84.6N、S84.6Q、S84.6K和S84.6R，优选选自S84.6D、S84.6E、S84.6N和S84.6Q；和/或
    (5)所述α链恒定区中I114位置处的氨基酸取代选自I114E、I114D、I114K和I114R，优选选自I114E和I114D。
  3. 根据权利要求1或2所述的TCRα链恒定区，其中：
    (1)所述α链恒定区中Q6位置处的氨基酸取代为Q6R；和/或
    (2)所述α链恒定区中S21位置处的氨基酸取代为S21I；和/或
    (3)所述α链恒定区中K83位置处的氨基酸取代为K83P；和/或
    (4)所述α链恒定区中S84.6位置处的氨基酸取代选自S84.6D、S84.6E和S84.6N；和/或
    (5)所述α链恒定区中I114位置处的氨基酸取代为I114E。
  4. 根据权利要求1-3中任一项所述的TCRα链恒定区，其中所述α链恒定区包含S21、K83和S84.6处的氨基酸取代。
  5. 根据权利要求1-4中任一项所述的TCRα链恒定区，其中所述α链恒定区包含以下氨基酸取代中的一种或多种：S21I、K83P和S84.6D；优选包含氨基酸取代S21I、K83P和S84.6D中的至少两种；更优选包含氨基酸取代S21I、K83P和S84.6D。
  6. 根据权利要求1-5中任一项所述的TCRα链恒定区，其中所述α链恒定区包含选自以下(1)-(4)中任一项的氨基酸取代：
    (1)S21I、K83P和S84.6D；
    (2)K83P和S84.6D；
    (3)S21I和S84.6D；
    (4)S21I和K83P。
  7. 根据权利要求1-6中任一项所述的TCRα链恒定区，其中所述α链恒定区还包含以下氨基酸突变中的一种或多种：N1.2K、T84C和120-124位FFPSP缺失突变，
    优选包含T84C以及可选的N1.2K和/或120-124位FFPSP缺失突变；
    更优选包含N1.2K、T84C和120-124位FFPSP缺失突变。
  8. 根据权利要求1-7中任一项所述的TCRα链恒定区，其中所述α链恒定区包含SEQ ID NO:3-13中任一项所示的氨基酸序列或与SEQ ID NO:3-13中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
  9. TCRα链恒定区(TRAC)，所述TCRα链恒定区(TRAC)包含氨基酸取代S21I、K83P和S84.6D，其中氨基酸位置根据SEQ ID NO:1所示的TCRα链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号；
    优选地，所述TCRα链恒定区包含SEQ ID NO:13所示的氨基酸序列或与SEQ ID NO:13具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
  10. TCRβ链恒定区(TRBC)，其中所述β链恒定区(TRBC)在以下一个或多个位置处包含氨基酸取代：S92、T94和Q107；
    其中氨基酸位置根据SEQ ID NO:14所示的TCRβ恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号。
  11. 根据权利要求10所述的TCRβ链恒定区，其中：
    (1)所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V、S92Q、S92K和S92R，优选选自S92P、S92E、S92D、S92L、S92N、S92G、S92A、S92I、S92V和S92Q；和/或
    (2)所述β链恒定区中T94位置处的氨基酸取代为T94D、T94E、T94K和T94R，优选选自T94D和T94E；和/或
    (3)所述β链恒定区中Q107位置处的氨基酸取代为Q107L、Q107I、Q107V、Q107G、Q107A和Q107P，优选选自Q107L、Q107I和Q107V。
  12. 根据权利要求10或11所述的TCRβ链恒定区，其中：
    (1)所述β链恒定区中S92位置处的氨基酸取代选自S92P、S92E、S92D、S92L和S92N；和/或
    (2)所述β链恒定区中T94位置处的氨基酸取代为T94D；和/或
    (3)所述β链恒定区中Q107位置处的氨基酸取代为Q107L。
  13. 根据权利要求10-12中任一项所述的TCRβ链恒定区，其中所述β链恒定区包含T94和Q107处的氨基酸取代。
  14. 根据权利要求10-13中任一项所述的TCRβ链恒定区，其中所述β链恒定区包含以下氨基酸取代中的一种或多种：T94D和Q107L；优选包含氨基酸取代T94D和Q107L。
  15. 根据权利要求10-14中任一项所述的TCRβ链恒定区，其中所述β链恒定区包含选自以下(1)-(3)中任一项的氨基酸取代：
    (1)T94D和Q107L；
    (2)Q107L；
    (3)T94D。
  16. 根据权利要求10-15中任一项所述的TCRβ链恒定区，其中所述TCRβ链恒定区还包含以下氨基酸突变中的一种或多种：S79C、C85.1A和N97D；
    优选包含S79C以及可选的C85.1A和/或N97D；
    更优选包含S79C、C85.1A和N97D。
  17. 根据权利要求10-16中任一项所述的TCRβ链恒定区，其中所述TCRβ链恒定区包含SEQ ID NO:16-23中任一项所示的氨基酸序列或与SEQ ID NO:16-23中任一项具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
  18. TCRβ链恒定区(TRBC)，所述TCRβ链恒定区(TRBC)包含氨基酸取代T94D和Q107L，其中氨基酸位置根据SEQ ID NO:14所示的TCRβ链恒定区，按照国际免疫遗传学信息系统(IMGT)的命名规则进行编号；
    优选地，所述TCRβ链恒定区包含SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
  19. TCR恒定区，其包含权利要求1-9中任一项所述的TCRα链恒定区(TRAC)和权利要求10-18中任一项所述的TCRβ链恒定区(TRBC)；
    优选地，所述TCR恒定区包含选自以下(1)-(6)中任一项的氨基酸取代：
    (1)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的T94D和Q107L；
    (2)所述α链恒定区中的K83P和S84.6D以及所述β链恒定区中的T94D和Q107L；
    (3)所述α链恒定区中的S21I和S84.6D以及所述β链恒定区中的T94D和Q107L；
    (4)所述α链恒定区中的S21I和K83P以及所述β链恒定区中的T94D和Q107L；
    (5)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的Q107L；
    (6)所述α链恒定区中的S21I、K83P和S84.6D以及所述β链恒定区中的T94D；
    更优选地，所述α链恒定区包含氨基酸取代S21I、K83P和S84.6D，并且所述β链恒定区包含氨基酸取代T94D和Q107L；
    进一步优选地，所述TCRα链恒定区包含SEQ ID NO:13所示的氨基酸序列或与SEQ ID NO:13具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列；和所述TCRβ链恒定区包含SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、至少85％、至少90％、至少95％或至少99％序列同一性的氨基酸序列。
  20. 多肽分子，其包含权利要求1-9中任一项所述的TCRα链恒定区、权利要求10-18中任一项所述的TCRβ链恒定区、或权利要求19所述的TCR恒定区。
  21. 根据权利要求20所述的多肽分子，其还包含结合至少一个抗原的抗原结合区。
  22. 根据权利要求21所述的多肽分子，其中所述抗原结合区选自来源于抗体的抗原结合区和来源于TCR的抗原结合区。
  23. T细胞受体(TCR)，所述TCR包含α链和β链，其中所述α链包含权利要求1-9中任一项所述的TCRα链恒定区，和/或所述β链包含权利要求10-18中任一项所述的TCRβ链恒定区；或所述TCR包含权利要求19所述的TCR恒定区。
  24. 根据权利要求23所述的TCR，其中所述TCR结合选自以下的抗原：黑色素瘤相关抗原(例如gp100、MAGEA1)、HPV抗原(例如HPV E6或E7)和KRAS抗原(例如KRAS G12V、KRAS G12D)。
  25. 根据权利要求23或24所述的TCR，其中所述TCR包含来源于鼠TCR或人TCR的可变区。
  26. 根据权利要求23-25中任一项所述的TCR，其中所述TCR是分离或纯化的。
  27. 多特异性抗原结合分子，其包含权利要求1-9中任一项所述的TCRα链恒定区、权利要求10-18中任一项所述的TCRβ链恒定区、权利要求19所述的TCR恒定区、权利要求20-22中任一项所述的多肽分子、或权利要求23-26中任一项所述的TCR。
  28. 多特异性抗原结合分子，其包含至少一个来源于TCR的抗原结合区以及以下任意一项：
    (1)权利要求1-9中任一项所述的TCRα链恒定区(TRAC)和/或权利要求10-18中任一项所述的TCRβ链恒定区(TRBC)；
    (2)权利要求19所述的TCR恒定区。
  29. 根据权利要求28所述的多特异性抗原结合分子，其中所述多特异性抗原结合分子包含结合两个或更多个抗原的两个或更多个抗原结合区，并且所述两个或更多个抗原各自独立地选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原、自身抗原、和免疫细胞表面分子；
    优选地，所述两个或更多个抗原中的一个选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原；另一个为免疫细胞表面分子；
    更优选地，所述TAA为黑色素瘤相关抗原(例如gp100、MAGEA1)；和/或
    所述TSA为KRAS抗原(例如KRAS G12V、KRAS G12D)；和/或
    所述病毒抗原为HPV抗原(例如HPV E6或E7)；和/或
    所述免疫细胞表面分子选自CD3、CD28、4-1BB(CD137)、PD-1和PD-L1。
  30. 根据权利要求28或29所述的多特异性抗原结合分子，其中所述多特异性抗原结合分子在至少两条多肽链上包含结合第一抗原的第一结合区和结合第二抗原的第二结合区，
    其中所述第一结合区包含来源于与所述第一抗原-MHC复合物结合的TCR的α链可变区(TRAV)和β链可变区(TRBV)；
    其中所述第二结合区包含来源于与所述第二抗原结合的抗体的重链可变区(VH)和轻链可变区(VL)；
    其中所述第一抗原选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原，且所述第二抗原为免疫细胞表面分子；
    其中所述多特异性抗原结合分子还包含以下至少一种功能结构域：
    1)源于抗体的铰链区；
    2)源于抗体的Fc结构域或其二聚化部分；
    3)源于抗体的CH3结构域；
    4)白蛋白(Alb)或其结合部分；
    5)源于TCR的恒定区或其片段。
  31. 根据权利要求30所述的多特异性抗原结合分子，其中所述TRAC和所述TRBC分别通过任选的接头连接至所述TRAV和所述TRBV的C端；或所述TRAC和所述TRBC分别通过任选的接头连接至所述TRBV和所述TRAV的C端；和/或
    所述抗体的N端连接在所述TCR的C端。
  32. 根据权利要求30或31所述的多特异性抗原结合分子，其中所述功能结构域来源于抗体的铰链区和/或抗体的Fc结构域或其二聚化部分和/或抗体的CH3结构域和/或源于TCR的恒定区或其片段；并且所述多特异性抗原结合分子的任意两条多肽链通过所述功能结构域两条链之间的共价结合或非共价结合而连接；
    优选地，所述功能结构域的两条链通过任选的接头分别连接在所述多特异性抗原结合分子的任意两条多肽链的C末端或N末端，优选C末端；或
    所述第一结合区和所述第二结合区通过所述功能结构域两条链之间的共价结合或非共价结合而连接；
    更优选地，所述多特异性抗原结合分子包含第一多肽链和第二多肽链，其中
    所述第一多肽链从N端到C端包含：TRBV-任选的接头-TRBC-接头-VL-接头-VH-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
    所述第一多肽链从N端到C端包含：TRAV-任选的接头-TRAC-接头-VL-接头-VH-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRBV-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3；或
    所述第一多肽链从N端到C端包含：TRBV-任选的接头-TRBC-接头-VH-接头-VL-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRAV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3；或
    所述第一多肽链从N端到C端包含：TRAV-任选的接头-TRBC-接头-VH-接头-VL-任选的接头-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：TRBV-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3；或
    所述多特异性抗原结合分子包含第一多肽链、第二多肽链和第三多肽链，其中所述第一多肽链从N端到C端包含：TRBV-任选的接头-TRBC-接头-VL，所述第二多肽链从N端到C端包含：VH-任选的接头-铰链区-CH2-CH3，并且所述第三多肽链从N端到C端包含：TRAV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。
  33. 核酸，其编码权利要求1-9中任一项所述的TCRα链恒定区、权利要求10-18中任一项所述的TCRβ链恒定区、权利要求19所述的TCR恒定区、权利要求20-22中任一项所述的多肽分子、权利要求23-26中任一项所述的TCR、或权利要求27-32中任一项所述的多特异性抗原结合分子。
  34. 载体，其包含根据权利要求33的核酸。
  35. 根据权利要求34所述的载体，其中所述载体选自慢病毒载体、逆转录病毒载体、质粒、DNA载体、mRNA载体、基于转座子的载体和人工染色体。
  36. 宿主细胞，其包含权利要求1-9中任一项所述的TCRα链恒定区、权利要求10-18中任一项所述的TCRβ链恒定区、权利要求19所述的TCR恒定区、权利要求20-22中任一项所述的多肽分子、权利要求23-26中任一项所述的TCR、权利要求27-32中任一项所述的多特异性抗原结合分子、根据权利要求33所述的核酸、或根据权利要求34或35所述的载体。
  37. 缀合物，其包含权利要求20-22中任一项所述的多肽分子、权利要求23-26中任一项所述的TCR或根据权利要求27-32中任一项所述的多特异性抗原结合分子，以及与所述多肽分子、所述TCR或所述多特异性抗原结合分子缀合的化学部分。
  38. 根据权利要求37所述的缀合物，其中所述化学部分选自可检测标记、免疫刺激分子和治疗剂；
    优选地，所述可检测标记选自生物素、链霉抗生物素蛋白、酶或其催化活性片段、放射性核素、纳米颗粒、顺磁性金属离子、核酸探针、造影剂、和萤光、磷光或化学发光分子；
    优选地，所述免疫刺激分子选自细胞因子、趋化因子、血小板因子和补体启动剂；
    优选地，所述治疗剂选自免疫调节剂、放射性化合物、酶、化学治疗剂和毒素。
  39. 组合物，其包含权利要求20-22中任一项所述的多肽分子、权利要求23-26中任一项所述的TCR、权利要求27-32中任一项所述的多特异性抗原结合分子、根据权利要求33所述的核酸、根据权利要求34或35所述的载体、根据权利要求36所述的宿主细胞、或根据权利要求37或38所述的缀合物；以及药学上可接受的载体或赋形剂。
  40. 根据权利要求39的组合物，其中所述组合物还包含第二治疗剂，优选地，所述第二治疗剂选自抗体、化疗剂和小分子药物。
  41. 治疗受试者的疾病的方法，其包括向所述受试者施用有效量的权利要求20-22中任一项所述的多肽分子、权利要求23-26中任一项所述的TCR、权利要求27-32中任一项所述的多特异性抗原结合分子、根据权利要求33所述的核酸、根据权利要求34或35所述的载体、根据权利要求36所述的宿主细胞、根据权利要求37或38所述的缀合物、或根据权利要求39或40所述的组合物。
  42. 根据权利要求41所述的方法，其中所述疾病选自癌症、感染性疾病、自身免疫病和炎性疾病。
  43. 根据权利要求41或42所述的方法，其中所述方法还包括施用第二治疗剂，优选地，所述第二治疗剂选自抗体、化疗剂和小分子药物。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1279690A (zh) * 1997-10-02 2001-01-10 苏诺尔分子公司 可溶性单链t细胞受体蛋白
WO2004074322A1 (en) * 2003-02-22 2004-09-02 Avidex Ltd Modified soluble t cell receptor
CN102574906A (zh) * 2009-07-03 2012-07-11 英美偌科有限公司 T细胞受体
WO2012131555A2 (en) * 2011-03-25 2012-10-04 Glenmark Pharmaceuticals S.A. Hetero-dimeric immunoglobulins
US20180355012A1 (en) * 2014-11-07 2018-12-13 Guangdong Xiangxue Life Science, Ltd. Soluble heterodimeric t cell receptor, and preparation method and use thereof
CN109328196A (zh) * 2016-05-09 2019-02-12 奥斯陆大学医院Hf 识别TGFβRII的移码突变体的T细胞受体

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1279690A (zh) * 1997-10-02 2001-01-10 苏诺尔分子公司 可溶性单链t细胞受体蛋白
WO2004074322A1 (en) * 2003-02-22 2004-09-02 Avidex Ltd Modified soluble t cell receptor
CN1745099A (zh) * 2003-02-22 2006-03-08 阿维德克斯有限公司 修饰的可溶性t细胞受体
CN102574906A (zh) * 2009-07-03 2012-07-11 英美偌科有限公司 T细胞受体
WO2012131555A2 (en) * 2011-03-25 2012-10-04 Glenmark Pharmaceuticals S.A. Hetero-dimeric immunoglobulins
US20180355012A1 (en) * 2014-11-07 2018-12-13 Guangdong Xiangxue Life Science, Ltd. Soluble heterodimeric t cell receptor, and preparation method and use thereof
CN109328196A (zh) * 2016-05-09 2019-02-12 奥斯陆大学医院Hf 识别TGFβRII的移码突变体的T细胞受体

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