WO2023246911A1 - T cell receptor-based bispecific polypeptide molecule and use thereof - Google Patents

T cell receptor-based bispecific polypeptide molecule and use thereof Download PDF

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WO2023246911A1
WO2023246911A1 PCT/CN2023/101881 CN2023101881W WO2023246911A1 WO 2023246911 A1 WO2023246911 A1 WO 2023246911A1 CN 2023101881 W CN2023101881 W CN 2023101881W WO 2023246911 A1 WO2023246911 A1 WO 2023246911A1
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polypeptide
terminus
antigen
amino acid
linker
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Chinese (zh)
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蒋栋
谢兴旺
李永红
王江华
王雪艳
闫宝琪
毕晶磊
党子懿
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北京可瑞生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/18—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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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

Definitions

  • the present invention relates to T cell receptor-based bispecific polypeptide molecules and their uses, particularly in the treatment of cancer, infectious diseases, autoimmune diseases and/or inflammatory diseases.
  • T cells are an important component of the adaptive immune system of vertebrates and play a crucial role in viral infection, cancer, and autoimmunity.
  • TCR recognition of major histocompatibility complex (MHC)-antigen peptide complex (pMHC) is an important step in T cell-mediated immune response.
  • MHC major histocompatibility complex
  • pMHC antigen peptide complex
  • the non-covalent binding of TCR to the CD3 signaling apparatus consisting of ⁇ , ⁇ , ⁇ and ⁇ subunits forms the TCR–CD3 complex. Binding of pMHC to the TCR activates LCK-induced phosphorylation of the intracellular immunoreceptor tyrosine-based activation motif (ITAM) in the CD3 ⁇ subunit of the TCR.
  • ITAM immunoreceptor tyrosine-based activation motif
  • CD3 ⁇ phosphorylation then triggers a cascade of phosphorylation signaling involving ⁇ chain-associated protein kinase 70 (ZAP70) and the activated T cell adapter (LAT) in T cells, causing the recruitment of multiple downstream adapters and signaling molecules as well as LAT Formation of the signal body.
  • the assembled LAT signalosome activates a variety of signaling pathways involving transcription factors, such as activating protein 1 (AP-1), nuclear factor- ⁇ B (NF- ⁇ B), and nuclear factor of activated T cells (NFAT).
  • AP-1 activating protein 1
  • NF- ⁇ B nuclear factor- ⁇ B
  • NFAT nuclear factor of activated T cells
  • Activated transcription factors can cause subsequent T cell activation, proliferation, cytokine production, and effector functions.
  • Bispecific antibody refers to an antibody molecule that targets two antigens at the same time or targets two different epitopes of one antigen. Compared with ordinary antibodies, bispecific antibodies have stronger specificity, can more accurately target tumor cells, and reduce off-target toxicity. With the development of recombinant protein expression technology and antibody engineering technology, many different antibody forms have been produced. Multispecific antibodies are used for a variety of purposes, including (1) receptor activation (2) blocking (3) internalization (4) aggregation, (5) binding of membrane-associated proteins, or (6) cytotoxic effector cells Orientation target.
  • T cell repositioning bsAb (also known as T cell engager, TcE) is an important form of cytotoxic effector cell repositioning and is the central pillar of current cancer immunotherapy.
  • This bsAb recognizes the target on the surface of tumor cells and also recognizes a molecule on the surface of T cells (the molecule is CD3 in most cases), so that the bsAb can couple tumor cells and cytotoxic T cells together to Causes the activation of T cell TCR downstream signaling pathways and kills tumor cells through T cell cytotoxicity.
  • Blincyto (blinatumomab) is a CD19/CD3 bispecific antibody that leads to complete remission in 69% of patients with relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL).
  • ALL relapsed/refractory B-precursor acute lymphoblastic leukemia
  • a large number of new T cell relocalizing antibody forms have emerged, such as BITE, BITE-Fc, DART-Fc, TriTAC, etc.
  • TCR can recognize the antigen peptides of intracellular and cell surface tumor-specific antigens that are processed and presented on the cell surface MHC molecules, and the recognition range is wider.
  • the target cell recognition region in TCR-based T cell repositioning bispecific antibodies is changed from traditional antibodies to TCR, so that the broad recognition properties of TCR can be used to increase target selectivity.
  • the potency of TcE against TCR of target cell targets can also be increased, and the potency of TcE against surface molecules on effector cells can also be increased to enhance T cell killing activity and proliferation ability.
  • the molecular size of TcE needs to be maintained within a certain range.
  • the glomerular filtration limit is generally around 60kDa.
  • Nanobodies 15kDa
  • scFv 28kDa
  • Fab 50kDa
  • Nanobodies have the smallest molecular weight among all antibody types, and their half-lives are often only tens of minutes. Therefore, in order to increase the efficacy of protein drugs, it is necessary to improve protein drugs to extend their half-life and increase activity.
  • Appropriately adding other functional domains to the polypeptide molecule can not only extend the length of the polypeptide molecule
  • the half-life can also enhance the killing activity of polypeptide molecules.
  • the present invention provides TCR-based bispecific polypeptide molecules.
  • the bispecific polypeptide molecule of the present invention adopts a specific antigen-binding region connection method and is combined with the TCR constant region (TRAC and TRBC). This combination with the TCR constant region significantly improves the stability of the molecule. This combination of each structural domain The specific combination achieved highly efficient immune cell activation and was significantly more effective than using the constant region of the antibody (CH3, hinge region-CH2-CH3, or CH1/CL).
  • the term “comprises” and variations thereof such as “includes” and “contains” shall be understood to mean the inclusion of stated elements or steps but not the exclusion of any other elements or steps.
  • the term “consisting of” is considered to be a preferred embodiment of the term “comprising”. If a group is defined below as including or containing at least a certain number of embodiments, this is also to be understood as disclosing a group that preferably consists exclusively of these embodiments.
  • T cell receptor or "TCR” as used herein includes native TCR as well as TCR variants, fragments and constructs. The term therefore includes heterodimers as well as multimeric and single-chain constructs comprising TCR alpha and TCR beta chains; optionally including other domains and/or portions, so long as the TCR retains its ability to recognize the antigenic target.
  • TCR In its native form, the TCR exists as a complex of several proteins on the surface of T cells.
  • T cell receptors are composed of two (separate) protein chains produced by separate T cell receptor alpha and beta (TCR ⁇ and TCR ⁇ ) genes and are called alpha and beta chains.
  • Each chain of the TCR has an N-terminal immunoglobulin-like (Ig)-variable (V) region/domain and an Ig-constant (C) region/domain that anchors the chain across the membrane in the plasma membrane. /membrane spanning region, and a short cytoplasmic tail at the C-terminus.
  • Ig immunoglobulin-like
  • V immunoglobulin-like
  • C Ig-constant
  • variable regions of the alpha and beta chains (TRAV and TRBV). Both variable regions of the TCR ⁇ chain and ⁇ chain contain three hypervariable or complementarity determining regions (CDR1 ⁇ / ⁇ , CDR2 ⁇ / ⁇ and CDR3 ⁇ / ⁇ ) surrounded by framework (FR) regions.
  • CDR3 is the major determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 interact primarily with MHC molecules presenting antigenic peptides.
  • the TCR recognizes an antigenic peptide that binds to a major histocompatibility complex (MHC) molecule at the surface of the antigen-presenting cell ("presented/displayed on the MHC molecule").
  • MHC major histocompatibility complex
  • Antigenic peptides presented on MHC molecules are also referred to herein as “antigenic peptides” "Epitope-MHC complex”, “epitope-MHC complex”, "antigen-MHC complex” or "target antigen peptide-MHC complex”.
  • MHC I and MHC II which presents peptides from different cellular 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. In humans, MHC is also called human leukocyte antigen (HLA). There are three main types of MHC class I: HLA-A, HLA-B, and HLA-C. Once the TCR binds to its specific epitope-MHC complex, the T cell is activated and functions Biological effect function.
  • TCR constant region includes the TCR alpha chain constant region (TRAC) and the beta chain constant region (TRBC), which may be human constant regions or derived from another species, such as murine.
  • the sequence of the wild-type TCR constant region can be found in the public database of the International Immunogenetic Information System (IMGT).
  • IMGT International Immunogenetic Information System
  • the constant domain sequence of the ⁇ chain of the TCR molecule is "TRAC*01”
  • the constant domain sequence of the ⁇ chain of the TCR molecule is " TRBC1*01” or "TRBC2*01”.
  • the positions of the amino acid sequences of the wild-type TCR in the present invention are numbered according to the naming rules of the International Immunogenetic Information System (IMGT). For example, if a certain amino acid in the TCR ⁇ chain variable region (TRAV) has a position number of 104 listed in IMGT, it is described in the present invention as the 104th amino acid of TRAV; in the TCR ⁇ chain variable region (TRBV) For a certain amino acid, if the position number listed in IMGT is 84, it will be described as the 84th amino acid of TRBV in the present invention; for others, the same applies.
  • IMGT International Immunogenetic Information System
  • an amino acid in the constant region of TCR ⁇ chain is 48, then it is described in this article as the 48th amino acid of TRAC;
  • an amino acid in the constant region of TCR ⁇ chain (TRBC) is 57, then it is described in this article as the 57th amino acid of TRBC; others are deduced by analogy.
  • TRBC constant region of TCR ⁇ chain
  • the special instructions will apply.
  • antibody refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen.
  • Such molecules typically contain two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (or domain) (herein abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (or domain) (herein abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the third step in the classical pathway of complement activation). one component).
  • the heavy chain of immunoglobulins can be divided into three functional regions: Fd region, hinge region and Fc region (crystallizable fragment).
  • the Fd region contains VH and CH1 domains and combines with the light chain to form Fab (antigen-binding fragment).
  • the Fc region also called an Fc domain, contains two or three heavy chain constant regions (CH2, CH3, CH4) in each heavy chain. In IgG class antibodies, the Fc region contains CH2 and CH3 domains.
  • the Fc regions of the two heavy chains dimerize to form the dimerization part.
  • the Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to cognate Fc receptors on effector cells.
  • the hinge region of IgG antibodies refers to the short amino acid sequence region between the CH1 and CH2 parts of the heavy chain, which is relatively flexible in the natural state of the antibody.
  • the hinge region found in the IgG, IgA and IgD immunoglobulin classes acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region.
  • Hinge domains are structurally diverse, varying in sequence and length between immunoglobulin classes and subclasses. Based on crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions structurally and functionally: upper hinge, core hinge, and lower hinge (Shin et al., Immunological Reviews 130:87, 1992).
  • the upper hinge includes from the carboxyl terminus of CH1 to the first residue in the hinge that limits movement.
  • Amino acid usually the first cysteine residue that forms an interchain disulfide bond between two heavy chains.
  • the length of the upper hinge region correlates with the fragment flexibility of the antibody.
  • the core hinge region contains inter-heavy chain disulfide bonds.
  • the lower hinge region connects the amino terminus of the CH2 domain and includes residues in the CH2 domain.
  • the core hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO:7) which when dimerized through disulfide bonds results in a cyclic octapeptide, which is believed to act as a pivot, Thus imparting flexibility.
  • the structure and flexibility of the immunoglobulin hinge region polypeptide sequence allow for conformational changes that can affect the effector function of the Fc portion of the antibody.
  • a “light chain variable region (VL)” or “heavy chain variable region (VH)” consists of a “framework” region interspersed with three “complementarity determining regions” or “CDRs".
  • the framework region is used to adjust the CDR for specific binding to the antigenic epitope.
  • the CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number.
  • the present disclosure may use CDRs defined according to any of these numbering systems, but the preferred embodiment uses CDRs defined by Kabat.
  • antibody is to be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments and multispecific antibodies containing at least two antigen-binding regions (e.g., bispecific antibodies). Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing the antigenic determinants of the antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, so long as these antibodies exhibit the intended biological activity.
  • antigen-binding fragment of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • antigen-binding fragments include, but are not limited to (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments, including Two Fab fragments connected by a disulfide bond in the hinge region; (iii) Fab' fragment, which is essentially a Fab but has a partial hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed.
  • the two domains of the Fv fragment, VL and VH are encoded by independent genes, They can be linked using recombinant methods via synthetic linkers that enable them to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (termed a single-chain Fv (ScFv); see e.g. Bird et al. Human (1988) Science 242, 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883).
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody Within.
  • the term also includes "linear antibodies” containing a pair of tandem Fd fragments (VH-CH1-VH-CH1) formed with a complementary light chain polypeptide and modified versions of any of the foregoing fragments that retain antigen-binding activity Antigen binding region.
  • antigen refers to any substance capable of inducing an immune response in the body. That is, it can be specifically recognized and combined with the antigen receptor (TCR/BCR) on the surface of T/B lymphocytes, activate T/B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can Substances that specifically bind to corresponding products in vivo and in vitro.
  • TCR/BCR antigen receptor
  • the antigen may be 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
  • tumor-associated antigen refers to an antigen that is differentially expressed (eg, significantly increased in expression on tumor cells) compared to normal cells.
  • TSA tumor-specific antigen
  • viral antigen refers to a substance in a virus that can induce an immune response in the body.
  • autoantigen refers to antigenic substances that cause changes in the structure and components of self-tissue cells caused by biological, physical, and chemical factors, thereby causing the body's immune system to produce an immune response against these self-tissue cell components.
  • the release of cryptic antigens generates an immune response.
  • Changes in its own composition may produce autoantigens.
  • denatured IgG can stimulate the body to produce anti-denatured IgG antibodies (rheumatoid factor), causing rheumatoid arthritis.
  • the clinical use of certain drugs can change the antigenicity of blood cell surface, causing autoimmune hemolytic anemia or neutropenia. Cross-reactions triggered by common antigens can generate immune responses.
  • Certain bacteria and viruses have similar antigenic determinants to certain tissue cells of the normal human body. Autoantibodies and sensitized lymphocytes produced against the antigenic determinants of these bacteria and viruses can cross-react with their own tissue cells, causing autoimmunity. disease.
  • Immuno cell surface molecules may include, for example, immune cell surface antigens (eg, T cell antigens) and costimulatory molecules.
  • Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed by consecutive amino acids (also called linear epitopes) are usually retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also called conformational epitopes) are usually lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the minimal binding site of the TCR or antibody and is therefore the specific target of the TCR or antibody or its antigen-binding fragment.
  • albumin encompasses any naturally occurring albumin from any vertebrate source, including mammals, such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). mouse). Albumin also encompasses the full-length, unprocessed preproalbumin protein as well as any form of albumin resulting from processing in the cell. Albumin also encompasses variants of naturally occurring albumin, such as splice variants or allelic variants. The term also covers any recombinant form of albumin. Albumin sequences are known in the art. Information on the human serum albumin gene (including genomic DNA sequence) can be found, for example, at NCBI.
  • the amino acid sequence of an exemplary full-length human serum albumin preproprotein can be found under NCBI accession number NP_000468.1.
  • Human serum albumin is synthesized in the form of prealbumin.
  • Mature albumin is a single-chain polypeptide of 585 amino acid residues with a molecular weight of 66,458. The molecule contains 17 disulfide bonds and does not contain sugar components.
  • albumin is a negative ion, and each molecule can carry more than 200 negative charges.
  • About 10% of exogenously infused albumin enters the extravascular tissue space after 2 hours, and reaches equilibrium in 20 days. The half-life of albumin in plasma is 15-19 days.
  • sequence identity refers to the extent to which two sequences (amino acids) aligned have identical residues at the same positions.
  • amino acid sequence is X% identical to SEQ ID NO:Y
  • sequence residues in the amino acid sequence are identical to SEQ ID NO :
  • sequence residues disclosed in Y are identical.
  • Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al. People, 1984).
  • Such conservative amino acid substitutions are well known in the art, for example WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferably) these substitutions
  • the type and/or combination may be selected based on the relevant teachings from WO 04/037999 and WO 98/49185 and further references cited therein.
  • Such conservative substitutions are preferably substitutions in which one amino acid of the following groups (a) to (e) is replaced by another amino acid residue of the same group: (a) small aliphatic, non-polar or weakly polar Residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His To Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.
  • vector is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell in which said nucleic acid molecule as a vector can, for example, be replicated and/or expressed.
  • host cell refers to any type of cell into which an expression vector has been introduced thereby capable of expressing foreign genetic material.
  • pharmaceutically acceptable means that the carrier or adjuvant is compatible with the other ingredients of the composition and is not substantially toxic to the recipient thereof, and/or that such carrier or adjuvant is approved or available for inclusion in parenteral administration to humans. in pharmaceutical compositions of medicines.
  • treatment refers to administering an agent or performing a procedure in order to obtain an effect. These effects may be prophylactic in the sense of completely or partially preventing the disease or its symptoms, and/or may be therapeutic insofar as affecting the partial or complete cure of the disease and/or the symptoms of the disease.
  • treating may include treating a disease or condition in a mammal, particularly a human (eg, cancer, infectious disease, autoimmune disease or inflammatory disease), and includes This includes: (a) preventing the occurrence of a disease or symptoms of a disease in subjects who are susceptible to the disease but have not yet been diagnosed with the disease (e.g., including diseases that may be associated with or caused by the primary disease); (b) ) inhibits the disease, i.e. prevents its progression; (c) alleviates the disease, i.e. causes the regression of the disease.
  • a mammal particularly a human (eg, cancer, infectious disease, autoimmune disease or inflammatory disease)
  • Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as elimination; remission; reduction of symptoms or making disease symptoms more tolerable for the patient; slowing of progression or decline ; or reduce the endpoint of worsening frailty. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination.
  • treating includes administering a multispecific polypeptide molecule or pharmaceutical composition or conjugate disclosed herein to prevent or delay, alleviate, or prevent or inhibit the development of symptoms or conditions associated with a disease, such as cancer.
  • therapeutic effect refers to the reduction, elimination, or prevention of disease, disease symptoms, or disease side effects in a subject.
  • the term "effective amount” as used herein means an amount of a therapeutic agent that when administered to a subject for the treatment or prevention of a disease is sufficient to effect such treatment or prevention.
  • the “effective amount” may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated.
  • “Therapeutically effective amount” refers to an amount effective for therapeutic treatment.
  • a “prophylactically effective amount” refers to an amount effective for prophylactic treatment.
  • the term "subject” refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired.
  • "Mammal” for therapeutic purposes means any animal classified as a mammal, including humans, domestic animals, and laboratory, zoo, sporting or pet animals, such as dogs, horses, cats, cattle, sheep, Goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.
  • the invention provides a bispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on one or more polypeptide chains. district,
  • the first binding region comprises an alpha chain variable region (TRAV) and a beta chain variable region (TRBV) derived from a T cell receptor (TCR) bound to the first antigen-MHC complex;
  • TRAV alpha chain variable region
  • TRBV beta 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 TRAV, TRBV, VH and VL are distributed on the one or more polypeptide chains, so that when the one or more polypeptide chains are folded, the TRAV and TRBV are spatially close to form the first a binding area, and the VH and VL are spatially close to form a second binding area;
  • the first antigen is selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens, and the second antigen is an immune cell surface molecule.
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • viral antigens and autoantigens
  • the second antigen is an immune cell surface molecule.
  • the TRAV, TRBV, VH and VL are distributed on the one or more polypeptide chains and adjacent variable regions on the same chain are separated by linker sequences.
  • the bispecific polypeptide molecule comprises a first binding region that binds a first antigen and a second binding region that binds a second antigen on both polypeptide chains,
  • the first binding region comprises an alpha chain variable region (TRAV) and a beta chain variable region (TRBV) derived from a T cell receptor (TCR) bound to the first antigen-MHC complex;
  • TRAV alpha chain variable region
  • TRBV beta 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 the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens, and the second antigen is an immune cell surface molecule;
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • viral antigens and autoantigens
  • the second antigen is an immune cell surface molecule
  • the two polypeptide chains of the bispecific polypeptide molecule respectively include:
  • the bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof linked to the C-terminus of each polypeptide chain via an optional linker.
  • the two polypeptide chains of the bispecific polypeptide molecule comprise: TRAV-VH and VL-TRBV respectively.
  • the two polypeptide chains of the bispecific polypeptide molecule respectively comprise: TRAV-VH and VL-TRBV, and adjacent variable regions in the two polypeptide chains are connected by a linker; and
  • the bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof linked to the C-terminus of each polypeptide chain via an optional linker.
  • the TCR constant region is selected from the group consisting of TCR alpha chain constant region (TRAC) and TCR beta chain constant region (TRBC).
  • TRAC TCR alpha chain constant region
  • TRBC TCR beta chain constant region
  • one polypeptide chain of the bispecific polypeptide molecule comprises TRAC and the other polypeptide chain comprises TRBC.
  • 1-5 amino acids at the N-terminus of TRAC may be substituted.
  • 1-5 amino acids at the N-terminus of TRBC may be substituted.
  • TRAC and TRBC can be of human or murine origin.
  • TRAC and TRBC can be wild type or variants thereof.
  • the variant TRAC may comprise one or more of T48C, N113K, PESS deletion mutation, FFPSPESS deletion mutation relative to the wild-type sequence.
  • the variant TRBC may comprise one or more of S57C, C187A, N210D, and FG loop deletion mutations relative to the wild-type sequence.
  • TRAC and/or TRBC comprise at least one cysteine mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, more preferably, said cysteine mutation 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.
  • the polypeptide molecule comprises TRAC or a fragment thereof, which is not cis-linked to TRAV through a linker.
  • TRAC or a fragment thereof, which is not cis-linked to TRAV through a linker.
  • 1-5 amino acids at the N-terminus of TRAC can be substituted.
  • the polypeptide molecule comprises TRBC or a fragment thereof that is not cis-linked to TRBV through a linker.
  • TRBC or a fragment thereof that is not cis-linked to TRBV through a linker.
  • 1-5 amino acids at the N terminus of TRBC can be substituted.
  • cis-linked refers to the connection form of the TCR constant region and the variable region in the natural state.
  • 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-any The optional linker-TRAC, and the second polypeptide chain from the N-terminus to the C-terminus includes: VL-linker-TRBV-optional linker-TRBC (format 22-2).
  • 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-linker -TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-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-any The optional linker-TRBC, and the second polypeptide chain from N-terminus to C-terminus includes: 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-any The optional linker-TRAC, and the second polypeptide chain includes 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-any The optional linker-TRBC, and the second polypeptide chain from N-terminus to C-terminus includes: 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-any optional linker-TRAC, and the second polypeptide chain from N-terminus to C-terminus includes: 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-any The optional linker-TRBC, and the second polypeptide chain includes 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-any The optional linker-TRAC, and the second polypeptide chain includes 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-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC.
  • the bispecific polypeptide molecule further comprises at least one of the following functional domains:
  • the introduction of the functional domain can not only extend the half-life of the polypeptide molecule, but also enhance the killing activity of the polypeptide molecule.
  • the functional domain is linked to the C-terminus of one or both polypeptide chains of the bispecific polypeptide molecule via an optional linker. In some embodiments, the functional domain is linked to the C-terminus of either polypeptide chain of the bispecific polypeptide molecule through an optional linker. In some embodiments, the functional domain is linked to the C-termini of both polypeptide chains of the bispecific polypeptide molecule via an optional linker.
  • the functional domain is derived from albumin or a fragment thereof.
  • albumin or a fragment thereof is linked to the C-terminus of one or both polypeptide chains of the bispecific polypeptide molecule via an optional linker.
  • albumin or a fragment thereof is linked to the C-terminus of either polypeptide chain of the bispecific polypeptide molecule through an optional linker.
  • the albumin can be any mammalian source of albumin, such as human albumin, bovine albumin, mouse albumin, etc.
  • the albumin is serum albumin, such as human serum albumin or bovine serum albumin.
  • the functional domain may be a fragment of albumin, for example, 100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550, 450 in length -550, 500-550, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, 450-500, 100-450, 150-450, 200-450 ,250-450,300-450,350-450,400-450,100-400,150-400,200-400,250-400,300-400,350-400,100-350,150-350,200 -350, 250-350, 300-350, 100-300, 150-300, 200-300, 250-300, 100-250, 150-250, 200-250, 100-200, 150-200, 100-150 fragment of amino acids.
  • the albumin is human serum albumin, whose amino acid sequence is shown in SEQ ID NO: 9.
  • the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain contains from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain contains from N-terminus to C-terminus: VL-linker-TRBV- Optional Linker-TRBC-Optional Linker-Alb (Format64).
  • 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-linker -TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC-linker-Alb.
  • 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-any optional linker-TRAC-optional linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: 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-any Optional linker-TRBC-Optional linker-Alb, and the second polypeptide chain includes 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-VH-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC-optional linker-Alb.
  • 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-any optional linker-TRAC-optional linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: 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-any The optional linker-TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC-optional linker-Alb.
  • 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-any Optional linker-TRBC-Optional linker-Alb, and the second polypeptide chain includes 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: TRAV-linker-VL-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC-optional linker-Alb.
  • 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-any optional linker-TRAC-optional linker-Alb, and the second polypeptide chain includes 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-any optional linker-TRAC, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-TRBC-optional linker-Alb.
  • 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-any optional linker-TRBC-optional linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: 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-VH-any optional linker-TRBC, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-TRAC-optional linker-Alb.
  • 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-any optional linker-TRAC-optional linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: 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-any optional linker-TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC-optional linker-Alb.
  • 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-any Optional Linker-TRBC-Optional Linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: VH-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-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC-optional linker-Alb.
  • the functional domain is derived from the hinge region of an antibody and/or the Fc domain of an antibody or a dimerization portion thereof; and the functional domain is linked to the bispecific via an optional linker.
  • the hinge region may comprise part or all of the wild-type hinge sequence of the antibody or a variant thereof with one or more substitutions.
  • the hinge region of the antibody is that of a human antibody.
  • the hinge region of an antibody can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4.
  • the hinge region of an antibody may be derived from the hinge domain of human IgGl, IgG2 or IgG4, or a portion thereof.
  • the hinge region may comprise one of the following amino acid sequences: EPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO:69), EPKSSDKTHTCPPCP (SEQ ID NO:70), PKSSDKTHTCPPCPAPPVAGP (SEQ ID NO:71), KSSDKTHTCPPCPAPPVAGP (SEQ ID NO:72) ), SSDKTHTCPPCPAPPVAGP(SEQ ID NO:73), SDKTHTCPPCPAPPVAGP(SEQ ID NO:74), DKTHTCPPCPAPPVAGP(SEQ ID NO:75), KTHTCPPCPAPPVAGP(SEQ ID NO:76), THTCPPCPAPPVAGP(SEQ ID NO:77), HTCPPCPAPPVAGP(SEQ ID NO:78), TCPPCPAPPVAGP(SEQ ID NO:79), CPPCPAPPVAGP(SEQ ID NO:80), PPCPAPPVAGP(SEQ ID NO:81), CPAPPVAGP(SEQ ID NO:82), or PAPPVAGP(SEQ ID NO:69
  • the Fc domain of the antibody contains CH2 and CH3.
  • the Fc domain of an antibody, or a dimerization portion thereof is that of a human antibody, or a dimerization portion thereof.
  • the Fc domain of an antibody can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can contain one or more mutations or modifications.
  • the Fc domain of the antibody, or a dimerization portion thereof is derived from the Fc domain of human IgG1, IgG2, or IgG4, or a portion thereof, and preferably contains at least two cysteine residue mutations, such as S354C and Y349C or L242C and K334C.
  • the Fc domain is of the IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In another embodiment, the Fc domain is or derived from the IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc domain is human IgG1 Fc or human IgG4 Fc.
  • the functional domain comprises the hinge region-CH2-CH3 of an antibody.
  • both polypeptide chains of the bispecific polypeptide molecule comprise hinge regions -CH2-CH3.
  • CH3 in both polypeptide chains of the bispecific polypeptide molecule contains at least one mutation capable of promoting heterodimer formation of the polypeptide molecule.
  • the CH3s in both polypeptide chains each independently comprise mutations 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, preferably , one of the CH3s contains the T366W mutation and the other contains the T366S, L368A and Y407V mutations.
  • the amino acid sequence of the hinge region-CH2-CH3 is shown in SEQ ID NO: 33 or 34.
  • 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-any Optional Connector-TRAC-Optional Connector-Hinge region-CH2-CH3, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3 (format 63) .
  • 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-linker -TRAC-hinge region-CH2-CH3, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC-hinge region-CH2-CH3.
  • 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-any Selected linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRBV-optional linker-TRAC-optional Joint-hinge region-CH2-CH3.
  • 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-any selected linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRBV-optional linker-TRBC-optional Joint-hinge region-CH2-CH3.
  • 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-any Selected linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRBV-optional linker-TRAC-optional Joint-hinge region-CH2-CH3.
  • 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-any selected linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-TRBC-optional Joint-hinge region-CH2-CH3.
  • 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-any selected linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-TRAC-optional Joint-hinge region-CH2-CH3.
  • 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-any selected linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRAV-optional linker-TRBC-optional Joint-hinge region-CH2-CH3.
  • 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-any Selected linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRAV-optional linker-TRAC-optional Joint-hinge region-CH2-CH3.
  • the linkers when the polypeptide molecule comprises more than one linker, the linkers may each be the same or different. In some embodiments, the linkers in the polypeptide molecule are each independently selected from a linker consisting of 1-35 amino acids.
  • the linkers are each independently selected from the group consisting of S (SEQ ID NO:41), GGGS (SEQ ID NO:62), GGGGS (SEQ ID NO:42), GGGSGGGG (SEQ ID NO:50), GGSGGS (SEQ ID NO: 47), GGSGGGGGS (SEQ ID NO: 48), GGGSGGGGS (SEQ ID NO: 46), GGGGSGGGGSGGGGS (SEQ ID NO: 44), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 43), GGGGSGGGGSGGGGGGSGGGGS (SEQ ID NO: 43) :45), GQPKAAP (SEQ ID NO:49), TVLRT (SEQ ID NO:53), TVSSAS (SEQ ID NO:54), GGEGG (SEQ ID NO:55), GSEGGGS (SEQ ID NO:56), RTSGPGDGGKGGPGKGPGGEGTKGTGPGG (SEQ ID NO:57), GKGPGGEGTKGTGP
  • the variant of EDLKN is the amino acid sequence formed by EDLKN through substitution, deletion or addition of one or several amino acids
  • the variant of EDLNK is the amino acid sequence of EDLNK through substitution, deletion or addition of one or several amino acids.
  • the formed amino acid sequence; and/or the variant of ANIQK is the amino acid sequence formed by substituting, deleting or adding one or several amino acids of ANIQK.
  • the variant of EDLKN is the amino acid sequence formed by substituting one or several amino acids of EDLKN; and/or the variant of EDLNK is the amino acid sequence formed by substituting one or several amino acids of EDLNK; and/or the variation of ANIQK
  • the body is the amino acid sequence formed by ANIQK by substituting one or several amino acids.
  • the variant of EDLKN is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLNK.
  • An amino acid sequence consisting of 1-3 amino acids; and/or a variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids of ANIQK.
  • the variants of EDLKN are EDL, DL, L, ED, E, D; and/or the variants of ANIQK are ANI, NI, I, AN, N, A, PNI, P, PN.
  • the variant of EDLKN is an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to EDLKN (preferably at the N-terminus); and/or the variant of EDLNK is EDLNK after adding (preferably at the N-terminus) N-terminus) an amino acid sequence consisting of 6-35 amino acids formed by one or several amino acids; and/or the variant of ANIQK is ANIQK formed by adding (preferably at the N-terminus) one or several amino acids consisting of 6- An amino acid sequence consisting of 35 amino acids.
  • the linkers in the polypeptide molecule are each independently selected from a linker consisting of no more than 12 amino acids.
  • the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, EDLKN or variants thereof, EDLNK or variants thereof and ANIQK or variants thereof.
  • the variant of EDLKN is the amino acid sequence formed by EDLKN through substitution, deletion or addition of one or several amino acids; and/or the variant of EDLNK is the amino acid sequence of EDLNK through substitution, deletion or addition of one or several amino acids.
  • the formed amino acid sequence; and/or the variant of ANIQK is the amino acid sequence formed by substituting, deleting or adding one or several amino acids of ANIQK.
  • the variant of EDLKN is the amino acid sequence formed by substituting one or several amino acids of EDLKN; and/or the variant of EDLNK is the amino acid sequence formed by substituting one or several amino acids of EDLNK; and/or the variation of ANIQK
  • the body is the amino acid sequence formed by ANIQK by substituting one or several amino acids.
  • the variant of EDLKN is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLNK.
  • An amino acid sequence consisting of 1-3 amino acids; and/or a variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids of ANIQK.
  • the variant of EDLKN is an amino acid sequence consisting of 6-12 amino acids formed by adding one or several amino acids to EDLKN (preferably at the N-terminus); and/or the variant of EDLNK is EDLNK after adding (preferably at the N-terminus) N-terminus) an amino acid sequence consisting of 6-12 amino acids formed by one or several amino acids; and/or the variant of ANIQK is ANIQK formed by adding (preferably at the N-terminus) one or several amino acids consisting of 6- An amino acid sequence consisting of 12 amino acids.
  • the variants of EDLKN are EDL, DL, L, ED, E, D; and/or the variants of ANIQK are ANI, NI, I, AN, N, A, PNI, P, PN.
  • the linkers in the polypeptide molecule are each independently 1 or 2-3 or more of the linkers. Head combination. In some embodiments, each linker in the polypeptide molecule is independently one or more of the linkers. In some embodiments, each linker in the polypeptide molecule is independently a combination of two or more of the linkers. In some embodiments, each linker in the polypeptide molecule is independently a combination of three or more of the linkers.
  • the C-terminal of TRAV is connected to VH or VL through 2 linkers, and the proximal linker of TRAV is ANIQK or a variant thereof.
  • a variant of ANIQK is an amino acid sequence formed by substituting, deleting, or adding one or several amino acids to ANIQK.
  • the variant of ANIQK is the amino acid sequence formed by substituting one or several amino acids of ANIQK.
  • the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids of ANIQK, for example, ANI, NI, I, AN, N, A, PNI, P, PN.
  • the C-terminal of TRBV is connected to VH or VL through two linkers, and the proximal linker of TRBV is EDLKN or a variant thereof, EDLNK or a variant thereof, ANIQK or a variant thereof.
  • the variant of EDLKN is the amino acid sequence formed by EDLKN through substitution, deletion or addition of one or several amino acids; and/or the variant of EDLNK is the amino acid sequence of EDLNK through substitution, deletion or addition of one or several amino acids. formed amino acid sequence.
  • the variant of EDLKN is an amino acid sequence formed by substituting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence formed by substituting one or several amino acids of EDLNK.
  • the variant of EDLKN is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLNK.
  • Amino acid sequence consisting of 1-3 amino acids for example, EDL, DL, L, ED, E, D.
  • the C-terminus of TRAV is connected to TRAC through a linker, and the linker is ANIQK or a variant thereof.
  • a variant of ANIQK is an amino acid sequence formed by substituting, deleting, or adding one or several amino acids to ANIQK.
  • the variant of ANIQK is the amino acid sequence formed by substituting one or several amino acids of ANIQK.
  • ANIQK variants are ANIQK amino acid sequences consisting of 1-3 amino acids formed by deleting one or several amino acids, for example, ANI, NI, I, AN, N, A, PNI, P, PN.
  • the C-terminus of TRBV is connected to TRBC through a linker, and the linker is EDLKN or a variant thereof, or EDLNK or a variant thereof.
  • the variant of EDLKN is the amino acid sequence formed by EDLKN through substitution, deletion or addition of one or several amino acids; and/or the variant of EDLNK is the amino acid sequence of EDLNK through substitution, deletion or addition of one or several amino acids. formed amino acid sequence.
  • the variant of EDLKN is an amino acid sequence formed by substituting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence formed by substituting one or several amino acids of EDLNK.
  • the variant of EDLKN is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLNK.
  • Amino acid sequence consisting of 1-3 amino acids for example, EDL, DL, L, ED, E, D.
  • VH and VL comprise at least one cysteine mutation to form a disulfide bond between VH and VL, and the cysteine is introduced into FR4 in the case of VL and is introduced in the case of VH Introducing FR2.
  • the cysteine mutations are at positions 44 of VH and 100 of VL.
  • the TRAV and VL comprise at least one cysteine mutation to form a disulfide bond between TRAV and VL, preferably the cysteine mutation is at position 104 of TRAV bit and the 80th bit of VL.
  • the TRBV and VL comprise at least one cysteine mutation to form a disulfide bond between TRBV and VL, preferably the cysteine mutation is at position 84 of TRBV bit and the 80th bit of VL.
  • the TRAV and VH comprise at least one cysteine mutation to form a disulfide bond between TRAV and VH, preferably the cysteine mutation is at position 104 of TRAV position and VH's 89th.
  • the TRBV and VH comprise at least one cysteine mutation To form a disulfide bond between TRBV and VH, preferably, the cysteine mutation is at the following positions: position 104 of TRBV and position 89 of VH.
  • the TRAV may be native TRAV or a functional variant thereof, as long as the functional variant retains the ability to bind the target antigen.
  • TRAV may be a truncated variant of native TRAV, such as a variant with 8 amino acids truncated at the N- or C-terminus.
  • the TRBV may be native TRBV or a functional variant thereof, as long as the functional variant retains the ability to bind the target antigen.
  • the TRAV and TRBV are those of an ⁇ TCR.
  • the polypeptide molecule further contains a compound that can enhance its affinity or biological activity (e.g., enhance its solubility, enhance its aggregation, enhance its stability, extend its half-life, reduce its immunogenicity) or reduce its One or more amino acid mutations, insertions or deletions of post-translational modifications (eg glycosylation modifications), such as 1, 2, 3, 4, 5 or more amino acid mutations, insertions or deletions.
  • Mutated, inserted or deleted amino acids include but are not limited to: (a) neutral amino acids: Ser, Gln; (b) acidic amino acids: Asp; (c) long-chain aliphatic amino acids: Ile; (d) short aliphatic amino acids: Alas, Gly.
  • the amino acid mutations, insertions or deletions are located at the following amino acid positions: TRAV, TRBV, VL, beginning of VH, TRAV/TRAC junction amino acid, TRBV/TRBC junction amino acid, hinge region and adjacent amino acids , the boundary amino acid of CH2/CH3, the C terminus of CH3, and the amino acids near the linker.
  • the amino acid mutations include mutations that enhance binding to FcRn and/or effector function silencing mutations.
  • mutations capable of enhancing binding to FcRn include, for example, one or more of the following mutations: L234A, L235A, M252Y, S254T, T256E, M428L, N434S, T250R, M428L in the CH2 and CH3 domains , N434A, preferably L234AL235A, M252YS254TT256E, M428LN434S and T250RM428LN434A.
  • the effector function silencing mutation is at one or more of the following positions of the CH2-CH3 domain: 233, 234, 235, 236, 297, and 331; preferably, the effector function silencing mutation is obtained by using Derived from substitution of at least one residue at positions 233, 234, 235, 236 and 331 with corresponding residues from IgG2 or IgG4.
  • amino acid mutations that reduce post-translational modifications of the polypeptide molecule include, for example, N113K in TRAC and N210D in TRBC.
  • the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or both polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain, for example a signal peptide derived from albumin or immunoglobulin Sequence, preferably MGWSCIILFLVATATGVHS.
  • the first antigen is a TAA 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,
  • the first antigen is a TSA selected from: KRAS (eg, G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (eg, R175H, R173H, R273C, R248W, R248Q , R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (such as E542K, E545K, H1047R), CTNNB1 (such as S45P, T41A), EGFR (such as L858R, T790M), BRAF (such as V600E) and GNAS (such as R201C , R201H).
  • KRAS eg, G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125
  • TP53 eg, R175H, R173H, R273C
  • the first antigen is a viral antigen selected from the group consisting of: HPV E6 or E7 antigen, CMV antigen, HBV antigen, EBV antigen, herpes virus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen and coronavirus antigens.
  • the first antigen is an autoantigen selected from: 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, topological heterogeneity Structural enzyme 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, PR
  • the second antigen is selected from the group consisting of CD3 (e.g., CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ chains), 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 ⁇ RI, TCR ⁇ / ⁇ , TCR ⁇ / ⁇ , 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. CD80, BTLA, TIM3, TIGIT and LAG-3.
  • CD3
  • the first antigen is a TAA 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 antigen; and the second antigen is selected From CD3 (such as CD3 ⁇ , CD3 ⁇ and CD3 ⁇ chains), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42a
  • CD3
  • the first antigen is a TSA selected from: KRAS (eg, G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (eg, R175H, R173H, R273C, R248W, R248Q , R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (such as E542K, E545K, H1047R), CTNNB1 (such as S45P, T41A), EGFR (such as L858R, T790M), BRAF (such as V600E) and GNAS (such as R201C , R201H); and the second antigen is selected from the group consisting of CD3 (e.g., CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ chains), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18
  • CD3
  • the first antigen is a viral antigen selected from the group consisting of: HPV E6 or E7 antigen, CMV antigen, HBV antigen, EBV antigen, herpes virus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen and a coronavirus antigen; and the second antigen is selected from the group consisting of CD3 (e.g., CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ chains), 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 ⁇ RI, TCR ⁇ / ⁇ , TCR ⁇ / ⁇ , HLA-DR, CD28, 4-1BB(CD137), OX40 (CD134), ICOS (CD278), 2B
  • the first antigen is an autoantigen selected from: 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; and the second autoantigen selected
  • the first antigen is selected from gp100, MAGEA1, KRAS, and HPVE7.
  • the second antigen is selected from CD3, CD28, and 4-1BB (CD137). In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is CD28. In some embodiments, the second antigen is 4-1BB (CD137).
  • the second antigen includes, but is not limited to, OKT3, UCHT-1, BMA031, and 12F6.
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC, and the second polypeptide chain from N-terminus to C-terminus End contains: VL-connector-TRBV-connector TRBC.
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC-hinge region-CH2-CH3, and the second polypeptide chain
  • the peptide chain contains from N-terminus to C-terminus: VL-linker-TRBV-linker TRBC-hinge region-CH2-CH3.
  • the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC, and the second polypeptide chain from N-terminus to C-terminus The end contains: VL-connector-TRBV-connector TRBC-connector-ALB.
  • the first antigen is gp100.
  • the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO: 1 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 1 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO:2 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO:2 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity.
  • the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 1, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 2 chain.
  • the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO:3 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:3 % sequence identity of the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO:4 or having at least 80%, at least 85%, at least 90%, at least 95% with SEQ ID NO:4 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity.
  • the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 3, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 4 chain.
  • the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO:5 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:5 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO: 6 or having at least 80%, at least 85%, at least 90%, at least 95% with SEQ ID NO: 6 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity.
  • the polypeptide molecule comprises: having an amino group as shown in SEQ ID NO:5 A first polypeptide chain having an amino acid sequence, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 6.
  • the first antigen is KRAS.
  • TRAV in the polypeptide molecule, includes CDR1 represented by NSASQS (SEQ ID NO:89), CDR2 represented by VYSSGN (SEQ ID NO:90), and VVPGGTGGGNKLT (SEQ ID NO:91) CDR3 shown; TRBV contains CDR1 shown by LGHDT (SEQ ID NO:92), CDR2 shown by YNNKEL (SEQ ID NO:93), and CDR3 shown by ASSHWGAQETQY (SEQ ID NO:94).
  • TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGTQLKVEL (SEQ ID NO:95). In some embodiments, TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGGTQLPVPL (SEQ ID NO:96).
  • TRAV comprises RKIVEQDPGPFEVPEGATVAFICTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQDIHLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGGTQLPVPL (SEQ ID NO:97).
  • TRBV comprises DTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMDWYKQDSKKFLKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSHWGAQETQYFPGGTRLLVL (SEQ ID NO:98).
  • the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO:99 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:99 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO: 100 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 100 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity.
  • the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 99, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 100 chain.
  • the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO: 101 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 101 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO: 102 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 102 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity.
  • the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 101, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 102 chain.
  • the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO: 103 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 103 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO: 104 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 104 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity.
  • the polypeptide molecule comprises: a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 103, and a second polypeptide having the amino acid sequence shown in SEQ ID NO: 104 chain.
  • the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO: 105 or being at least 80%, at least 85%, at least 90%, at least 95%, or at least 99 identical to SEQ ID NO: 105 % sequence identity to a first polypeptide chain of an amino acid sequence, and having an amino acid sequence as set forth in SEQ ID NO: 106 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 106 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity.
  • the polypeptide molecule comprises: having SEQ ID NO:105 A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 106, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 106.
  • the first antigen is MAGE-A1.
  • TRAV comprises RGEDVEQSLFLSVREGDSSVINCTYTDSSSSTYLYWYKQEPGAGLQLLTYIFSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGGTRLQVFPN (SEQ ID NO: 107).
  • TRAV comprises RGEDVEQSLFLSVREGDSSVINCTYTDSSSSTYLYWYKQEPGAGLQLLTYTWPHMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGGTRLQVFPN (SEQ ID NO: 108).
  • TRBV comprises GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSDYQTCVTYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPFEQYFPGGTRLTVTE (SEQ ID NO: 109).
  • TRBV comprises GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPYEQYFPGGTRLTVTE (SEQ ID NO: 110).
  • the first antigen is an HPV antigen, such as HPV E7.
  • TRAV in the polypeptide molecule, includes CDR1 represented by NSASQS (SEQ ID NO:111), CDR2 represented by VYSSGN (SEQ ID NO:112), and AVISAGTALI ( CDR3 shown in SEQ ID NO:113); TRBV contains CDR1 shown in SGHDT (SEQ ID NO:114), CDR2 shown in YYEEEE (SEQ ID NO:115), and ASSLGWRGGLYTEAF (SEQ ID NO:116) CDR3.
  • NSASQS SEQ ID NO:111
  • CDR2 represented by VYSSGN
  • AVISAGTALI CDR3 shown in SEQ ID NO:113
  • TRBV contains CDR1 shown in SGHDT (SEQ ID NO:114), CDR2 shown in YYEEEE (SEQ ID NO:115), and ASSLGWRGGLYTEAF (SEQ ID NO:116) CDR3.
  • TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDG RFTAQLNRASQYISLLIRDSKLSDSATYLCAVISAGTALIFGKGTTLSVSS (SEQ ID NO: 117).
  • TRBV comprises DAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGWRGGLYTEAFFGQGTRLTVV (SEQ ID NO: 118).
  • the invention provides a T cell receptor (TCR) comprising a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV), wherein the TRAV comprises a TCR alpha chain variable region (TRAV), respectively, as shown in SEQ ID NO: 89 , CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO:90 and SEQ ID NO:91, or functional variants formed by inserting, deleting or replacing one or several amino acids; and/or the TRBV contains respectively ⁇ -chain CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, or functional variants formed by inserting, deleting or replacing one or several amino acids .
  • TCR T cell receptor
  • TRBV TCR alpha chain variable region
  • the TRAV comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91 respectively; and/or the TRBV comprises respectively Beta chain CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94.
  • 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: 95, and/or the TRBV comprises SEQ ID NO:98 An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity.
  • 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: 96, and/or the TRBV comprises SEQ ID NO:98 An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity.
  • 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: 97, and/or the TRBV comprises SEQ ID NO:98 An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity.
  • the invention provides a nucleic acid comprising a nucleotide sequence encoding each strand of a bispecific polypeptide molecule of the invention.
  • the invention also provides nucleic acids comprising a nucleotide sequence encoding the TCR of the invention.
  • the invention provides a vector comprising a nucleic acid according to the invention.
  • vectors include, but are not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyomavirus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, Cosmids and artificial chromosomes (including BAC and YAC).
  • viral vectors including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyomavirus vectors, and adenovirus-associated vectors (AAV)
  • phages phagemids
  • Cosmids and artificial chromosomes including BAC and YAC.
  • the vector itself is usually a nucleotide sequence, usually a DNA sequence containing the insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
  • Engineered vectors typically contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS).
  • the vector may additionally contain a promoter, genetic marker, reporter gene, targeting sequence, and/or 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 preferably selected from the group consisting of lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes.
  • the invention provides a vector system comprising on one or more vectors encoding the invention.
  • the nucleotide sequence of each chain of the bispecific polypeptide molecule is provided.
  • the invention provides a host cell comprising a nucleic acid, vector or vector system of the invention.
  • the cells may be eukaryotic cells, such as plants (without the potential to develop into plants), animals, fungi or algae, or may be prokaryotic cells, such as bacteria or protozoa.
  • the cells may be cultured cells or primary cells, ie, isolated directly from an organism, such as a human. Cells can be adherent cells or suspension cells, that is, cells grown in suspension. Suitable host cells are known in the art and include, for example, DH5 ⁇ E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like.
  • the cells are preferably mammalian cells. Most preferably, the host cells are human cells.
  • the cells are selected from lymphocytes (eg, T cells, NK cells), monocytes (eg, PBMCs), and stem cells.
  • the stem cells may be lymphoid progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells (HSCs).
  • iPSCs induced pluripotent stem cells
  • HSCs hematopoietic stem cells
  • stem cells do not include embryonic stem cells obtained by destroying human embryos, and/or do not include totipotent stem cells used to develop and form an animal individual.
  • the invention provides conjugates comprising a bispecific polypeptide molecule of the invention, and a chemical moiety conjugated to the bispecific polypeptide molecule.
  • the invention also provides conjugates comprising the TCR of the invention, and a chemical moiety conjugated to the TCR.
  • the chemical moiety is selected from the group consisting of therapeutic agents, immunostimulatory molecules, and detectable labels.
  • therapeutic agents include, but are not limited to, immunomodulators, radioactive compounds, enzymes (eg, perforin), chemotherapeutic agents (eg, cisplatin), or toxins.
  • the therapeutic agent may be, for example, maytansine, geldanamycin, a tubulin inhibitor such as a tubulin binding agent (such as auristatins) or a minor groove binding agent such as calicheamicin (calicheamicin).
  • cytotoxic agents include, for example, small molecule cytotoxic agents, ie, compounds with a molecular weight of less than 700 daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that break down or transform under physiological conditions to release the cytotoxic agent.
  • agents include cisplatin, maytansine derivatives, racithromycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, Mitoxantrone, sorfimer porphyrin sodium II, temozolomide, topotecan, metformin, auristatin E, vinca alkaloids, and doxorubicin; peptide cytotoxins, i.e., proteins with the ability to kill mammalian cells or Fragments thereof, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides, i.e., with the simultaneous emission of one or more alpha or beta particles or gamma rays Unstable isotopes of decaying elements, such as iodine-131, rhenium-186, indium-111,
  • the immunostimulatory molecule is an immune effector molecule that stimulates an immune response.
  • the immunostimulatory molecule may be a cytokine such as IL-2 and IFN- ⁇ , a chemokine such as IL-8, platelet factor 4, melanoma growth stimulating protein, complement activator; viral/bacterial protein domain, or viral/ Bacterial peptides.
  • the immunostimulatory molecule is selected from the group consisting of cytokines, chemokines, platelet factors and complement initiators.
  • the detectable label can be selected from biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, or fluorescent, phosphorescent, or chemiluminescent molecules.
  • Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific polypeptide molecule, nucleic acid, vector, vector system, host cell, or conjugate of the invention, and a pharmaceutically acceptable excipient.
  • compositions of the present invention are particularly suitable for administration to humans; however, they are also suitable for administration to non-human animals.
  • the compounds and their components i.e., active agents and optional excipients
  • Pharmaceutical compositions of the invention may, for example, be sterile.
  • excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, preservatives, antioxidants, flavoring agents, colorants, sweetening agents agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tonicity regulators. It is known to those skilled in the art to select suitable excipients for the preparation of pharmaceutical compositions of the invention.
  • Exemplary carriers for use in pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water.
  • saline aline
  • buffered saline aline
  • the pharmaceutical composition of the present invention can be prepared into various forms, such as solid, liquid, gaseous or freeze-dried forms, especially ointments, creams, transdermal patches, gels, in the form of a powder, tablet, solution, aerosol, granule, pill, suspension, emulsion, capsule, syrup, liquid, elixir, extract, tincture or liquid extract, or Particularly suitable for the form of application required.
  • Processes for producing pharmaceuticals known in this invention are shown in Remington's Pharmaceutical Sciences, 22nd Edition (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar-coating, Grinding, emulsifying, encapsulating, embedding or freeze-drying processes.
  • the pharmaceutical composition further comprises a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs.
  • 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 (sorfimer sodium photofrin II), temozolomide, topotecan, trimetreate glucuronate (trimetreate glucuronate), olefin auristatin E, vincristine, and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and asta
  • the invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the bispecific polypeptide molecule, TCR, nucleic acid, vector, vector of the invention A system, host cell, conjugate, or pharmaceutical composition, wherein the disease is selected from the group consisting of cancer, infectious diseases, autoimmune diseases, and inflammatory diseases.
  • the cancer may be any cancer, such as cancer of the blood system, cancer of the central and peripheral nervous system, cancer of the lymphatic lineage, cancer of the myeloid lineage, cancer of mesenchymal origin, solid tumors, etc.
  • cancers include, but are not limited to, acute lymphoblastic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct Cancer, joint cancer, neck cancer, gallbladder or pleural cancer, nose cancer, nasal cavity or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer Carcinoma, gastrointestinal carcinoid tumors, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer
  • Infectious diseases are diseases caused by pathogenic infections, including communicable diseases and non-communicable diseases. cause
  • the pathogens of infectious diseases include viruses, bacteria, mycoplasma, chlamydia, rickettsiae, 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, diabetes caused by autoimmune destruction of pancreatic islets, Sjogren's syndrome, Hashimoto's thyroid inflammatory bowel disease, Graves' thyroiditis, toxic diffuse goiter, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease (such as 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, autoimmunity of the central nervous system Diseases, celiac disease, autoimmune gastritis, primary bili
  • the dosage administered to a subject may vary depending on the embodiment, the agent used, the method of administration, and the site and subject being treated. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine an effective amount for administration to a human or other subject to treat a medical condition. The precise amount required for the treatment to be effective may depend on many factors, such as the activity of the active agent and the route of administration.
  • the dose of the polypeptide molecule, conjugate or pharmaceutical composition of the invention can be administered to the mammal once or in a series of sub-doses over a suitable period of time, for example, daily, every half week, every week, every day as needed. Apply biweekly, semimonthly, bimonthly, semiannually, or annually. Dosage units containing an effective amount of a polypeptide molecule, conjugate or pharmaceutical composition may be administered in a single daily dose, or the total daily dose may be administered in two, three, four or more doses per day as desired. Give in divided doses.
  • the appropriate method of administration can be chosen by the physician.
  • the route of administration may be parenteral, for example by injection, nasal administration, pulmonary administration or transdermal administration.
  • Systemic or local administration can be by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.
  • the polypeptide molecule, conjugate, or pharmaceutical composition is selected for parenteral delivery, inhalation, or delivery through the gastrointestinal tract, such as orally.
  • the dosage and method of administration may vary according to the subject's weight, age, condition, etc., and may be appropriately selected.
  • the method further includes administering a second therapeutic agent to the subject.
  • a polypeptide molecule, conjugate, or pharmaceutical composition of the invention is administered before, substantially simultaneously with, or after the second therapeutic agent.
  • the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs. Preferred examples of second therapeutic agents are as described above.
  • the present disclosure provides a method of detecting (e.g., diagnosing) a disease in a subject, wherein the method comprises (i) conjugating a sample obtained from the subject with a polypeptide molecule, TCR or conjugate of the invention contact; and (ii) detecting the presence of a target antigen in the sample, wherein the presence of the target antigen indicates that the subject suffers from the corresponding disease.
  • Target antigens can be selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens.
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • the disease may be selected from cancer, infectious diseases, autoimmune diseases and inflammatory diseases.
  • the target antigen is KRAS.
  • the antigen is MGAE-A1.
  • the cancers, infectious diseases, autoimmune diseases and inflammatory diseases are as defined above.
  • the sample obtained from the subject may be a blood sample, urine sample, tissue sample, or cell sample.
  • the methods are performed in vitro.
  • the method includes (i) contacting a sample obtained from a subject with a conjugate of the invention, wherein the conjugate comprises a detectable label; and (ii) by detecting the detectable label. The sample is detected for the presence of the target antigen.
  • detectable labels include, but are not limited to, biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, Nucleic acid probes, contrast agents, and fluorescent, phosphorescent or chemiluminescent molecules; preferably enzymes or catalytically active fragments thereof, radionuclides, fluorescent, phosphorescent or chemiluminescent molecules.
  • the disclosure provides a kit comprising a polypeptide molecule, TCR or conjugate of the disclosure for detecting the presence of a target antigen in a sample to be tested.
  • the kit is a kit for detecting (eg, diagnosing) a disease in a subject, comprising a polypeptide molecule or conjugate of the disclosure.
  • Target antigens can be selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens.
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • the disease may be selected from cancer, infectious diseases, autoimmune diseases and inflammatory diseases.
  • the cancers, infectious diseases, autoimmune diseases and inflammatory diseases are as defined above.
  • the conjugate includes a detectable label.
  • detectable labels include, but are not limited to, biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent Or chemiluminescent molecules; preferably enzymes or catalytically active fragments thereof, radionuclides, fluorescent, phosphorescent or chemiluminescent molecules.
  • the kit may also include instructions on how to use the kit.
  • the present disclosure provides bispecific polypeptide molecules, TCRs, nucleic acids, vectors, vector systems, host cells, conjugates or pharmaceutical compositions of the invention for use in the treatment or prevention of disease in a subject. Uses in medicines.
  • the disclosure provides a bispecific polypeptide molecule, TCR, nucleic acid, vector, vector system, host cell, conjugate or pharmaceutical composition of the invention for use in treating or preventing a disease in a subject .
  • the disclosure provides the use of a bispecific polypeptide molecule, TCR or conjugate of the invention in the preparation of a kit for detecting the presence of a target antigen in a sample to be tested.
  • the present disclosure provides the use of a bispecific polypeptide molecule, TCR, or conjugate of the invention in the preparation of a kit for detecting (eg, diagnosing) a disease in a subject.
  • the disclosure provides a bispecific polypeptide molecule, TCR or conjugate of the invention for use in detecting the presence of a target antigen in a sample to be tested.
  • the target antigen may be selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens, and autoantigens.
  • TAA tumor-associated antigens
  • TSA tumor-specific antigens
  • viral antigens and autoantigens.
  • the disease may be selected from cancer, infectious diseases, autoimmune diseases and inflammatory diseases.
  • the cancers, infectious diseases, autoimmune diseases and inflammatory diseases are as defined above.
  • Figure 1 shows the structural schematic diagram, AlphaFold2 diagram, immunoblotting result diagram and ELISPOT assay result diagram of a specific form of format 22-2 of the present invention.
  • Figure 2 shows a schematic structural diagram, an immunoblotting result diagram and an ELISPOT assay result diagram of a specific form of format 64 of the present invention.
  • Figure 3 shows a schematic structural diagram of a specific form of format 63 of the present invention.
  • Figure 4 shows a schematic structural diagram of a specific form of control format 17, protein staining and immunoblotting results, and ELISPOT assay results.
  • Figure 5 shows a schematic structural diagram of a specific form of control format 22, protein staining and immunoblotting results, and ELISPOT assay results.
  • Figure 6 shows the ELISPOT measurement results of format 22-2 of the present invention when KRAS is used as the target peptide.
  • TCR-based bispecific peptide molecules format 22-2 (such as the first chain shown in SEQ ID NO: 1 and the second chain shown in SEQ ID NO: 2), format 64 (such as The first strand as shown in SEQ ID NO:3 and the second strand as shown in SEQ ID NO:4) and format 63 (the first strand as shown in SEQ ID NO:5 and the first strand as shown in SEQ ID NO:6 The second chain), the structures of these formats are shown in Figure 1-3. The three-dimensional structure diagrams of these formats are also displayed through AlphaFold2.
  • formats 17 and 22 were also constructed as controls.
  • the TRAC and TRBC of the format 22-2 of the present invention are replaced with the hinge region-CH2-CH3, which is Format 17; the TRAC and TRBC of the format 22-2 of the present invention are replaced with CH3, which is Format 22.
  • the structure of Format 17 is: the first polypeptide chain contains from N-terminus to C-terminus: TRAV-linker-VH-optional linker-hinge region-CH2-CH3, and the second polypeptide chain contains from N-terminus to C-terminus :VL-linker-TRBV-optional linker-hinge region-CH2-CH3.
  • the structure of Format 22 is: the first polypeptide chain from N-terminus to C-terminus contains: TRAV-linker-VH-optional linker-CH3, and the second polypeptide chain from N-terminus to C-terminus contains: VL-linker- TRBV-optional linker-CH3.
  • the structural diagrams of Format 17 and Format 22 are shown in Figure 4 and Figure 5 respectively.
  • amino acid sequence components used in these formats are as follows:
  • >TRBC1 contains S57C, C187A, N210D mutations
  • >TRBC1 contains S57C, C187A, N210D, and FG loop deletion mutations
  • CH2-CH3 Hole (contains T366S, L368A and Y407V hole mutations and Y349C mutation)
  • amino acid sequences of the two polypeptide chains of control format 17 are as follows:
  • amino acid sequences of the two polypeptide chains of control format 22 are as follows:
  • the signal peptide sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 66) is added to the N-terminus of each polypeptide chain of these formats to increase the secretion of the polypeptide molecules, and a His6 tag is added to the C-terminus of any chain of the format for purification.
  • the format in this example can bind to the complex of gp100 epitope YLEPGPVTA (SEQ ID NO: 67) or its mutant form (YLEPGPVTV, SEQ ID NO: 68)) and HLA-A*02.
  • the multispecific polypeptide molecule format targeting KRAS was constructed as described in Example 1.1.
  • KRAS-targeting foramt 22-2 molecules KVA11-N03_B22-2_WT (the amino acid sequences of its two chains are shown in SEQ ID NO:99 and 100 respectively), KVA11-N03_B22-2_Mut1 (these The amino acid sequences of the two chains are shown in SEQ ID NO:101 and 102 respectively), KVA11-N03_B22-2_Mut2 (the amino acid sequences of the two chains are shown in SEQ ID NO:103 and 104 respectively), KVA11-N03_B22-2_Mut3 (The amino acid sequences of its two chains are shown in SEQ ID NO: 105 and 106 respectively).
  • the format in this example can bind to the complex of KRAS epitope VVGAVGVGK (SEQ ID NO: 119) and HLA-A*11.
  • the multispecific polypeptide molecule format targeting MAGEA1 was constructed as described in Example 1.1.
  • the structure of the format is the same as that in Example 1.1. The only difference is that the gp100-specific TCR in Example 1.1 was replaced with TRAV and TRBV of MAGEA1-specific TCR. TRAV and TRBV.
  • the amino acid sequences of TRAV and TRBV of the MAGEA1-specific TCR are as follows:
  • the format in this example can bind to the complex of MAGEA1 epitope KVLEYVIKV (SEQ ID NO: 120) and HLA-A*02.
  • the nucleotide sequence encoding one or more polypeptide chains of the format in Example 1 is directly cloned into the expression vector pTT5.
  • the obtained vector clone is confirmed by sequencing and then amplified, cultured, and plasmid extracted.
  • transfection solution (1ml): Dilute 10 ⁇ g DNA with about 800 ⁇ L of 150mM sterilized NaCl solution, mix well and place it on the workbench for 5 minutes; add about 50 ⁇ l of transfection reagent to the DNA diluent and mix well to obtain the final transfection solution. The total volume is 1mL.
  • the collected culture supernatant was used for nickel column purification. Prepare a 1ml packed nickel column, rinse with 10ml sterile water, and balance with 10ml 1XBind Buffer. The culture supernatant was centrifuged at 3000 rpm for 5 min, then filtered with chlorine gas at 450 nm, and loaded. After loading the sample, wash the column with 10ml 1XBind Buffer, wash the column with 10ml 1XBind Buffer, wash the column with 10ml 1XWash Buffer (collect 10 tubes of eluate, the flow rate is around 0.3-0.4ml/min), and elute the protein with 1.2ml 1XElution Buffer. Collect the eluate to obtain the purified protein, aliquot and store at -80°C.
  • the purified protein was denatured (R) or non-denatured (NR) and then subjected to SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining to evaluate the protein expression purity and degree of aggregation.
  • Another set of parallel samples were subjected to gel electrophoresis and then subjected to western blotting.
  • Mouse anti-His6 was used as the primary antibody to detect the His6 tag of the purified protein to confirm the reliability of Coomassie brilliant blue staining. The results show that the format provided by the invention can be successfully expressed.
  • Exemplary western blot results are shown in Figures 1 and 2.
  • the concentration of the target protein is predicted based on the harvested target protein yield, and the purity is determined based on the SDS-PAGE Coomassie Brilliant Blue staining results. It can be seen that the format provided by the present invention can be successfully expressed.
  • Example 2 of the present invention The culture supernatant collected in Example 2 of the present invention is subjected to gradient dilution (usually 2-fold dilution and 5-fold dilution are used). Then the corresponding target peptide and non-related peptide were added to DMSO to dissolve, and diluted with water to a usage concentration of 10-4M. T2 cells were used to load 10-6M of target peptide and irrelevant peptide respectively. Add 1640 complete medium containing 10% FBS to the ELISPOT plate and block for 30 min at room temperature.
  • the secretion of IFN- ⁇ was determined by ELISPOT detection to evaluate the immune cell activation induced by the format of the present invention through the target antigen peptide-MHC complex.
  • results show that the format of the present invention can activate immune cells in the presence of target antigen, but cannot activate immune cells when adding irrelevant target peptides or in the negative control group without adding polypeptides.
  • FIG. 6 Exemplary results of the format of the present invention are shown in Figure 6, in which the culture supernatant of format 22-2 with different amino acid sequences can activate immune cells at both 2-fold and 5-fold dilution.
  • Example 1 of the present invention was gradient diluted (10 -7 to 10 -13 M) with 1640 complete culture medium containing 10% FBS. Add gp100 polypeptide, KRAS polypeptide, and MAGE-A1 polypeptide to DMSO to dissolve respectively, and then dilute with water to a usage concentration of 10 -4 M. T2 cells were used to load 10 -6 M gp100 polypeptide, HPV E7 polypeptide, and MAGE-A1 polypeptide respectively. Add 1640 complete medium containing 10% FBS to the ELISPOT plate and block for 30 min at room temperature.
  • Exemplary results of the format of the present invention are shown in Figures 1 and 2 (the reference mark “+” indicates the experimental group in which gp100 polypeptide was added, “-” indicates the negative control group in which gp100 polypeptide was not added, and “irrelevant” indicates the addition of gp100 polypeptide.
  • the results of control formats 17 and 22 are shown in Figures 4 and 5 respectively (the reference mark “+” indicates the experimental group in which gp100 polypeptide was added, and "-” indicates the negative control group in which gp100 polypeptide was not added).
  • format 22-2 and format 64 of the present invention can achieve effective activation of immune cells in the concentration range of 10 -7 -10 -10 M and 10 -7 -10 -9 M respectively, especially format 22-2 Immune cells can be effectively activated at a low concentration of 10 -10 M ( Figure 1), while control formats 17 and 22 require a higher concentration of 10 -8 M to activate immune cells ( Figures 4 and 5), indicating that this The invented format has a significantly better activation effect on immune cells than the control formats 17 and 22. This suggests that TRAC and TRAC used in the format of the present invention have significantly better effects than using the CH3 or hinge region-CH2-CH3 of the antibody when combined with specific first and second antigen-binding regions.
  • Example 1 of the present invention was serially diluted (10 -7 to 10 -13 M) with 1640 complete culture medium containing 10% FBS. Add gp100 polypeptide, KRAS polypeptide, and MAGE-A1 polypeptide to DMSO to dissolve respectively, and then dilute with water to a usage concentration of 10 -4 M. T2 cells were loaded with 10 -6 M of target polypeptide. Add 5x10 5 cells/mL PBMC (100 ⁇ L/well), 5x10 4 cells/mL polypeptide-loaded T2 cells (100 ⁇ L/well), and different concentrations of format (10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 M).
  • T2 cells and PBMC not loaded with gp100 polypeptide were added. After adding all samples, cover the plate and place it in a 37°C, 5% CO2 incubator for 20-24 hours. Discard the supernatant and add 100 ⁇ L of the detection solution of the luciferase reporter gene quantitative assay kit to detect the target cells. Luciferase activity to assess killing of target cells. Based on the target cell killing data, nonlinear regression was used to calculate the EC50 of each format for target cell killing using GraphPad software.
  • the results show that the format provided by the present invention can kill target cells in the concentration range of 10 -7 -10 -10 M or even lower.
  • the prepared expression plasmids containing TCR ⁇ -chain and ⁇ -chain were each transformed into E. coli strain Rosetta (DE3) pLysS, protein expression was induced, inclusion bodies were harvested, inclusion bodies were dissolved with a denaturant, and then dialyzed, renatured, and purified. Purified TCR-based multispecific polypeptide molecules provided by the invention are obtained.
  • the TCR-based multispecific polypeptide molecules provided by the present invention have a KD that binds to MHC-polypeptide complexes less than or equal to 1 ⁇ M and/or a koff of 1 ⁇ 10-3S-1 or slower, and a KD that binds to T cell surface molecules is less than or equal to 1 ⁇ M and/or koff is 1 ⁇ 10-3S-1 or slower.
  • TCR-based multispecific polypeptide molecules of the present invention The killing of transplanted tumors by the TCR-based multispecific polypeptide molecules of the present invention is determined to evaluate its tumor suppressive effect in vivo.
  • the TCR-based multispecific polypeptide molecules provided by the present invention have anti-tumor efficacy in vivo.

Abstract

The present invention relates to a T cell receptor-based bispecific polypeptide molecule and use thereof. The present invention further provides a nucleic acid encoding the polypeptide molecule of the present invention, a vector or a vector system comprising the nucleic acid of the present invention, a host cell comprising the polypeptide molecule of the present invention, a conjugate and a pharmaceutical composition comprising the polypeptide molecule of the present invention, and a method for preventing or treating a disease in a subject using the polypeptide molecule of the present invention.

Description

基于T细胞受体的双特异性多肽分子及其用途Bispecific polypeptide molecules based on T cell receptors and their uses
本国际专利申请要求2022年6月24日提交中国专利申请CN202210730436.0的优先权权益，其全部内容通过引用并入本文。This international patent application claims the priority rights of Chinese patent application CN202210730436.0 filed on June 24, 2022, the entire content of which is incorporated herein by reference.
技术领域Technical field
本发明涉及基于T细胞受体的双特异性多肽分子及其用途，特别是在治疗癌症、感染性疾病、自身免疫病和/或炎性疾病中的用途。The present invention relates to T cell receptor-based bispecific polypeptide molecules and their uses, particularly in the treatment of cancer, infectious diseases, autoimmune diseases and/or inflammatory diseases.
背景技术Background technique
T细胞是脊椎动物适应性免疫系统的重要组成部分，在病毒感染、癌症和自身免疫中起着至关重要的作用。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与由γ、δ、ε和ζ亚基组成的CD3信号装置的非共价结合形成TCR–CD3复合物。pMHC与TCR的结合激活LCK诱导的TCR CD3ζ亚基中细胞内免疫受体酪氨酸基激活基序(ITAM)的磷酸化。然后，CD3ζ磷酸化触发一系列磷酸化信号转导，涉及ζ链相关蛋白激酶70(ZAP70)和T细胞中的激活T细胞连接体(LAT)，引起多个下游适配器和信号分子的招募以及LAT信号体的形成。组装的LAT信号体激活多种涉及转录因子的信号通路，如激活蛋白1(AP-1)、核因子-κB(NF-κB)和活化T细胞的核因子(NFAT)。活化的转录因子可引起后续的T细胞激活、增殖、细胞因子产生和效应器功能。T cells are an important component of the adaptive immune system of vertebrates and play a crucial role in viral infection, cancer, and autoimmunity. TCR recognition of major histocompatibility complex (MHC)-antigen peptide complex (pMHC) is an important step in T cell-mediated immune response. The non-covalent binding of TCR to the CD3 signaling apparatus consisting of γ, δ, ε and ζ subunits forms the TCR–CD3 complex. Binding of pMHC to the TCR activates LCK-induced phosphorylation of the intracellular immunoreceptor tyrosine-based activation motif (ITAM) in the CD3ζ subunit of the TCR. CD3ζ phosphorylation then triggers a cascade of phosphorylation signaling involving ζ chain-associated protein kinase 70 (ZAP70) and the activated T cell adapter (LAT) in T cells, causing the recruitment of multiple downstream adapters and signaling molecules as well as LAT Formation of the signal body. The assembled LAT signalosome activates a variety of signaling pathways involving transcription factors, such as activating protein 1 (AP-1), nuclear factor-κB (NF-κB), and nuclear factor of activated T cells (NFAT). Activated transcription factors can cause subsequent T cell activation, proliferation, cytokine production, and effector functions.
双特异性抗体(bispecific antibody,BsAb，简称“双抗”)是指一个抗体分子同时靶向两个抗原或者靶向一个抗原的两个不同的抗原表位。双特异性抗体与普通抗体相比，特异性更强、更能准确靶向肿瘤细胞以及降低脱靶毒性。随着重组蛋白表达技术和抗体工程技术的发展，产生了许多不同的抗体形式。多特异性抗体用于各种目的，包括(1)受体激活(2)阻断(3)内化(4)聚集，(5)膜相关蛋白的结合，或(6)细胞毒性效应细胞的定向靶点。Bispecific antibody (BsAb, referred to as "double antibody") refers to an antibody molecule that targets two antigens at the same time or targets two different epitopes of one antigen. Compared with ordinary antibodies, bispecific antibodies have stronger specificity, can more accurately target tumor cells, and reduce off-target toxicity. With the development of recombinant protein expression technology and antibody engineering technology, many different antibody forms have been produced. Multispecific antibodies are used for a variety of purposes, including (1) receptor activation (2) blocking (3) internalization (4) aggregation, (5) binding of membrane-associated proteins, or (6) cytotoxic effector cells Orientation target.
T细胞重定位的bsAb(又名T cell engager，TcE)是重要的细胞毒性效应细胞重定位形式，是目前癌症免疫治疗的中心支柱。这种bsAb可识别肿瘤细胞表面的靶点，同时识别T细胞表面的分子(该分子大多数情况下是CD3)，这样bsAb就可以将肿瘤细胞和具有细胞毒性的T细胞偶联在一起，以引起T细胞TCR下游信号通路的激活，通过T细胞的细胞毒性杀伤肿瘤细胞。Blincyto(blinatumomab)是一种CD19/CD3双特异性抗体，其导致69％的复发/难治性B前体急性淋巴细胞白血病(ALL)患者完全缓解。目前已经出现了大量新的T细胞重定位抗体形式，如BITE、BITE-Fc、DART-Fc、TriTAC等。T cell repositioning bsAb (also known as T cell engager, TcE) is an important form of cytotoxic effector cell repositioning and is the central pillar of current cancer immunotherapy. This bsAb recognizes the target on the surface of tumor cells and also recognizes a molecule on the surface of T cells (the molecule is CD3 in most cases), so that the bsAb can couple tumor cells and cytotoxic T cells together to Causes the activation of T cell TCR downstream signaling pathways and kills tumor cells through T cell cytotoxicity. Blincyto (blinatumomab) is a CD19/CD3 bispecific antibody that leads to complete remission in 69% of patients with relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL). A large number of new T cell relocalizing antibody forms have emerged, such as BITE, BITE-Fc, DART-Fc, TriTAC, etc.
基于抗体识别靶细胞表面分子的bsAb，仅仅识别肿瘤细胞表面抗原，不能识别将近90％的细胞内蛋白。而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。bsAb, which is based on antibodies recognizing target cell surface molecules, only recognizes tumor cell surface antigens and cannot recognize nearly 90% of intracellular proteins. The TCR can recognize the antigen peptides of intracellular and cell surface tumor-specific antigens that are processed and presented on the cell surface MHC molecules, and the recognition range is wider. The target cell recognition region in TCR-based T cell repositioning bispecific antibodies is changed from traditional antibodies to TCR, so that the broad recognition properties of TCR can be used to increase target selectivity.
发明内容Contents of the invention
为增加TcE的体内效果，除了提升TCR对pMHC的亲和力以外，还可以增加TcE针对靶细胞靶点的TCR的效价，也可以增加TcE针对效应细胞表面分子的效价，以提升 T细胞杀伤活性和增殖能力。同时，为了维持TcE进入肿瘤的能力，同时保持TcE在体内合适的半衰期，需要将TcE的分子大小维持在一定的范围内。此外，对于蛋白药物来说，肾小球的滤过界限一般在60kDa左右。也就是说小分子量的抗体片段或衍生物，例如纳米抗体(15kDa)和scFv(28kDa)甚至Fab(50kDa)都会通过肾小球的滤过作用从体内清除，导致其拥有较短的半衰期。其中，纳米抗体在所有抗体类型中分子量最小，半衰期往往只有几十分钟。因此，为了增加蛋白药物的疗效，有必要对蛋白药物进行改进，以延长其半衰期并增加活性。在多肽分子中适当地增加其他功能结构域，例如来源于抗体的铰链区、抗体的Fc结构域或其二聚化部分和/或白蛋白(Alb)的功能结构域，不但能延长多肽分子的半衰期，还能增强多肽分子的杀伤活性。In order to increase the in vivo effect of TcE, in addition to increasing the affinity of TCR for pMHC, the potency of TcE against TCR of target cell targets can also be increased, and the potency of TcE against surface molecules on effector cells can also be increased to enhance T cell killing activity and proliferation ability. At the same time, in order to maintain the ability of TcE to enter tumors and maintain the appropriate half-life of TcE in the body, the molecular size of TcE needs to be maintained within a certain range. In addition, for protein drugs, the glomerular filtration limit is generally around 60kDa. In other words, small molecular weight antibody fragments or derivatives, such as Nanobodies (15kDa), scFv (28kDa) and even Fab (50kDa), will be cleared from the body through glomerular filtration, resulting in a shorter half-life. Among them, nanobodies have the smallest molecular weight among all antibody types, and their half-lives are often only tens of minutes. Therefore, in order to increase the efficacy of protein drugs, it is necessary to improve protein drugs to extend their half-life and increase activity. Appropriately adding other functional domains to the polypeptide molecule, such as the hinge region derived from the antibody, the Fc domain of the antibody or its dimerization part and/or the functional domain of albumin (Alb), can not only extend the length of the polypeptide molecule The half-life can also enhance the killing activity of polypeptide molecules.
相应地，本发明提供了基于TCR的双特异性多肽分子。本发明的双特异性多肽分子采用了特定的抗原结合区连接方式并与TCR恒定区(TRAC和TRBC)组合，这种与TCR恒定区的组合显著改善分子的稳定性，这种各结构域的特定组合实现了高效的免疫细胞激活作用，并且相比于使用抗体的恒定区(CH3、铰链区-CH2-CH3、或CH1/CL)具有显著更好的效果。Accordingly, the present invention provides TCR-based bispecific polypeptide molecules. The bispecific polypeptide molecule of the present invention adopts a specific antigen-binding region connection method and is combined with the TCR constant region (TRAC and TRBC). This combination with the TCR constant region significantly improves the stability of the molecule. This combination of each structural domain The specific combination achieved highly efficient immune cell activation and was significantly more effective than using the constant region of the antibody (CH3, hinge region-CH2-CH3, or CH1/CL).
术语the term
除非另有说明或定义，否则所使用的所有术语都具有技术人员所熟知的本领域通常的含义。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-4010 Basel,Switzerland中所述的定义。Unless otherwise stated or defined, all terms used have their ordinary meanings known to those skilled in the art. For example, the terms used in this article are such as "Immunobiology" by Janeway CA Jr, Travers P, Walport M, et al., Fifth Edition, New York: GarlandScience (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Definitions as stated in H. Editor (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.
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。It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Accordingly, the terms "a", "an", "one or more" and "at least one" may be used interchangeably. Similarly, the terms "comprising," "including," and "having" may be used interchangeably.
除非另有说明或定义，否则术语“包含”及其变体诸如“包括”和“含有”应理解为意味着包括所述元素或步骤，但不排除任何其他元素或步骤。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。Unless otherwise stated or defined, the term "comprises" and variations thereof such as "includes" and "contains" shall be understood to mean the inclusion of stated elements or steps but not the exclusion of any other elements or steps. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined below as including or containing at least a certain number of embodiments, this is also to be understood as disclosing a group that preferably consists exclusively of these embodiments.
如本文所用的术语“T细胞受体”或“TCR”包括天然TCR以及TCR变体、片段和构建体。因此该术语包括包含TCRα链和TCRβ链的异二聚体以及多聚体和单链构建体；任选地包含其它结构域和/或部分，只要所述TCR保留其识别抗原靶标的能力。The term "T cell receptor" or "TCR" as used herein includes native TCR as well as TCR variants, fragments and constructs. The term therefore includes heterodimers as well as multimeric and single-chain constructs comprising TCR alpha and TCR beta chains; optionally including other domains and/or portions, so long as the TCR retains its ability to recognize the antigenic target.
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。In its native form, the TCR exists as a complex of several proteins on the surface of T cells. T cell receptors are composed of two (separate) protein chains produced by separate T cell receptor alpha and beta (TCRα and TCRβ) genes and are called alpha and beta chains. Each chain of the TCR has an N-terminal immunoglobulin-like (Ig)-variable (V) region/domain and an Ig-constant (C) region/domain that anchors the chain across the membrane in the plasma membrane. /membrane spanning region, and a short cytoplasmic tail at the C-terminus.
抗原特异性是由α和β链的可变区(TRAV和TRBV)赋予的。TCRα链和β链的两个可变区均包含由框架(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。Antigen specificity is conferred by the variable regions of the alpha and beta chains (TRAV and TRBV). Both variable regions of the TCR α chain and β chain contain three hypervariable or complementarity determining regions (CDR1α/β, CDR2α/β and CDR3α/β) surrounded by framework (FR) regions. CDR3 is the major determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 interact primarily with MHC molecules presenting antigenic peptides.
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”、“抗原-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。The TCR recognizes an antigenic peptide that binds to a major histocompatibility complex (MHC) molecule at the surface of the antigen-presenting cell ("presented/displayed on the MHC molecule"). Antigenic peptides presented on MHC molecules are also referred to herein as "antigenic peptides" "Epitope-MHC complex", "epitope-MHC complex", "antigen-MHC complex" or "target antigen peptide-MHC complex". There are two different classes of MHC molecules: MHC I and MHC II , which presents peptides from different cellular 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. In humans, MHC is also called human leukocyte antigen (HLA). There are three main types of MHC class I: HLA-A, HLA-B, and HLA-C. Once the TCR binds to its specific epitope-MHC complex, the T cell is activated and functions Biological effect function.
如本文所用的术语“TCR恒定区”包括TCRα链恒定区(TRAC)和β链恒定区(TRBC)，其可以是人恒定区或衍生自另一物种，例如鼠。The term "TCR constant region" as used herein includes the TCR alpha chain constant region (TRAC) and the beta chain constant region (TRBC), which may be human constant regions or derived from another species, such as murine.
据报道，在恒定区添加二硫键可促进TCRα和β链的正确配对(Kuball J等Blood.2007 Mar 15；109(6)：2331-8)。因此，本文还设想在恒定区中添加一个或多个半胱氨酸修饰，以在TCRα链和TCRβ链之间形成二硫键。It has been reported that the addition of disulfide bonds in the constant region promotes the correct pairing of TCRα and β chains (Kuball J et al. Blood. 2007 Mar 15;109(6):2331-8). Therefore, this article also envisages adding one or more cysteine modifications in the constant region to form a disulfide bond between the TCRα chain and the TCRβ chain.
野生型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链的恒定域序列为“TRAC*01”，TCR分子β链的恒定域序列为“TRBC1*01”或“TRBC2*01”。The sequence of the wild-type TCR constant region can be found in the public database of the International Immunogenetic Information System (IMGT). For example, the constant domain sequence of the α chain of the TCR molecule is "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule is " TRBC1*01" or "TRBC2*01".
本发明中野生型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链可变区(TRAV)中的某个氨基酸，IMGT中列出的位置编号为104，则本发明中将其描述为TRAV第104位氨基酸；TCRβ链可变区(TRBV)中的某个氨基酸，IMGT中列出的位置编号为84，则本发明中将其描述为TRBV第84位氨基酸；其他以此类推。例如，TCRα链恒定区(TRAC)中的某个氨基酸，IMGT中列出的位置编号为48，则本文中将其描述为TRAC第48位氨基酸；TCRβ链恒定区(TRBC)中的某个氨基酸，IMGT中列出的位置编号为57，则本文中将其描述为TRBC第57位氨基酸；其他以此类推。本发明中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。The positions of the amino acid sequences of the wild-type TCR in the present invention are numbered according to the naming rules of the International Immunogenetic Information System (IMGT). For example, if a certain amino acid in the TCRα chain variable region (TRAV) has a position number of 104 listed in IMGT, it is described in the present invention as the 104th amino acid of TRAV; in the TCRβ chain variable region (TRBV) For a certain amino acid, if the position number listed in IMGT is 84, it will be described as the 84th amino acid of TRBV in the present invention; for others, the same applies. For example, an amino acid in the constant region of TCRα chain (TRAC), the position number listed in IMGT is 48, then it is described in this article as the 48th amino acid of TRAC; an amino acid in the constant region of TCRβ chain (TRBC) , the position number listed in IMGT is 57, then it is described in this article as the 57th amino acid of TRBC; others are deduced by analogy. In the present invention, if there are special instructions for the sequence position number of other amino acids, the special instructions will apply.
如本文所用的术语“抗体”是指免疫球蛋白分子，其具有特异性结合特定抗原的能力。此类分子通常包含通过二硫键相互连接的两条重(H)链和两条轻(L)链。每条重链由重链可变区(或结构域)(本文缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(或结构域)(本文缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。抗体重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，宿主组织或因子包括免疫系统的各种细胞(如效应细胞)和补体系统的组分如C1q(补体激活经典途径中的第一组分)。The term "antibody" as used herein refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Such molecules typically contain two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (or domain) (herein abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (or domain) (herein abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the third step in the classical pathway of complement activation). one component).
免疫球蛋白的重链可分为三个功能区：Fd区、铰链区和Fc区(可结晶片段)。Fd区包含VH和CH1结构域，并与轻链结合形成Fab(抗原结合片段)。Fc区也称为Fc结构域，其在每条重链中包含两个或三个重链恒定区(CH2、CH3、CH4)。在IgG类抗体中，Fc区包含CH2和CH3结构域。在免疫球蛋白的天然结构中，两条重链的Fc区发生二聚化形成二聚化部分。Fc片段负责免疫球蛋白效应功能，包括例如补体结合和与效应细胞的同源Fc受体结合。The heavy chain of immunoglobulins can be divided into three functional regions: Fd region, hinge region and Fc region (crystallizable fragment). The Fd region contains VH and CH1 domains and combines with the light chain to form Fab (antigen-binding fragment). The Fc region, also called an Fc domain, contains two or three heavy chain constant regions (CH2, CH3, CH4) in each heavy chain. In IgG class antibodies, the Fc region contains CH2 and CH3 domains. In the native structure of immunoglobulins, the Fc regions of the two heavy chains dimerize to form the dimerization part. The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to cognate Fc receptors on effector cells.
IgG类抗体的铰链区是指重链CH1和CH2部分之间的短氨基酸序列区域，在抗体天然状态下相对柔性。在IgG、IgA和IgD免疫球蛋白类别中发现的铰链区充当柔性间隔区，允许Fab部分相对于Fc区在空间中自由移动。铰链结构域在结构上多种多样，在免疫球蛋白类别和亚类之间的序列和长度都不同。根据晶体学研究，免疫球蛋白铰链区在结构和功能上可进一步细分为三个区域：上铰链、核心铰链和下铰链(Shin等人,Immunological Reviews 130:87,1992)。上铰链包括从CH1的羧基末端到铰链中限制运动的第一个残基的 氨基酸，通常是在两条重链之间形成链间二硫键的第一个半胱氨酸残基。上铰链区的长度与抗体的片段柔性相关。核心铰链区含有重链间二硫键。下铰链区连接CH2结构域的氨基末端，并包括CH2结构域中的残基。人IgG1的核心铰链区含有当通过二硫键形成二聚化时会产生环状八肽的序列Cys-Pro-Pro-Cys(SEQ ID NO:7)，据信环状八肽充当枢轴，从而赋予柔性。免疫球蛋白铰链区多肽序列的结构和柔性允许的构象变化可以影响抗体Fc部分的效应功能。The hinge region of IgG antibodies refers to the short amino acid sequence region between the CH1 and CH2 parts of the heavy chain, which is relatively flexible in the natural state of the antibody. The hinge region found in the IgG, IgA and IgD immunoglobulin classes acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region. Hinge domains are structurally diverse, varying in sequence and length between immunoglobulin classes and subclasses. Based on crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions structurally and functionally: upper hinge, core hinge, and lower hinge (Shin et al., Immunological Reviews 130:87, 1992). The upper hinge includes from the carboxyl terminus of CH1 to the first residue in the hinge that limits movement. Amino acid, usually the first cysteine residue that forms an interchain disulfide bond between two heavy chains. The length of the upper hinge region correlates with the fragment flexibility of the antibody. The core hinge region contains inter-heavy chain disulfide bonds. The lower hinge region connects the amino terminus of the CH2 domain and includes residues in the CH2 domain. The core hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO:7) which when dimerized through disulfide bonds results in a cyclic octapeptide, which is believed to act as a pivot, Thus imparting flexibility. The structure and flexibility of the immunoglobulin hinge region polypeptide sequence allow for conformational changes that can affect the effector function of the Fc portion of the antibody.
“轻链可变区(VL)”或“重链可变区(VH)”由间插三个“互补决定区”或“CDR”的“框架”区组成。框架区用于调整CDR，以用于特异性结合抗原表位。CDR包含抗体中主要负责抗原结合的氨基酸残基。从氨基末端到羧基末端，VL和VH结构域都包含以下框架(FR)区和CDR区：FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。A "light chain variable region (VL)" or "heavy chain variable region (VH)" consists of a "framework" region interspersed with three "complementarity determining regions" or "CDRs". The framework region is used to adjust the CDR for specific binding to the antigenic epitope. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
每个VL和VH结构域的氨基酸分配按照CDR的任何常规定义。常规定义包括Kabat定义(Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,MD,1987和1991))；Chothia定义(Chothia&Lesk,J.Mol.Biol.196：901-917,1987；Chothia等人,Nature 342：878-883,1989)；Chothia Kabat CDR的复合，其中CDR-H1是Chothia和Kabat CDR的复合；Oxford Molecular的抗体建模软件所使用的AbM定义；以及Martin等人的CONTACT定义(万维网bioinfo.org.uk/abs)。Kabat提供了广泛使用的编号惯例(Kabat编号系统)，其中不同重链之间或不同轻链之间的对应残基被赋予相同的编号。本公开可以使用根据这些编号系统中的任一种定义的CDR，但是优选的实施方案使用Kabat定义的CDR。The assignment of amino acids to each VL and VH domain follows any conventional definition of a CDR. Conventional definitions include Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)); Chothia definition (Chothia & Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of Chothia Kabat CDRs, where CDR-H1 is a complex of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT of Martin et al. Definition (www.bioinfo.org.uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. The present disclosure may use CDRs defined according to any of these numbering systems, but the preferred embodiment uses CDRs defined by Kabat.
本文所用的术语“抗体”应以其最广泛的意义来理解，并且包括单克隆抗体(包含全长单克隆抗体)、多克隆抗体、抗体片段和包含至少两个抗原结合区的多特异性抗体(例如，双特异性抗体)。抗体可含有另外的修饰，例如非天然存在的氨基酸、Fc区中的突变、以及糖基化位点的突变。抗体还包括翻译后修饰的抗体、含有抗体的抗原决定簇的融合蛋白以及含有对抗原识别位点的任何其他修饰的免疫球蛋白分子，只要这些抗体表现出预期的生物活性即可。As used herein, the term "antibody" is to be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments and multispecific antibodies containing at least two antigen-binding regions (e.g., bispecific antibodies). Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing the antigenic determinants of the antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, so long as these antibodies exhibit the intended biological activity.
如本文所用，术语抗体的“抗原结合片段”是指保持特异性结合抗原能力的一种或多种抗体片段。已经表明，抗体的抗原结合功能可以由全长抗体的片段来执行。As used herein, the term "antigen-binding fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
抗原结合片段的实例包括但不限于(i)Fab片段，其为由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab')2片段，其为二价片段，包含通过铰链区二硫键连接的两个Fab片段；(iii)Fab'片段，其基本上是Fab，但具有部分铰链区(参见，FUNDAMENTAL IMMUNOLOGY(Paul ed.,3.sup.rd ed.1993)；(iv)由VH和CH1结构域组成的Fd片段；(v)具有VH和CH1结构域以及位于CH1结构域的C端的一个或多个半胱氨酸残基的Fd'片段；(vi)由抗体单臂的VL和VH结构域组成的Fv片段；(vii)dAb片段(Ward等人(1989)Nature 341：544-546)，其由VH结构域组成；(viii)单独的互补决定区(CDR)；和(ix)纳米抗体，其为包含单个可变域和两个恒定域的重链可变区。此外，尽管Fv片段的两个结构域VL和VH通过独立的基因编码，但它们可以使用重组方法通过合成接头连接，该合成接头能够使它们形成单个蛋白质链，在该蛋白质链中，VL和VH区配对以形成单价分子(称为单链Fv(ScFv)；参见例如Bird等人(1988)Science 242,423-426；以及Huston等人(1988)Proc.Natl.Acad.Sci.USA 85,5879-5883)。此类单链抗体也意在包含在术语抗体的“抗原结合片段”内。此外，该术语还包括含有一对串联Fd片段(VH-CH1-VH-CH1)的“线性抗体”，其与互补的轻链多肽以及保留抗原结合活性的任何前述片段的修饰版本共同形成抗原结合区。 Examples of antigen-binding fragments include, but are not limited to (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments, including Two Fab fragments connected by a disulfide bond in the hinge region; (iii) Fab' fragment, which is essentially a Fab but has a partial hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993) ; (iv) Fd fragment consisting of VH and CH1 domains; (v) Fd' fragment having VH and CH1 domains and one or more cysteine residues located at the C-terminus of the CH1 domain; (vi) Fv fragment consisting of the VL and VH domains of one arm of the antibody; (vii) dAb fragment (Ward et al. (1989) Nature 341:544-546) consisting of the VH domain; (viii) separate complementarity determining regions (CDR); and (ix) Nanobodies, which are heavy chain variable regions containing a single variable domain and two constant domains. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, They can be linked using recombinant methods via synthetic linkers that enable them to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (termed a single-chain Fv (ScFv); see e.g. Bird et al. Human (1988) Science 242, 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody Within. In addition, the term also includes "linear antibodies" containing a pair of tandem Fd fragments (VH-CH1-VH-CH1) formed with a complementary light chain polypeptide and modified versions of any of the foregoing fragments that retain antigen-binding activity Antigen binding region.
如本文所用的术语“抗原”是指任何能够诱导机体产生免疫应答物质。即能被T/B淋巴细胞表面的抗原受体(TCR/BCR)特异性识别并结合，活化T/B细胞.使之增殖分化，产生免疫应答产物(致敏淋巴细胞或抗体)，并能与相应产物在体内外发生特异性结合的物质。The term "antigen" as used herein refers to any substance capable of inducing an immune response in the body. That is, it can be specifically recognized and combined with the antigen receptor (TCR/BCR) on the surface of T/B lymphocytes, activate T/B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can Substances that specifically bind to corresponding products in vivo and in vitro.
在本文中，抗原可以是肿瘤相关抗原(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。In this context, the antigen may be a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, an autoantigen, and an immune cell surface molecule.
如本文所用的术语“肿瘤相关抗原(TAA)”是指与正常细胞相比，在肿瘤细胞中差异表达(例如在肿瘤细胞上表达明显增加)的抗原。The term "tumor-associated antigen (TAA)" as used herein refers to an antigen that is differentially expressed (eg, significantly increased in expression on tumor cells) compared to normal cells.
如本文所用的术语“肿瘤特异性抗原(TSA)”是指肿瘤细胞所特有，不存在于正常组织和细胞中的抗原。The term "tumor-specific antigen (TSA)" as used herein refers to antigens that are unique to tumor cells and are not present in normal tissues and cells.
如本文所用的术语“病毒抗原”是指病毒中能够诱导机体产生免疫应答的物质。The term "viral antigen" as used herein refers to a substance in a virus that can induce an immune response in the body.
如本文所用的术语“自身抗原”是指生物、物理、化学因素引起的自身组织细胞结构与成分发生改变，从而引起机体免疫系统针对这些自身组织细胞成分产生免疫应答反应的抗原物质。隐蔽抗原的释放可产生免疫应答。自身成分的改变可能产生自身抗原。例如，变性的IgG可刺激机体产生抗变性IgG抗体(类风湿因子)，引起类风湿关节炎。临床上使用某些药物后，可改变血细胞表面抗原性，引起自身免疫性溶血性贫血或粒细胞减少。共同抗原引发的交叉反应可产生免疫应答。某些细菌、病毒与正常人体某些组织细胞上有相类似的抗原决定簇，针对这些细菌、病毒抗原决定簇产生的自身抗体和致敏淋巴细胞可与自身组织细胞发生交叉反应，引起自身免疫性疾病。The term "autoantigen" as used herein refers to antigenic substances that cause changes in the structure and components of self-tissue cells caused by biological, physical, and chemical factors, thereby causing the body's immune system to produce an immune response against these self-tissue cell components. The release of cryptic antigens generates an immune response. Changes in its own composition may produce autoantigens. For example, denatured IgG can stimulate the body to produce anti-denatured IgG antibodies (rheumatoid factor), causing rheumatoid arthritis. The clinical use of certain drugs can change the antigenicity of blood cell surface, causing autoimmune hemolytic anemia or neutropenia. Cross-reactions triggered by common antigens can generate immune responses. Certain bacteria and viruses have similar antigenic determinants to certain tissue cells of the normal human body. Autoantibodies and sensitized lymphocytes produced against the antigenic determinants of these bacteria and viruses can cross-react with their own tissue cells, causing autoimmunity. disease.
“免疫细胞表面分子”可以包括例如免疫细胞表面抗原(例如T细胞抗原)和共刺激分子。"Immune cell surface molecules" may include, for example, immune cell surface antigens (eg, T cell antigens) and costimulatory molecules.
抗原上被TCR或抗体结合的位点被称为“表位”。表位可以由连续氨基酸或通过一种或多种蛋白质的三级折叠而并置的非连续氨基酸形成。由连续氨基酸形成的表位(也称为线性表位)通常在暴露于变性溶剂后保留，而通过三级折叠形成的表位(也称为构象表位)通常在变性溶剂的处理中丢失。表位通常包含处于独特空间构象的至少3个，更通常地至少5个或8-10个氨基酸。表位限定了TCR或抗体的最小结合位点，因此是TCR或抗体或其抗原结合片段的特异性靶标。The site on the antigen that is bound by TCR or antibody is called an "epitope." Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed by consecutive amino acids (also called linear epitopes) are usually retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also called conformational epitopes) are usually lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the minimal binding site of the TCR or antibody and is therefore the specific target of the TCR or antibody or its antigen-binding fragment.
本文中，术语“白蛋白”涵盖任何脊椎动物来源的任何天然白蛋白，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人和猴)和啮齿类(例如，小鼠和大鼠)。白蛋白还涵盖全长、未加工的白蛋白前原蛋白以及由细胞中的加工所产生的任何形式的白蛋白。白蛋白还涵盖天然存在的白蛋白的变体，例如剪接变体或等位基因变体。该术语还涵盖任何重组形式的白蛋白。白蛋白序列是本领域已知的。例如可在NCBI中找到关于人血清白蛋白基因(包括基因组DNA序列)的信息。示例性的全长人血清白蛋白前原蛋白的氨基酸序列可在NCBI登录号NP_000468.1下找到。人血清白蛋白以前白蛋白的形式合成，成熟的白蛋白是585个氨基酸残基的单链多肽，分子量为66458，分子中含17个二硫键，不含有糖的组分。在体液pH7.4的环境中，白蛋白为负离子，每分子可以带有200个以上负电荷。外源性输注的白蛋白在2h后约有10％进入血管外的组织间隙，20天达平衡。白蛋白在血浆中的半衰期为15-19天。As used herein, the term "albumin" encompasses any naturally occurring albumin from any vertebrate source, including mammals, such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). mouse). Albumin also encompasses the full-length, unprocessed preproalbumin protein as well as any form of albumin resulting from processing in the cell. Albumin also encompasses variants of naturally occurring albumin, such as splice variants or allelic variants. The term also covers any recombinant form of albumin. Albumin sequences are known in the art. Information on the human serum albumin gene (including genomic DNA sequence) can be found, for example, at NCBI. The amino acid sequence of an exemplary full-length human serum albumin preproprotein can be found under NCBI accession number NP_000468.1. Human serum albumin is synthesized in the form of prealbumin. Mature albumin is a single-chain polypeptide of 585 amino acid residues with a molecular weight of 66,458. The molecule contains 17 disulfide bonds and does not contain sugar components. In the environment of body fluid pH 7.4, albumin is a negative ion, and each molecule can carry more than 200 negative charges. About 10% of exogenously infused albumin enters the extravascular tissue space after 2 hours, and reaches equilibrium in 20 days. The half-life of albumin in plasma is 15-19 days.
>gi|4502027|ref|NP_000468.1|血清白蛋白前原蛋白[智人]>gi|4502027|ref|NP_000468.1|Serum albumin preproprotein [Homo sapiens]
下划线指示信号肽，粗体指示前肽，斜体指示成熟人血清白蛋白)。 Underlined indicates signal peptide, bolded indicates propeptide, italicized indicates mature human serum albumin).
如本发明所用，术语“序列同一性”是指两条序列(氨基酸)对齐后在相同位置具有相同残基的程度。例如，“氨基酸序列与SEQ ID NO：Y是X％相同的”是指该氨基酸序列与SEQ ID NO：Y的同一性百分比，并被阐述为该氨基酸序列中X％的残基与SEQ ID NO：Y中公开的序列残基相同。As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acids) aligned have identical residues at the same positions. For example, "the amino acid sequence is X% identical to SEQ ID NO:Y" refers to the percent identity of the amino acid sequence to SEQ ID NO:Y and is stated as X% of the residues in the amino acid sequence are identical to SEQ ID NO : The sequence residues disclosed in Y are identical.
通常使用计算机程序进行此类计算。比较和对齐序列对的示例性程序包括ALIGN(Myers和Miller,1988)、FASTA(Pearson和Lipman,1988；Pearson,1990)以及gapped BLAST(Altschul等人,1997)、BLASTP、BLASTN或者GCG(Devereux等人,1984)。Computer programs are often used to perform such calculations. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al. People, 1984).
此外，在确定两条氨基酸序列之间的序列同一性的程度时，技术人员可以考虑所谓的“保守性”氨基酸取代，其通常可以描述为氨基酸残基被替换为具有类似化学结构的另一种氨基酸残基的氨基酸取代，其对多肽的功能、活性或其他生物学性质几乎没有影响或基本上没有影响。这种保守性氨基酸取代在本领域中是众所周知的，例如WO 04/037999，GB-A-2 357 768，WO 98/49185，WO 00/46383和WO 01/09300；并且(优选地)这些取代的类型和/或组合可以根据来自WO 04/037999以及WO 98/49185以及其中引用的另外的参考文献的相关教导来选择。Furthermore, when determining the degree of sequence identity between two amino acid sequences, the skilled artisan may consider so-called "conservative" amino acid substitutions, which can generally be described as an amino acid residue being replaced by another with a similar chemical structure Amino acid substitutions of amino acid residues that have little or essentially no effect on the function, activity, or other biological properties of a polypeptide. Such conservative amino acid substitutions are well known in the art, for example WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferably) these substitutions The type and/or combination may be selected based on the relevant teachings from WO 04/037999 and WO 98/49185 and further references cited therein.
这种保守性取代优选地是以下组(a)到(e)中的一个氨基酸被同组中的另一个氨基酸残基取代的取代：(a)小的脂肪族、非极性或弱极性残基：Ala、Ser、Thr、Pro和Gly；(b)极性、带负电的残基及其(不带电的)酰胺：Asp、Asn、Glu和Gln；(c)极性、带正电的残基：His、Arg和Lys；(d)大的脂肪族、非极性残基：Met、Leu、He、Val和Cys；以及(e)芳香族残基：Phe、Tyr和Trp。Such conservative substitutions are preferably substitutions in which one amino acid of the following groups (a) to (e) is replaced by another amino acid residue of the same group: (a) small aliphatic, non-polar or weakly polar Residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
特别优选的保守性取代如下：Ala到Gly或到Ser；Arg到Lys；Asn到Gln或到His；Asp到Glu；Cys到Ser；Gln到Asn；Glu到Asp；Gly到Ala或到Pro；His到Asn或到Gln；Ile到Leu或到Val；Leu到Ile或到Val；Lys到Arg、到Gln或到Glu；Met到Leu、到Tyr或到Ile；Phe到Met、到Leu或到Tyr；Ser到Thr；Thr到Ser；Trp到Tyr；Tyr到Trp；和/或Phe到Val、到Ile或到Leu。Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His To Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.
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。The term "vector" as used herein is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell in which said nucleic acid molecule as a vector can, for example, be replicated and/or expressed.
如本文所用的术语“宿主细胞”是指已引入表达载体从而能够表达外援遗传材料的任何类型的细胞。The term "host cell" as used herein refers to any type of cell into which an expression vector has been introduced thereby capable of expressing foreign genetic material.
术语“药学上可接受”是指载体或佐剂与组合物的其他成分相容并且对其接受者没有大量毒害，和/或这些载体或佐剂被批准或可用于包含在对人类肠胃外给药的药物组合物中。The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and is not substantially toxic to the recipient thereof, and/or that such carrier or adjuvant is approved or available for inclusion in parenteral administration to humans. in pharmaceutical compositions of medicines.
如本文所用的术语“治疗”、“处理”等，指施用药剂或进行程序，以便获得效果。这些效果可以就完全或部分地预防疾病或其症状而言是预防性的，和/或可以就影响疾病和/或疾病症状的部分或完全治愈而言是治疗性的。如本发明所用，“治疗”可包括治疗哺乳动物，特别是人类的疾病或病症(例如癌症、感染性疾病、自身免疫病或炎性疾病)，并且包 括：(a)在对疾病易感而尚未被诊断为患病的受试者中预防该疾病或疾病症状的发生(例如，包括可能与原代疾病相关或由其引起的疾病)；(b)抑制疾病，即阻止其发展；(c)缓解疾病，即导致疾病的消退。治疗可指在治疗或改善或预防癌症方面取得成功的任何指代，包括任何客观或主观参数，例如消除；缓解；减少症状或使疾病病症对患者而言更容易忍受；减慢恶化或衰退速度；或使恶化的终点衰弱减少。症状的治疗或改善基于一个或多个客观或主观参数；包括医生检查的结果。因此，术语“治疗”包括施用本发明公开的多特异性多肽分子或药物组合物或缀合物，以预防或延迟、缓解或阻止或抑制与疾病(例如癌症)相关的症状或病症的发展。术语“治疗效果”是指受试者中疾病、疾病症状或疾病副作用的减少、消除或预防。The terms "treatment,""treatment," etc., as used herein, refer to administering an agent or performing a procedure in order to obtain an effect. These effects may be prophylactic in the sense of completely or partially preventing the disease or its symptoms, and/or may be therapeutic insofar as affecting the partial or complete cure of the disease and/or the symptoms of the disease. As used herein, "treating" may include treating a disease or condition in a mammal, particularly a human (eg, cancer, infectious disease, autoimmune disease or inflammatory disease), and includes This includes: (a) preventing the occurrence of a disease or symptoms of a disease in subjects who are susceptible to the disease but have not yet been diagnosed with the disease (e.g., including diseases that may be associated with or caused by the primary disease); (b) ) inhibits the disease, i.e. prevents its progression; (c) alleviates the disease, i.e. causes the regression of the disease. Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as elimination; remission; reduction of symptoms or making disease symptoms more tolerable for the patient; slowing of progression or decline ; or reduce the endpoint of worsening frailty. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination. Accordingly, the term "treating" includes administering a multispecific polypeptide molecule or pharmaceutical composition or conjugate disclosed herein to prevent or delay, alleviate, or prevent or inhibit the development of symptoms or conditions associated with a disease, such as cancer. The term "therapeutic effect" refers to the reduction, elimination, or prevention of disease, disease symptoms, or disease side effects in a subject.
如本文所用的术语“有效量”意指当施用到受试者用于治疗或预防疾病时足以实现这样的治疗或预防的治疗剂的量。“有效量”可根据化合物、疾病及其严重度、以及待治疗的受试者的年龄、体重等改变。“治疗有效量”是指用于治疗性治疗的有效量。“预防有效量”是指用于预防性治疗的有效量。The term "effective amount" as used herein means an amount of a therapeutic agent that when administered to a subject for the treatment or prevention of a disease is sufficient to effect such treatment or prevention. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated. "Therapeutically effective amount" refers to an amount effective for therapeutic treatment. A "prophylactically effective amount" refers to an amount effective for prophylactic treatment.
如本文所用，术语“受试者”是指期望诊断、医治或治疗的任何哺乳动物受试者。用于治疗目的的“哺乳动物”是指任何归类为哺乳动物的动物，包括人、家畜、以及实验室动物、动物园动物、运动动物或宠物动物，如狗、马、猫、牛、绵羊、山羊、猪、小鼠、大鼠、兔、豚鼠、猴子等。As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes means any animal classified as a mammal, including humans, domestic animals, and laboratory, zoo, sporting or pet animals, such as dogs, horses, cats, cattle, sheep, Goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.
本发明的优选实施方案Preferred embodiments of the invention
通过结合附图对以下实施方案的详细描述，本发明的特征和优点及其附加特征和优点将在下文中得到更清楚的理解。本文参照附图描述的实施方案是解释性的，说明性的，并用于普遍理解本发明。实施方案不应解释为限制本发明的范围。相同或相似的要素和具有相同或相似功能的要素在整个描述中使用相同的附图标记表示。The features and advantages of the present invention and its additional features and advantages will be more clearly understood in the following by the detailed description of the following embodiments in conjunction with the accompanying drawings. The embodiments described herein with reference to the accompanying drawings are illustrative, illustrative, and provide a general understanding of the invention. The embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are designated with the same reference signs throughout the description.
在一方面，本发明提供了一种双特异性多肽分子，所述双特异性多肽分子在一条或多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，In one aspect, the invention provides a bispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on one or more polypeptide chains. district,
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(TRAV)和β链可变区(TRBV)；wherein the first binding region comprises an alpha chain variable region (TRAV) and a beta chain variable region (TRBV) derived from a T cell receptor (TCR) bound to the first antigen-MHC complex;
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；wherein 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;
其中，所述TRAV、TRBV、VH和VL分布在所述一条或多条多肽链上，使得当所述一条或多条多肽链折叠时，所述TRAV和TRBV在空间上靠近以形成所述第一结合区，且所述VH和VL在空间上靠近以形成第二结合区；Wherein, the TRAV, TRBV, VH and VL are distributed on the one or more polypeptide chains, so that when the one or more polypeptide chains are folded, the TRAV and TRBV are spatially close to form the first a binding area, and the VH and VL are spatially close to form a second binding area;
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。The first antigen is selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens, and the second antigen is an immune cell surface molecule.
在一些实施方案中，所述TRAV、TRBV、VH和VL分布在所述一条或多条多肽链上且同一条链上的相邻可变区之间通过接头序列分隔开。In some embodiments, the TRAV, TRBV, VH and VL are distributed on the one or more polypeptide chains and adjacent variable regions on the same chain are separated by linker sequences.
在一些实施方案中，所述双特异性多肽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，In some embodiments, the bispecific polypeptide molecule comprises a first binding region that binds a first antigen and a second binding region that binds a second antigen on both polypeptide chains,
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(TRAV)和β链可变区(TRBV)；wherein the first binding region comprises an alpha chain variable region (TRAV) and a beta chain variable region (TRBV) derived from a T cell receptor (TCR) bound to the first antigen-MHC complex;
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； wherein 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;
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；wherein the first antigen is selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens, and the second antigen is an immune cell surface molecule;
其中所述双特异性多肽分子的两条多肽链分别包含：The two polypeptide chains of the bispecific polypeptide molecule respectively include:
TRAV-VH和VL-TRBV；TRAV-VH and VL-TRBV;
TRAV-VL和VH-TRBV；TRAV-VL and VH-TRBV;
TRBV-VH和VL-TRAV；或TRBV-VH and VL-TRAV; or
TRBV-VL和VH-TRAV；TRBV-VL and 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And the adjacent variable regions in the two polypeptide chains are connected through a linker; and
所述双特异性多肽分子还包含通过任选的接头连接在每条多肽链的C端的TCR恒定区或其片段。The bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof linked to the C-terminus of each polypeptide chain via an optional linker.
在优选的实施方案中，所述双特异性多肽分子的两条多肽链分别包含：TRAV-VH和VL-TRBV。In a preferred embodiment, the two polypeptide chains of the bispecific polypeptide molecule comprise: TRAV-VH and VL-TRBV respectively.
在优选的实施方案中，所述双特异性多肽分子的两条多肽链分别包含：TRAV-VH和VL-TRBV，并且所述两条多肽链中相邻可变区之间通过接头连接；并且所述双特异性多肽分子还包含通过任选的接头连接在每条多肽链的C端的TCR恒定区或其片段。In a preferred embodiment, the two polypeptide chains of the bispecific polypeptide molecule respectively comprise: TRAV-VH and VL-TRBV, and adjacent variable regions in the two polypeptide chains are connected by a linker; and The bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof linked to the C-terminus of each polypeptide chain via an optional linker.
在一些实施方案中，所述TCR恒定区选自TCRα链恒定区(TRAC)和TCRβ链恒定区(TRBC)。在优选的实施方案中，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。In some embodiments, the TCR constant region is selected from the group consisting of TCR alpha chain constant region (TRAC) and TCR beta chain constant region (TRBC). In a preferred embodiment, one polypeptide chain of the bispecific polypeptide molecule comprises TRAC and the other polypeptide chain comprises TRBC.
在一些实施方式中，TRAC中N端的1-5个氨基酸可以发生取代。In some embodiments, 1-5 amino acids at the N-terminus of TRAC may be substituted.
在一些实施方式中，TRBC中N端的1-5个氨基酸可以发生取代。In some embodiments, 1-5 amino acids at the N-terminus of TRBC may be substituted.
TRAC和TRBC可以是人源或鼠源的。TRAC和TRBC可以是野生型的或其变体。例如，变体TRAC可以相对于野生型序列包含T48C、N113K、PESS缺失突变、FFPSPESS缺失突变中的一种或多种。例如，变体TRBC可以相对于野生型序列包含S57C、C187A、N210D、FG环缺失突变中的一种或多种。在优选的实施方案中，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。TRAC and TRBC can be of human or murine origin. TRAC and TRBC can be wild type or variants thereof. For example, the variant TRAC may comprise one or more of T48C, N113K, PESS deletion mutation, FFPSPESS deletion mutation relative to the wild-type sequence. For example, the variant TRBC may comprise one or more of S57C, C187A, N210D, and FG loop deletion mutations relative to the wild-type sequence. In a preferred embodiment, TRAC and/or TRBC comprise at least one cysteine mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, more preferably, said cysteine mutation At the following positions: position 48 of the wild-type TCRα chain constant region and position 57 of the wild-type TCRβ chain constant region.
在一些实施方式中，所述多肽分子包含TRAC或其片段，其不通过接头与TRAV顺势连接。优选地，TRAC中N端的1-5个氨基酸可以发生取代。In some embodiments, the polypeptide molecule comprises TRAC or a fragment thereof, which is not cis-linked to TRAV through a linker. Preferably, 1-5 amino acids at the N-terminus of TRAC can be substituted.
在一些实施方式中，所述多肽分子包含TRBC或其片段，其不通过接头与TRBV顺势连接。优选地，TRBC中N端的1-5个氨基酸可以发生取代。In some embodiments, the polypeptide molecule comprises TRBC or a fragment thereof that is not cis-linked to TRBV through a linker. Preferably, 1-5 amino acids at the N terminus of TRBC can be substituted.
在没有相反说明的情况下，术语“顺势连接”是指在天然状态下TCR恒定区与可变区的连接形态。In the absence of instructions to the contrary, the term "cis-linked" refers to the connection form of the TCR constant region and the variable region in the natural state.
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(format 22-2)。In some embodiments, 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-any The optional linker-TRAC, and the second polypeptide chain from the N-terminus to the C-terminus includes: VL-linker-TRBV-optional linker-TRBC (format 22-2).
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-接头-TRAC，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-接头-TRBC。In some embodiments, 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-linker -TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-linker-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。 In some embodiments, 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-any The optional linker-TRBC, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRBV-optional linker-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。In some embodiments, 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-any The optional linker-TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRBV-optional linker-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。In some embodiments, 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-any The optional linker-TRBC, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRBV-optional linker-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。In some embodiments, 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-any optional linker-TRAC, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-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。In some embodiments, 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-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRAV-optional linker-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。In some embodiments, 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-any The optional linker-TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRAV-optional linker-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。In some embodiments, 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-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC.
在一些实施方案中，所述双特异性多肽分子还包含以下至少一种功能结构域：In some embodiments, the bispecific polypeptide molecule further comprises at least one of the following functional domains:
1)源于抗体的铰链区；1) Derived from the hinge region of the antibody;
2)源于抗体的Fc结构域或其二聚化部分；2) Derived from the Fc domain of an antibody or its dimerization part;
3)白蛋白(Alb)。3) Albumin (Alb).
所述功能结构域的引入不但能延长多肽分子的半衰期，还能增强多肽分子的杀伤活性。The introduction of the functional domain can not only extend the half-life of the polypeptide molecule, but also enhance the killing activity of the polypeptide molecule.
在一些实施方案中，所述功能结构域通过任选的接头连接在所述双特异性多肽分子的一条或两条多肽链的C末端。在一些实施方案中，所述功能结构域通过任选的接头连接在所述双特异性多肽分子的任一条多肽链的C末端。在一些实施方案中，所述功能结构域通过任选的接头连接在所述双特异性多肽分子的两条多肽链的C末端。In some embodiments, the functional domain is linked to the C-terminus of one or both polypeptide chains of the bispecific polypeptide molecule via an optional linker. In some embodiments, the functional domain is linked to the C-terminus of either polypeptide chain of the bispecific polypeptide molecule through an optional linker. In some embodiments, the functional domain is linked to the C-termini of both polypeptide chains of the bispecific polypeptide molecule via an optional linker.
在一些实施方案中，所述功能结构域来源于白蛋白或其片段。在一些实施方案中，白蛋白或其片段通过任选的接头连接在所述双特异性多肽分子的一条或两条多肽链的C末端。在优选的实施方案中，白蛋白或其片段通过任选的接头连接在所述双特异性多肽分子的任一条多肽链的C末端。在一些实施方案中，白蛋白可以为任何哺乳动物来源的白蛋白，例如人白蛋白、牛白蛋白、小鼠白蛋白等。在一些实施方案中，白蛋白为血清白蛋白，例如人血清白蛋白或牛血清白蛋白。在一些实施方案中，所述功能结构域可以是白蛋白的片段，例如长度为100-550、150-550、200-550、250-550、300-550、350-550、400-550、450-550、500-550、100-500、150-500、200-500、250-500、300-500、350-500、400-500、450-500、100-450、150-450、200-450、250-450、300-450、350-450、400-450、100-400、150-400、200-400、250-400、300-400、350-400、100-350、150-350、200-350、250-350、300-350、100-300、150-300、200-300、250-300、100-250、150-250、200-250、100-200、150-200、100-150个氨基酸的片段。在优选的实施方案中，白蛋白为人血清白蛋白，其氨基酸序列如SEQ ID NO:9所示。In some embodiments, the functional domain is derived from albumin or a fragment thereof. In some embodiments, albumin or a fragment thereof is linked to the C-terminus of one or both polypeptide chains of the bispecific polypeptide molecule via an optional linker. In a preferred embodiment, albumin or a fragment thereof is linked to the C-terminus of either polypeptide chain of the bispecific polypeptide molecule through an optional linker. In some embodiments, the albumin can be any mammalian source of albumin, such as human albumin, bovine albumin, mouse albumin, etc. In some embodiments, the albumin is serum albumin, such as human serum albumin or bovine serum albumin. In some embodiments, the functional domain may be a fragment of albumin, for example, 100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550, 450 in length -550, 500-550, 100-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, 450-500, 100-450, 150-450, 200-450 ,250-450,300-450,350-450,400-450,100-400,150-400,200-400,250-400,300-400,350-400,100-350,150-350,200 -350, 250-350, 300-350, 100-300, 150-300, 200-300, 250-300, 100-250, 150-250, 200-250, 100-200, 150-200, 100-150 fragment of amino acids. In a preferred embodiment, the albumin is human serum albumin, whose amino acid sequence is shown in SEQ ID NO: 9.
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-任选的接头-Alb(Format64)。In some embodiments, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain contains from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain contains from N-terminus to C-terminus: VL-linker-TRBV- Optional Linker-TRBC-Optional Linker-Alb (Format64).
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-接头-TRAC，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-接头-TRBC-接头-Alb。In some embodiments, 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-linker -TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC-linker-Alb.
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-任选的接头-Alb，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。In some embodiments, 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-any optional linker-TRAC-optional linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRBV-optional linker-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-任选的接头-Alb，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。In some embodiments, 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-any Optional linker-TRBC-Optional linker-Alb, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-Optional linker-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-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-任选的接头-Alb。In some embodiments, 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-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC-optional linker-Alb.
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-任选的接头-Alb，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。In some embodiments, 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-any optional linker-TRAC-optional linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRBV-optional linker-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-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-任选的接头-Alb。In some embodiments, 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-any The optional linker-TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC-optional linker-Alb.
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-任选的接头-Alb，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。In some embodiments, 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-any Optional linker-TRBC-Optional linker-Alb, and the second polypeptide chain includes from N-terminus to C-terminus: VH-Linker-TRBV-Optional linker-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-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-任选的接头-Alb。In some embodiments, 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-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC-optional linker-Alb.
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-任选的接头-Alb，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。In some embodiments, 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-any optional linker-TRAC-optional linker-Alb, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRAV-optional linker-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-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-任选的接头-Alb。In some embodiments, 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-any optional linker-TRAC, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-TRBC-optional linker-Alb.
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-任选的接头-Alb，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。In some embodiments, 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-any optional linker-TRBC-optional linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-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-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-任选的接头-Alb。In some embodiments, 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-any optional linker-TRBC, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-TRAC-optional linker-Alb.
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-任选的接头-Alb，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。 In some embodiments, 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-any optional linker-TRAC-optional linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRAV-optional linker-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-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-任选的接头-Alb。In some embodiments, 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-any optional linker-TRAC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC-optional linker-Alb.
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-任选的接头-Alb，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。In some embodiments, 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-any Optional Linker-TRBC-Optional Linker-Alb, and the second polypeptide chain from N-terminus to C-terminus includes: VH-Linker-TRAV-Optional Linker-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-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-任选的接头-Alb。In some embodiments, 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-any The optional linker-TRBC, and the second polypeptide chain includes from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC-optional linker-Alb.
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；并且所述功能结构域通过任选的接头连接在所述双特异性多肽分子的两条多肽链的C末端。In some embodiments, the functional domain is derived from the hinge region of an antibody and/or the Fc domain of an antibody or a dimerization portion thereof; and the functional domain is linked to the bispecific via an optional linker. The C termini of the two polypeptide chains of a polypeptide molecule.
铰链区可包含抗体的野生型铰链序列的部分或全部或其具有一处或多处取代的变体。在一些实施方案中，抗体的铰链区为人源抗体的铰链区。抗体的铰链区可以是任何同种型，包括但不限于IgG1、IgG2、IgG3和IgG4。例如，抗体的铰链区可以源自人IgG1、IgG2或IgG4的铰链结构域或其部分。在一些实施方案中，铰链区可包含以下氨基酸序列之一：EPKSSDKTHTCPPCPAPPVAGP(SEQ ID NO:69)、EPKSSDKTHTCPPCP(SEQ ID NO:70)、PKSSDKTHTCPPCPAPPVAGP(SEQ ID NO:71)、KSSDKTHTCPPCPAPPVAGP(SEQ ID NO:72)、SSDKTHTCPPCPAPPVAGP(SEQ ID NO:73)、SDKTHTCPPCPAPPVAGP(SEQ ID NO:74)、DKTHTCPPCPAPPVAGP(SEQ ID NO:75)、KTHTCPPCPAPPVAGP(SEQ ID NO:76)、THTCPPCPAPPVAGP(SEQ ID NO:77)、HTCPPCPAPPVAGP(SEQ ID NO:78)、TCPPCPAPPVAGP(SEQ ID NO:79)、CPPCPAPPVAGP(SEQ ID NO:80)、PPCPAPPVAGP(SEQ ID NO:81)、CPAPPVAGP(SEQ ID NO:82)、或PAPPVAGP(SEQ ID NO:83)；或其具有一个或多个取代(例如，1-6个取代，例如1-5、1-4、1、2、3、4、5或6个取代)的变体。The hinge region may comprise part or all of the wild-type hinge sequence of the antibody or a variant thereof with one or more substitutions. In some embodiments, the hinge region of the antibody is that of a human antibody. The hinge region of an antibody can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. For example, the hinge region of an antibody may be derived from the hinge domain of human IgGl, IgG2 or IgG4, or a portion thereof. In some embodiments, the hinge region may comprise one of the following amino acid sequences: EPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO:69), EPKSSDKTHTCPPCP (SEQ ID NO:70), PKSSDKTHTCPPCPAPPVAGP (SEQ ID NO:71), KSSDKTHTCPPCPAPPVAGP (SEQ ID NO:72) ), SSDKTHTCPPCPAPPVAGP(SEQ ID NO:73), SDKTHTCPPCPAPPVAGP(SEQ ID NO:74), DKTHTCPPCPAPPVAGP(SEQ ID NO:75), KTHTCPPCPAPPVAGP(SEQ ID NO:76), THTCPPCPAPPVAGP(SEQ ID NO:77), HTCPPCPAPPVAGP(SEQ ID NO:78), TCPPCPAPPVAGP(SEQ ID NO:79), CPPCPAPPVAGP(SEQ ID NO:80), PPCPAPPVAGP(SEQ ID NO:81), CPAPPVAGP(SEQ ID NO:82), or PAPPVAGP(SEQ ID NO:83 ); or variants thereof having one or more substitutions (eg, 1-6 substitutions, such as 1-5, 1-4, 1, 2, 3, 4, 5 or 6 substitutions).
抗体的Fc结构域包含CH2和CH3。在一些实施方案中，抗体的Fc结构域或其二聚化部分为人源抗体的Fc结构域或其二聚化部分。抗体的Fc结构域可以是任何同种型，包括但不限于IgG1、IgG2、IgG3和IgG4，并且可以包含一个或多个突变或修饰。在一些实施方案中，抗体的Fc结构域或其二聚化部分源自人IgG1、IgG2或IgG4的Fc结构域或其部分，并且优选地包含至少两个半胱氨酸残基突变，例如S354C和Y349C或L242C和K334C。在一个实施方案中，Fc结构域是IgG1同种型或由其衍生，任选地具有一个或多个突变或修饰。在另一个实施方案中，Fc结构域是IgG4同种型或由其衍生，任选地具有一个或多个突变或修饰。在一个实施方案中，Fc结构域是人IgG1Fc或人IgG4Fc。The Fc domain of the antibody contains CH2 and CH3. In some embodiments, the Fc domain of an antibody, or a dimerization portion thereof, is that of a human antibody, or a dimerization portion thereof. The Fc domain of an antibody can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can contain one or more mutations or modifications. In some embodiments, the Fc domain of the antibody, or a dimerization portion thereof, is derived from the Fc domain of human IgG1, IgG2, or IgG4, or a portion thereof, and preferably contains at least two cysteine residue mutations, such as S354C and Y349C or L242C and K334C. In one embodiment, the Fc domain is of the IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In another embodiment, the Fc domain is or derived from the IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc domain is human IgG1 Fc or human IgG4 Fc.
在一些实施方案中，所述功能结构域包含抗体的铰链区-CH2-CH3。在一些实施方案中，所述双特异性多肽分子的两条多肽链均包含铰链区-CH2-CH3。在一些实施方案中，所述双特异性多肽分子的两条多肽链中的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。在一些实施方案中，两条多肽链中的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，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。在优选的实施方案中，铰链区-CH2-CH3的氨基酸序列如SEQ ID NO:33或34所示。In some embodiments, the functional domain comprises the hinge region-CH2-CH3 of an antibody. In some embodiments, both polypeptide chains of the bispecific polypeptide molecule comprise hinge regions -CH2-CH3. In some embodiments, CH3 in both polypeptide chains of the bispecific polypeptide molecule contains at least one mutation capable of promoting heterodimer formation of the polypeptide molecule. In some embodiments, the CH3s in both polypeptide chains each independently comprise mutations 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, preferably , one of the CH3s contains the T366W mutation and the other contains the T366S, L368A and Y407V mutations. In a preferred embodiment, the amino acid sequence of the hinge region-CH2-CH3 is shown in SEQ ID NO: 33 or 34.
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-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：VL-接头-TRBV-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3(format 63)。In some embodiments, 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-any Optional Connector-TRAC-Optional Connector-Hinge region-CH2-CH3, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3 (format 63) .
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-接头-TRAC-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-接头-TRBC-铰链区-CH2-CH3。In some embodiments, 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-linker -TRAC-hinge region-CH2-CH3, and the second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC-hinge region-CH2-CH3.
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-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：VL-接头-TRBV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。In some embodiments, 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-any Selected linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRBV-optional linker-TRAC-optional Joint-hinge region-CH2-CH3.
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-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：VH-接头-TRBV-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3。In some embodiments, 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-any selected linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRBV-optional linker-TRBC-optional Joint-hinge region-CH2-CH3.
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-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：VH-接头-TRBV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。In some embodiments, 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-any Selected linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRBV-optional linker-TRAC-optional Joint-hinge region-CH2-CH3.
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-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：VL-接头-TRAV-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3。In some embodiments, 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-any selected linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-TRBC-optional Joint-hinge region-CH2-CH3.
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-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：VL-接头-TRAV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。In some embodiments, 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-any selected linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VL-linker-TRAV-optional linker-TRAC-optional Joint-hinge region-CH2-CH3.
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-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：VH-接头-TRAV-任选的接头-TRBC-任选的接头-铰链区-CH2-CH3。In some embodiments, 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-any selected linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRAV-optional linker-TRBC-optional Joint-hinge region-CH2-CH3.
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-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：VH-接头-TRAV-任选的接头-TRAC-任选的接头-铰链区-CH2-CH3。In some embodiments, 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-any Selected linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain from N-terminus to C-terminus includes: VH-linker-TRAV-optional linker-TRAC-optional Joint-hinge region-CH2-CH3.
在本发明的双特异多肽分子的实施方案中，在本发明的双特异性多肽分子的实施方案中，当所述多肽分子包含多于一个接头时，所述接头可以各自相同或不同。在一些实施方案中，所述多肽分子中的接头各自独立地选自由1-35个氨基酸组成的接头。In embodiments of the bispecific polypeptide molecules of the invention, when the polypeptide molecule comprises more than one linker, the linkers may each be the same or different. In some embodiments, the linkers in the polypeptide molecule are each independently selected from a linker consisting of 1-35 amino acids.
在一些实施方式中，所述接头各自独立地选自S(SEQ ID NO:41)、GGGS(SEQ ID NO:62)、GGGGS(SEQ ID NO:42)、GGGSGGGG(SEQ ID NO:50)、GGSGGS(SEQ ID NO:47)、GGSGGSGGS(SEQ ID NO:48)、GGGGSGGGGS(SEQ ID NO:46)、GGGGSGGGGSGGGGS(SEQ ID NO:44)、GGGGSGGGGSGGGGSGGGGSGGGS(SEQ ID NO:43)、GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:45)、GQPKAAP(SEQ ID NO:49)、TVLRT(SEQ ID NO:53)、TVSSAS(SEQ ID NO:54)、GGEGG(SEQ ID  NO:55)、GSEGGGS(SEQ ID NO:56)、RTSGPGDGGKGGPGKGPGGEGTKGTGPGG(SEQ ID NO:57)、GKGPGGEGTKGTGPGG(SEQ ID NO:58)、TVLSSAS(SEQ ID NO:59)、EDLKN(SEQ ID NO:63)或其变体、EDLNK(SEQ ID NO:64)或其变体和ANIQK(SEQ ID NO:65)或其变体，或其任意组合。In some embodiments, the linkers are each independently selected from the group consisting of S (SEQ ID NO:41), GGGS (SEQ ID NO:62), GGGGS (SEQ ID NO:42), GGGSGGGG (SEQ ID NO:50), GGSGGS (SEQ ID NO: 47), GGSGGGGGS (SEQ ID NO: 48), GGGSGGGGS (SEQ ID NO: 46), GGGGSGGGGSGGGGS (SEQ ID NO: 44), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 43), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43) :45), GQPKAAP (SEQ ID NO:49), TVLRT (SEQ ID NO:53), TVSSAS (SEQ ID NO:54), GGEGG (SEQ ID NO:55), GSEGGGS (SEQ ID NO:56), RTSGPGDGGKGGPGKGPGGEGTKGTGPGG (SEQ ID NO:57), GKGPGGEGTKGTGPGG (SEQ ID NO:58), TVLSSAS (SEQ ID NO:59), EDLKN (SEQ ID NO:63) or Variants thereof, EDLNK (SEQ ID NO:64) or variants thereof, and ANIQK (SEQ ID NO:65) or variants thereof, or any combination thereof.
在一些实施方式中，EDLKN的变体为EDLKN经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或ANIQK的变体为ANIQK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列。In some embodiments, the variant of EDLKN is the amino acid sequence formed by EDLKN through substitution, deletion or addition of one or several amino acids; and/or the variant of EDLNK is the amino acid sequence of EDLNK through substitution, deletion or addition of one or several amino acids. The formed amino acid sequence; and/or the variant of ANIQK is the amino acid sequence formed by substituting, deleting or adding one or several amino acids of ANIQK.
优选地，EDLKN的变体为EDLKN经过取代一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代一个或几个氨基酸所形成的氨基酸序列；和/或ANIQK的变体为ANIQK经过取代一个或几个氨基酸所形成的氨基酸序列。Preferably, the variant of EDLKN is the amino acid sequence formed by substituting one or several amino acids of EDLKN; and/or the variant of EDLNK is the amino acid sequence formed by substituting one or several amino acids of EDLNK; and/or the variation of ANIQK The body is the amino acid sequence formed by ANIQK by substituting one or several amino acids.
优选地，EDLKN的变体为EDLKN经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列；和/或EDLNK的变体为EDLNK经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列；和/或ANIQK的变体为ANIQK经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列。Preferably, the variant of EDLKN is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLNK. An amino acid sequence consisting of 1-3 amino acids; and/or a variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids of ANIQK.
优选地，EDLKN的变体为EDL、DL、L、ED、E、D；和/或ANIQK的变体为ANI、NI、I、AN、N、A、PNI、P、PN。Preferably, the variants of EDLKN are EDL, DL, L, ED, E, D; and/or the variants of ANIQK are ANI, NI, I, AN, N, A, PNI, P, PN.
优选地，EDLKN的变体为EDLKN经过添加(优选在N端)一个或几个氨基酸所形成的由6-35个氨基酸组成的氨基酸序列；和/或EDLNK的变体为EDLNK经过添加(优选在N端)一个或几个氨基酸所形成的由6-35个氨基酸组成的氨基酸序列；和/或ANIQK的变体为ANIQK经过添加(优选在N端)一个或几个氨基酸所形成的由6-35个氨基酸组成的氨基酸序列。Preferably, the variant of EDLKN is an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to EDLKN (preferably at the N-terminus); and/or the variant of EDLNK is EDLNK after adding (preferably at the N-terminus) N-terminus) an amino acid sequence consisting of 6-35 amino acids formed by one or several amino acids; and/or the variant of ANIQK is ANIQK formed by adding (preferably at the N-terminus) one or several amino acids consisting of 6- An amino acid sequence consisting of 35 amino acids.
在一些实施方式中，所述多肽分子中的接头各自独立地选自由不超过12个氨基酸组成的接头。优选地，所述接头各自独立地选自S、GGGS、GGGGS、GGGSGGGG、GGSGGS、GGSGGSGGS、GGGGSGGGGS、EDLKN或其变体、EDLNK或其变体和ANIQK或其变体。在一些实施方式中，EDLKN的变体为EDLKN经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或ANIQK的变体为ANIQK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列。优选地，EDLKN的变体为EDLKN经过取代一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代一个或几个氨基酸所形成的氨基酸序列；和/或ANIQK的变体为ANIQK经过取代一个或几个氨基酸所形成的氨基酸序列。优选地，EDLKN的变体为EDLKN经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列；和/或EDLNK的变体为EDLNK经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列；和/或ANIQK的变体为ANIQK经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列。优选地，EDLKN的变体为EDLKN经过添加(优选在N端)一个或几个氨基酸所形成的由6-12个氨基酸组成的氨基酸序列；和/或EDLNK的变体为EDLNK经过添加(优选在N端)一个或几个氨基酸所形成的由6-12个氨基酸组成的氨基酸序列；和/或ANIQK的变体为ANIQK经过添加(优选在N端)一个或几个氨基酸所形成的由6-12个氨基酸组成的氨基酸序列。优选地，EDLKN的变体为EDL、DL、L、ED、E、D；和/或ANIQK的变体为ANI、NI、I、AN、N、A、PNI、P、PN。In some embodiments, the linkers in the polypeptide molecule are each independently selected from a linker consisting of no more than 12 amino acids. Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, EDLKN or variants thereof, EDLNK or variants thereof and ANIQK or variants thereof. In some embodiments, the variant of EDLKN is the amino acid sequence formed by EDLKN through substitution, deletion or addition of one or several amino acids; and/or the variant of EDLNK is the amino acid sequence of EDLNK through substitution, deletion or addition of one or several amino acids. The formed amino acid sequence; and/or the variant of ANIQK is the amino acid sequence formed by substituting, deleting or adding one or several amino acids of ANIQK. Preferably, the variant of EDLKN is the amino acid sequence formed by substituting one or several amino acids of EDLKN; and/or the variant of EDLNK is the amino acid sequence formed by substituting one or several amino acids of EDLNK; and/or the variation of ANIQK The body is the amino acid sequence formed by ANIQK by substituting one or several amino acids. Preferably, the variant of EDLKN is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLNK. An amino acid sequence consisting of 1-3 amino acids; and/or a variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids of ANIQK. Preferably, the variant of EDLKN is an amino acid sequence consisting of 6-12 amino acids formed by adding one or several amino acids to EDLKN (preferably at the N-terminus); and/or the variant of EDLNK is EDLNK after adding (preferably at the N-terminus) N-terminus) an amino acid sequence consisting of 6-12 amino acids formed by one or several amino acids; and/or the variant of ANIQK is ANIQK formed by adding (preferably at the N-terminus) one or several amino acids consisting of 6- An amino acid sequence consisting of 12 amino acids. Preferably, the variants of EDLKN are EDL, DL, L, ED, E, D; and/or the variants of ANIQK are ANI, NI, I, AN, N, A, PNI, P, PN.
在一些实施方式中，所述多肽分子中的接头各自独立地为1个或2-3个以上所述接 头的组合。在一些实施方式中，所述多肽分子中的接头各自独立地为1个以上所述接头。在一些实施方式中，所述多肽分子中的接头各自独立的为2个以上所述接头的组合。在一些实施方式中，所述多肽分子中的接头各自独立地为3个以上所述接头的组合。In some embodiments, the linkers in the polypeptide molecule are each independently 1 or 2-3 or more of the linkers. Head combination. In some embodiments, each linker in the polypeptide molecule is independently one or more of the linkers. In some embodiments, each linker in the polypeptide molecule is independently a combination of two or more of the linkers. In some embodiments, each linker in the polypeptide molecule is independently a combination of three or more of the linkers.
在一些实施方式中，所述TRAV的C端通过2个接头与VH或VL连接，TRAV的近端接头为ANIQK或其变体。在一些实施方式中，ANIQK的变体为ANIQK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列。优选地，ANIQK的变体为ANIQK经过取代一个或几个氨基酸所形成的氨基酸序列。优选地，ANIQK的变体为ANIQK经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列，例如，ANI、NI、I、AN、N、A、PNI、P、PN。In some embodiments, the C-terminal of TRAV is connected to VH or VL through 2 linkers, and the proximal linker of TRAV is ANIQK or a variant thereof. In some embodiments, a variant of ANIQK is an amino acid sequence formed by substituting, deleting, or adding one or several amino acids to ANIQK. Preferably, the variant of ANIQK is the amino acid sequence formed by substituting one or several amino acids of ANIQK. Preferably, the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids of ANIQK, for example, ANI, NI, I, AN, N, A, PNI, P, PN.
在一些实施方式中，所述TRBV的C端通过2个接头与VH或VL连接，TRBV的近端接头为EDLKN或其变体、EDLNK或其变体、ANIQK或其变体。在一些实施方式中，EDLKN的变体为EDLKN经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列。优选地，EDLKN的变体为EDLKN经过取代一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代一个或几个氨基酸所形成的氨基酸序列。优选地，EDLKN的变体为EDLKN经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列；和/或EDLNK的变体为EDLNK经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列，例如，EDL、DL、L、ED、E、D。In some embodiments, the C-terminal of TRBV is connected to VH or VL through two linkers, and the proximal linker of TRBV is EDLKN or a variant thereof, EDLNK or a variant thereof, ANIQK or a variant thereof. In some embodiments, the variant of EDLKN is the amino acid sequence formed by EDLKN through substitution, deletion or addition of one or several amino acids; and/or the variant of EDLNK is the amino acid sequence of EDLNK through substitution, deletion or addition of one or several amino acids. formed amino acid sequence. Preferably, the variant of EDLKN is an amino acid sequence formed by substituting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence formed by substituting one or several amino acids of EDLNK. Preferably, the variant of EDLKN is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLNK. Amino acid sequence consisting of 1-3 amino acids, for example, EDL, DL, L, ED, E, D.
在一些实施方式中，所述TRAV的C端通过接头与TRAC连接，所述接头为ANIQK或其变体。在一些实施方式中，ANIQK的变体为ANIQK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列。优选地，ANIQK的变体为ANIQK经过取代一个或几个氨基酸所形成的氨基酸序列。ANIQK的变体为ANIQK经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列，例如，ANI、NI、I、AN、N、A、PNI、P、PN。In some embodiments, the C-terminus of TRAV is connected to TRAC through a linker, and the linker is ANIQK or a variant thereof. In some embodiments, a variant of ANIQK is an amino acid sequence formed by substituting, deleting, or adding one or several amino acids to ANIQK. Preferably, the variant of ANIQK is the amino acid sequence formed by substituting one or several amino acids of ANIQK. ANIQK variants are ANIQK amino acid sequences consisting of 1-3 amino acids formed by deleting one or several amino acids, for example, ANI, NI, I, AN, N, A, PNI, P, PN.
在一些实施方式中，所述TRBV的C端通过接头与TRBC连接，所述接头为EDLKN或其变体、或EDLNK或其变体。在一些实施方式中，EDLKN的变体为EDLKN经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列。优选地，EDLKN的变体为EDLKN经过取代一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代一个或几个氨基酸所形成的氨基酸序列。优选地，EDLKN的变体为EDLKN经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列；和/或EDLNK的变体为EDLNK经过缺失一个或几个氨基酸所形成的由1-3个氨基酸组成的氨基酸序列，例如，EDL、DL、L、ED、E、D。In some embodiments, the C-terminus of TRBV is connected to TRBC through a linker, and the linker is EDLKN or a variant thereof, or EDLNK or a variant thereof. In some embodiments, the variant of EDLKN is the amino acid sequence formed by EDLKN through substitution, deletion or addition of one or several amino acids; and/or the variant of EDLNK is the amino acid sequence of EDLNK through substitution, deletion or addition of one or several amino acids. formed amino acid sequence. Preferably, the variant of EDLKN is an amino acid sequence formed by substituting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence formed by substituting one or several amino acids of EDLNK. Preferably, the variant of EDLKN is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLKN; and/or the variant of EDLNK is an amino acid sequence of 1-3 amino acids formed by deleting one or several amino acids of EDLNK. Amino acid sequence consisting of 1-3 amino acids, for example, EDL, DL, L, ED, E, D.
在一些实施方案中，VH和VL包含至少一个半胱氨酸突变，以在VH和VL之间形成二硫键，并且半胱氨酸在VL的情况下被引入FR4，在VH的情况下被引入FR2。在优选的实施方案中，所述半胱氨酸突变在以下位置处：VH的第44位和VL的第100位。In some embodiments, VH and VL comprise at least one cysteine mutation to form a disulfide bond between VH and VL, and the cysteine is introduced into FR4 in the case of VL and is introduced in the case of VH Introducing FR2. In a preferred embodiment, the cysteine mutations are at positions 44 of VH and 100 of VL.
在一些实施方案中，所述TRAV和VL包含至少一个半胱氨酸突变以在TRAV和VL之间形成二硫键，优选地，所述半胱氨酸突变在以下位置处：TRAV的第104位和VL的第80位。在一些实施方案中，所述TRBV和VL包含至少一个半胱氨酸突变以在TRBV和VL之间形成二硫键，优选地，所述半胱氨酸突变在以下位置处：TRBV的第84位和VL的第80位。在一些实施方案中，所述TRAV和VH包含至少一个半胱氨酸突变以在TRAV和VH之间形成二硫键，优选地，所述半胱氨酸突变在以下位置处：TRAV的第104位和VH的第89位。在一些实施方案中，所述TRBV和VH包含至少一个半胱氨酸突变 以在TRBV和VH之间形成二硫键，优选地，所述半胱氨酸突变在以下位置处：TRBV的第104位和VH的第89位。In some embodiments, the TRAV and VL comprise at least one cysteine mutation to form a disulfide bond between TRAV and VL, preferably the cysteine mutation is at position 104 of TRAV bit and the 80th bit of VL. In some embodiments, the TRBV and VL comprise at least one cysteine mutation to form a disulfide bond between TRBV and VL, preferably the cysteine mutation is at position 84 of TRBV bit and the 80th bit of VL. In some embodiments, the TRAV and VH comprise at least one cysteine mutation to form a disulfide bond between TRAV and VH, preferably the cysteine mutation is at position 104 of TRAV position and VH's 89th. In some embodiments, the TRBV and VH comprise at least one cysteine mutation To form a disulfide bond between TRBV and VH, preferably, the cysteine mutation is at the following positions: position 104 of TRBV and position 89 of VH.
在一些实施方案中，TRAV可以是天然的TRAV或其功能变体，只要该功能变体保留结合靶抗原的能力即可。例如TRAV可以是天然TRAV的截短变体，例如N端或C端截短8个氨基酸的变体。在一些实施方案中，TRBV可以是天然的TRBV或其功能变体，只要该功能变体保留结合靶抗原的能力即可。在一些实施方案中，TRAV和TRBV是αβTCR的TRAV和TRBV。In some embodiments, the TRAV may be native TRAV or a functional variant thereof, as long as the functional variant retains the ability to bind the target antigen. For example, TRAV may be a truncated variant of native TRAV, such as a variant with 8 amino acids truncated at the N- or C-terminus. In some embodiments, the TRBV may be native TRBV or a functional variant thereof, as long as the functional variant retains the ability to bind the target antigen. In some embodiments, the TRAV and TRBV are those of an αβ TCR.
在一些实施方案中，所述多肽分子还包含能够增强其亲和力或生物学活性(例如，增强其溶解性、增强其聚集、增强其稳定性、延长其半衰期、降低其免疫原性)或减少其翻译后修饰(例如糖基化修饰)的一个或多个氨基酸突变、插入或缺失，例如1、2、3、4、5或更多个氨基酸突变、插入或缺失。突变、插入或缺失的氨基酸包括但不限于：(a)中性氨基酸：Ser、Gln；(b)酸性氨基酸：Asp；(c)长链脂肪族氨基酸：Ile；(d)短脂肪族氨基酸：Ala、Gly。在一些实施方案中，所述氨基酸突变、插入或缺失位于以下氨基酸位置处：TRAV、TRBV、VL、VH的开始、TRAV/TRAC的交界氨基酸、TRBV/TRBC的交界氨基酸、铰链区及其临近氨基酸、CH2/CH3的交界氨基酸、CH3的C末端、接头附近的氨基酸。在一些实施方案中，所述氨基酸突变包括能够增强对FcRn的结合的突变和/或效应子功能沉默突变。在一些实施方案中，能够增强对FcRn的结合的突变包括例如以下突变中的一种或多种：CH2和CH3结构域中的L234A、L235A、M252Y、S254T、T256E、M428L、N434S、T250R、M428L、N434A，优选L234AL235A、M252YS254TT256E、M428LN434S和T250RM428LN434A。在一些实施方案中，效应子功能沉默突变在CH2-CH3结构域的以下一个或多个位置处：233、234、235、236、297和331；优选地，所述效应子功能沉默突变通过用衍生自IgG2或IgG4相应残基取代233、234、235、236和331位置的至少一个残基而产生。在一些实施方案中，能够减少所述多肽分子翻译后修饰的氨基酸突变包括例如TRAC中的N113K和TRBC中的N210D。In some embodiments, the polypeptide molecule further contains a compound that can enhance its affinity or biological activity (e.g., enhance its solubility, enhance its aggregation, enhance its stability, extend its half-life, reduce its immunogenicity) or reduce its One or more amino acid mutations, insertions or deletions of post-translational modifications (eg glycosylation modifications), such as 1, 2, 3, 4, 5 or more amino acid mutations, insertions or deletions. Mutated, inserted or deleted amino acids include but are not limited to: (a) neutral amino acids: Ser, Gln; (b) acidic amino acids: Asp; (c) long-chain aliphatic amino acids: Ile; (d) short aliphatic amino acids: Alas, Gly. In some embodiments, the amino acid mutations, insertions or deletions are located at the following amino acid positions: TRAV, TRBV, VL, beginning of VH, TRAV/TRAC junction amino acid, TRBV/TRBC junction amino acid, hinge region and adjacent amino acids , the boundary amino acid of CH2/CH3, the C terminus of CH3, and the amino acids near the linker. In some embodiments, the amino acid mutations include mutations that enhance binding to FcRn and/or effector function silencing mutations. In some embodiments, mutations capable of enhancing binding to FcRn include, for example, one or more of the following mutations: L234A, L235A, M252Y, S254T, T256E, M428L, N434S, T250R, M428L in the CH2 and CH3 domains , N434A, preferably L234AL235A, M252YS254TT256E, M428LN434S and T250RM428LN434A. In some embodiments, the effector function silencing mutation is at one or more of the following positions of the CH2-CH3 domain: 233, 234, 235, 236, 297, and 331; preferably, the effector function silencing mutation is obtained by using Derived from substitution of at least one residue at positions 233, 234, 235, 236 and 331 with corresponding residues from IgG2 or IgG4. In some embodiments, amino acid mutations that reduce post-translational modifications of the polypeptide molecule include, for example, N113K in TRAC and N210D in TRBC.
在一些实施方案中，所述多肽分子在一条或两条多肽链的N端还包含信号肽序列，优选在每一条链的N端包含信号肽序列，例如衍生自白蛋白或免疫球蛋白的信号肽序列，优选MGWSCIILFLVATATGVHS。In some embodiments, the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or both polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain, for example a signal peptide derived from albumin or immunoglobulin Sequence, preferably MGWSCIILFLVATATGVHS.
在一些实施方案中，所述第一抗原为选自以下的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。In some embodiments, the first antigen is a TAA 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.
在一些实施方案中，所述第一抗原为选自以下的TSA：KRAS(例如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)。In some embodiments, the first antigen is a TSA selected from: KRAS (eg, G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (eg, R175H, R173H, R273C, R248W, R248Q , R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (such as E542K, E545K, H1047R), CTNNB1 (such as S45P, T41A), EGFR (such as L858R, T790M), BRAF (such as V600E) and GNAS (such as R201C , R201H).
在一些实施方案中，所述第一抗原为选自以下的病毒抗原：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。In some embodiments, the first antigen is a viral antigen selected from the group consisting of: HPV E6 or E7 antigen, CMV antigen, HBV antigen, EBV antigen, herpes virus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen and coronavirus antigens.
在一些实施方案中，所述第一抗原为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。In some embodiments, the first antigen is an autoantigen selected from: 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, topological heterogeneity Structural enzyme 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 and MPO.
在一些实施方案中，所述第二抗原选自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、CD80、BTLA、TIM3、TIGIT和LAG-3。In some embodiments, the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and CD3ε chains), 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. CD80, BTLA, TIM3, TIGIT and LAG-3.
在一些实施方案中，所述第一抗原为选自以下的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；并且所述第二抗原选自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和B7-H3。In some embodiments, the first antigen is a TAA 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 antigen; and the second antigen is selected From CD3 (such as CD3γ, CD3δ and CD3ε chains), 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 and B7-H3.
在一些实施方案中，所述第一抗原为选自以下的TSA：KRAS(例如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)；并且所述第二抗原选自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和B7-H3。In some embodiments, the first antigen is a TSA selected from: KRAS (eg, G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (eg, R175H, R173H, R273C, R248W, R248Q , R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (such as E542K, E545K, H1047R), CTNNB1 (such as S45P, T41A), EGFR (such as L858R, T790M), BRAF (such as V600E) and GNAS (such as R201C , R201H); and the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and CD3ε chains), 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 and B7-H3.
在一些实施方案中，所述第一抗原为选自以下的病毒抗原：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；并且所述第二抗原选自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和B7-H3。In some embodiments, the first antigen is a viral antigen selected from the group consisting of: HPV E6 or E7 antigen, CMV antigen, HBV antigen, EBV antigen, herpes virus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen and a coronavirus antigen; and the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and CD3ε chains), 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 and B7-H3.
在一些实施方案中，所述第一抗原为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；并且所述第二抗原选自CTLA-4、PD-1、CD80、BTLA、TIM3、TIGIT和LAG-3。In some embodiments, the first antigen is an autoantigen selected from: 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-γ, CHRNA1, MUSK, LRP4, AQP4, MOG, GRIN1, COL4A3, PLA2R, GM-SCF, PR3 and MPO; and the second antigen is selected from CTLA-4, PD-1, CD80, BTLA, TIM3, TIGIT and LAG-3.
在一些实施方案中，第一抗原选自gp100、MAGEA1、KRAS和HPVE7。In some embodiments, the first antigen is selected from gp100, MAGEA1, KRAS, and HPVE7.
在一些实施方案中，第二抗原选自CD3、CD28和4-1BB(CD137)。在一些实施方案中，第二抗原为CD3。在一些实施方案中，第二抗原为CD28。在一些实施方案中，第二抗原为4-1BB(CD137)。In some embodiments, the second antigen is selected from CD3, CD28, and 4-1BB (CD137). In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is CD28. In some embodiments, the second antigen is 4-1BB (CD137).
在一些实施方式中，所述第二抗原包括但不限于OKT3、UCHT-1、BMA031和12F6。In some embodiments, the second antigen includes, but is not limited to, OKT3, UCHT-1, BMA031, and 12F6.
在一些实施方案中，所述第一多肽链从N端到C端包含：TRAV-第一接头-第二接头-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-接头TRBC。In some embodiments, the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC, and the second polypeptide chain from N-terminus to C-terminus End contains: VL-connector-TRBV-connector TRBC.
在一些实施方案中，所述第一多肽链从N端到C端包含：TRAV-第一接头-第二接头-VH-接头-TRAC-铰链区-CH2-CH3，并且所述第二多肽链从N端到C端包含：VL-接头-TRBV-接头TRBC-铰链区-CH2-CH3。In some embodiments, the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC-hinge region-CH2-CH3, and the second polypeptide chain The peptide chain contains from N-terminus to C-terminus: VL-linker-TRBV-linker TRBC-hinge region-CH2-CH3.
在一些实施方案中，所述第一多肽链从N端到C端包含：TRAV-第一接头-第二接头-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-接头TRBC-接头-ALB。In some embodiments, the first polypeptide chain comprises from N-terminus to C-terminus: TRAV-first linker-second linker-VH-linker-TRAC, and the second polypeptide chain from N-terminus to C-terminus The end contains: VL-connector-TRBV-connector TRBC-connector-ALB.
在一些实施方式中，所述第一抗原为gp100。In some embodiments, the first antigen is gp100.
在一些实施方案中，所述多肽分子包含：具有如SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1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第一多肽链，和具有如SEQ ID NO:2所示的氨基酸序列或与SEQ ID NO:2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第二多肽链。在优选的实施方案中，所述多肽分子包含：具有如SEQ ID NO:1所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:2所示的氨基酸序列的第二多肽链。In some embodiments, the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO: 1 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 1 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO:2 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO:2 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 1, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 2 chain.
SEQ ID NO:1SEQ ID NO:1
Format 22-2(GPA2_TCD3_B22-2_M0)第一链：
Format 22-2(GPA2_TCD3_B22-2_M0) first chain:
SEQ ID NO:2SEQ ID NO:2
Format 22-2(GPA2_TCD3_B22-2_M0)第二链：

Format 22-2(GPA2_TCD3_B22-2_M0) second chain:

在一些实施方案中，所述多肽分子包含：具有如SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3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第一多肽链，和具有如SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO:4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第二多肽链。在优选的实施方案中，所述多肽分子包含：具有如SEQ ID NO:3所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:4所示的氨基酸序列的第二多肽链。In some embodiments, the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO:3 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:3 % sequence identity of the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO:4 or having at least 80%, at least 85%, at least 90%, at least 95% with SEQ ID NO:4 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 3, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 4 chain.
SEQ ID NO:3SEQ ID NO:3
Format 64(GPA2_TCD3_B64)第一链：
Format 64 (GPA2_TCD3_B64) first link:
SEQ ID NO:4SEQ ID NO:4
Format 64(GPA2_TCD3_B64)第二链：
Format 64(GPA2_TCD3_B64) second chain:
在一些实施方案中，所述多肽分子包含：具有如SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO:5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第一多肽链，和具有如SEQ ID NO:6所示的氨基酸序列或与SEQ ID NO:6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第二多肽链。在优选的实施方案中，所述多肽分子包含：具有如SEQ ID NO:5所示的氨 基酸序列的第一多肽链，和具有如SEQ ID NO:6所示的氨基酸序列的第二多肽链。In some embodiments, the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO:5 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:5 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO: 6 or having at least 80%, at least 85%, at least 90%, at least 95% with SEQ ID NO: 6 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity. In a preferred embodiment, the polypeptide molecule comprises: having an amino group as shown in SEQ ID NO:5 A first polypeptide chain having an amino acid sequence, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 6.
SEQ ID NO:5SEQ ID NO:5
Format 63(GPA2_TCD3_B63)第一链：
Format 63 (GPA2_TCD3_B63) first link:
SEQ ID NO:6SEQ ID NO:6
Format 63(GPA2_TCD3_B63)第二链：
Format 63(GPA2_TCD3_B63) second chain:
在一些实施方式中，所述第一抗原为KRAS。在一些实施方式中，在所述多肽分子中，TRAV包含NSASQS(SEQ ID NO:89)所示的CDR1，VYSSGN(SEQ ID NO:90)所示的CDR2，和VVPGGTGGGNKLT(SEQ ID NO:91)所示的CDR3；TRBV包含LGHDT(SEQ ID NO:92)所示的CDR1，YNNKEL(SEQ ID NO:93)所示的CDR2，和ASSHWGAQETQY(SEQ ID NO:94)所示的CDR3。In some embodiments, the first antigen is KRAS. In some embodiments, in the polypeptide molecule, TRAV includes CDR1 represented by NSASQS (SEQ ID NO:89), CDR2 represented by VYSSGN (SEQ ID NO:90), and VVPGGTGGGNKLT (SEQ ID NO:91) CDR3 shown; TRBV contains CDR1 shown by LGHDT (SEQ ID NO:92), CDR2 shown by YNNKEL (SEQ ID NO:93), and CDR3 shown by ASSHWGAQETQY (SEQ ID NO:94).
在一些实施方案中，TRAV包含RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGTGTQLKVEL(SEQ ID NO:95)。在一些实施方案中，TRAV包含RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGTGTQLPVPL(SEQ ID NO:96)。在一些实施方案中，TRAV包含RKIVEQDPGPFEVPEGATVAFICTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQDIHLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGTGTQLPVPL(SEQ ID NO:97)。 In some embodiments, TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGTGTQLKVEL (SEQ ID NO:95). In some embodiments, TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGGTQLPVPL (SEQ ID NO:96). In some embodiments, TRAV comprises RKIVEQDPGPFEVPEGATVAFICTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQDIHLLIRDSKLSDSATYLCVVPGGTGGGNKLTFGGTQLPVPL (SEQ ID NO:97).
在一些实施方案中，TRBV包含DTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMDWYKQDSKKFLKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSHWGAQETQYFGPGTRLLVL(SEQ ID NO:98)。In some embodiments, TRBV comprises DTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMDWYKQDSKKFLKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSHWGAQETQYFPGGTRLLVL (SEQ ID NO:98).
在一些实施方案中，所述多肽分子包含：具有如SEQ ID NO:99所示的氨基酸序列或与SEQ ID NO:99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第一多肽链，和具有如SEQ ID NO:100所示的氨基酸序列或与SEQ ID NO:100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第二多肽链。在优选的实施方案中，所述多肽分子包含：具有如SEQ ID NO:99所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:100所示的氨基酸序列的第二多肽链。In some embodiments, the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO:99 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:99 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO: 100 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 100 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 99, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 100 chain.
SEQ ID NO:99SEQ ID NO:99
Format 22-2(KVA11-N03_B22-2_WT)第一链：
Format 22-2(KVA11-N03_B22-2_WT) first chain:
SEQ ID NO:100SEQ ID NO:100
Format 22-2(KVA11-N03_B22-2_WT)第二链：
Format 22-2(KVA11-N03_B22-2_WT) second chain:
在一些实施方案中，所述多肽分子包含：具有如SEQ ID NO:101所示的氨基酸序列或与SEQ ID NO:101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第一多肽链，和具有如SEQ ID NO:102所示的氨基酸序列或与SEQ ID NO:102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第二多肽链。在优选的实施方案中，所述多肽分子包含：具有如SEQ ID NO:101所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:102所示的氨基酸序列的第二多肽链。In some embodiments, the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO: 101 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 101 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO: 102 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 102 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 101, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 102 chain.
SEQ ID NO:101SEQ ID NO:101
Format 22-2(KVA11-N03_B22-2_Mut1)第一链：

Format 22-2(KVA11-N03_B22-2_Mut1) first chain:

SEQ ID NO:102SEQ ID NO:102
Format 22-2(KVA11-N03_B22-2_Mut1)第二链：
Format 22-2(KVA11-N03_B22-2_Mut1) second chain:
在一些实施方案中，所述多肽分子包含：具有如SEQ ID NO:103所示的氨基酸序列或与SEQ ID NO:103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第一多肽链，和具有如SEQ ID NO:104所示的氨基酸序列或与SEQ ID NO:104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第二多肽链。在优选的实施方案中，所述多肽分子包含：具有如SEQ ID NO:103所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:104所示的氨基酸序列的第二多肽链。In some embodiments, the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO: 103 or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 103 % sequence identity to the first polypeptide chain of the amino acid sequence, and having an amino acid sequence as shown in SEQ ID NO: 104 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 104 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity. In a preferred embodiment, the polypeptide molecule comprises: a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 103, and a second polypeptide having the amino acid sequence shown in SEQ ID NO: 104 chain.
SEQ ID NO:103SEQ ID NO:103
Format 22-2(KVA11-N03_B22-2_Mut2)第一链：
Format 22-2(KVA11-N03_B22-2_Mut2) first chain:
SEQ ID NO:104SEQ ID NO:104
Format 22-2(KVA11-N03_B22-2_Mut2)第二链：
Format 22-2(KVA11-N03_B22-2_Mut2) second chain:
在一些实施方案中，所述多肽分子包含：具有如SEQ ID NO:105所示的氨基酸序列或与SEQ ID NO:105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第一多肽链，和具有如SEQ ID NO:106所示的氨基酸序列或与SEQ ID NO:106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第二多肽链。在优选的实施方案中，所述多肽分子包含：具有如SEQ ID NO:105 所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:106所示的氨基酸序列的第二多肽链。In some embodiments, the polypeptide molecule comprises: having an amino acid sequence as set forth in SEQ ID NO: 105 or being at least 80%, at least 85%, at least 90%, at least 95%, or at least 99 identical to SEQ ID NO: 105 % sequence identity to a first polypeptide chain of an amino acid sequence, and having an amino acid sequence as set forth in SEQ ID NO: 106 or having at least 80%, at least 85%, at least 90%, at least 95% identity with SEQ ID NO: 106 or a second polypeptide chain having an amino acid sequence of at least 99% sequence identity. In a preferred embodiment, the polypeptide molecule comprises: having SEQ ID NO:105 A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 106, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 106.
SEQ ID NO:105SEQ ID NO:105
Format 22-2(KVA11-N03_B22-2_Mut3)第一链：
Format 22-2(KVA11-N03_B22-2_Mut3) first chain:
SEQ ID NO:106SEQ ID NO:106
Format 22-2(KVA11-N03_B22-2_Mut3)第二链：
Format 22-2(KVA11-N03_B22-2_Mut3) second chain:
在一些实施方案中，所述第一抗原为MAGE-A1。In some embodiments, the first antigen is MAGE-A1.
在一些实施方案中，在所述多肽分子中，TRAV包含RGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGTGTRLQVFPN(SEQ ID NO:107)。在一些实施方案中，在所述多肽分子中，TRAV包含RGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYTWPHMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGTGTRLQVFPN(SEQ ID NO:108)。In some embodiments, in the polypeptide molecule, TRAV comprises RGEDVEQSLFLSVREGDSSVINCTYTDSSSSTYLYWYKQEPGAGLQLLTYIFSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGGTRLQVFPN (SEQ ID NO: 107). In some embodiments, in the polypeptide molecule, TRAV comprises RGEDVEQSLFLSVREGDSSVINCTYTDSSSSTYLYWYKQEPGAGLQLLTYTWPHMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAGSGGGTDKLIFGGTRLQVFPN (SEQ ID NO: 108).
在一些实施方案中，在所述多肽分子中，TRBV包含GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSDYQTCVTYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPFEQYFGPGTRLTVTE(SEQ ID NO:109)。在一些实施方案中，在所述多肽分子中，TRBV包含GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPYEQYFGPGTRLTVTE(SEQ ID NO:110)。In some embodiments, in the polypeptide molecule, TRBV comprises GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSDYQTCVTYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPFEQYFPGGTRLTVTE (SEQ ID NO: 109). In some embodiments, in the polypeptide molecule, TRBV comprises GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAREPGQGPYEQYFPGGTRLTVTE (SEQ ID NO: 110).
在一些实施方案中，所述第一抗原为HPV抗原，例如HPV E7。In some embodiments, the first antigen is an HPV antigen, such as HPV E7.
在一些实施方式中，在一些实施方式中，在所述多肽分子中，TRAV包含NSASQS(SEQ ID NO:111)所示的CDR1，VYSSGN(SEQ ID NO:112)所示的CDR2，和AVISAGTALI(SEQ ID NO:113)所示的CDR3；TRBV包含SGHDT(SEQ ID NO:114)所示的CDR1，YYEEEE(SEQ ID NO:115)所示的CDR2，和ASSLGWRGGLYTEAF(SEQ ID NO:116)所示的CDR3。In some embodiments, in the polypeptide molecule, TRAV includes CDR1 represented by NSASQS (SEQ ID NO:111), CDR2 represented by VYSSGN (SEQ ID NO:112), and AVISAGTALI ( CDR3 shown in SEQ ID NO:113); TRBV contains CDR1 shown in SGHDT (SEQ ID NO:114), CDR2 shown in YYEEEE (SEQ ID NO:115), and ASSLGWRGGLYTEAF (SEQ ID NO:116) CDR3.
在一些实施方式中，TRAV包含RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDG RFTAQLNRASQYISLLIRDSKLSDSATYLCAVISAGTALIFGKGTTLSVSS(SEQ ID NO:117)。In some embodiments, TRAV comprises RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDG RFTAQLNRASQYISLLIRDSKLSDSATYLCAVISAGTALIFGKGTTLSVSS (SEQ ID NO: 117).
在一些实施方式中，TRBV包含DAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGWRGGLYTEAFFGQGTRLTVV(SEQ ID NO:118)。In some embodiments, TRBV comprises DAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGWRGGLYTEAFFGQGTRLTVV (SEQ ID NO: 118).
在另一方面，本发明提供了T细胞受体(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:89、SEQ ID NO:90和SEQ ID NO:91所示的氨基酸序列的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:92、SEQ ID NO:93和SEQ ID NO:94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。In another aspect, the invention provides a T cell receptor (TCR) comprising a TCR alpha chain variable region (TRAV) and a TCR beta chain variable region (TRBV), wherein the TRAV comprises a TCR alpha chain variable region (TRAV), respectively, as shown in SEQ ID NO: 89 , CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO:90 and SEQ ID NO:91, or functional variants formed by inserting, deleting or replacing one or several amino acids; and/or the TRBV contains respectively β-chain CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, or functional variants formed by inserting, deleting or replacing one or several amino acids .
在一些实施方案中，所述TRAV包含分别具有如SEQ ID NO:89、SEQ ID NO:90和SEQ ID NO:91所示的氨基酸序列的CDR1、CDR2和CDR3；和/或所述TRBV包含分别具有如SEQ ID NO:92、SEQ ID NO:93和SEQ ID NO:94所示的氨基酸序列的β链CDR1、CDR2和CDR3。In some embodiments, the TRAV comprises CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91 respectively; and/or the TRBV comprises respectively Beta chain CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94.
在一些实施方案中，所述TRAV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，和/或所述TRBV包含与SEQ ID NO:98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。In some embodiments, 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: 95, and/or the TRBV comprises SEQ ID NO:98 An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity.
在一些实施方案中，所述TRAV包含与SEQ ID NO:96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:98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。In some embodiments, 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: 96, and/or the TRBV comprises SEQ ID NO:98 An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity.
在一些实施方案中，所述TRAV包含与SEQ ID NO:97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:98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。In some embodiments, 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: 97, and/or the TRBV comprises SEQ ID NO:98 An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity.
在另一方面，本发明提供了核酸，其包含编码本发明所述的双特异性多肽分子的每一条链的核苷酸序列。本发明还提供了核酸，其包含编码本发明所述的TCR的核苷酸序列。In another aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding each strand of a bispecific polypeptide molecule of the invention. The invention also provides nucleic acids comprising a nucleotide sequence encoding the TCR of the invention.
在又一方面，本发明提供了载体，其包含本发明所述的核酸。In yet another aspect, the invention provides a vector comprising a nucleic acid according to the invention.
任何合适的载体均可用于本发明。载体的实例包括但不限于质粒、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。Any suitable carrier may be used in the present invention. Examples of vectors include, but are not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyomavirus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, Cosmids and artificial chromosomes (including BAC and YAC). The vector itself is usually a nucleotide sequence, usually a DNA sequence containing the insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Engineered vectors typically contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may additionally contain a promoter, genetic marker, reporter gene, targeting sequence, and/or protein purification tag. As is known to those skilled in the art, a large number of suitable vectors are known to those skilled in the art, and many are commercially available. Examples of 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.
在一些实施方案中，载体优选选自慢病毒载体、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。In some embodiments, the vector is preferably selected from the group consisting of lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes.
在又一方面，本发明提供了载体系统，其在一个或多个载体上包含编码本发明所述 的双特异性多肽分子的每一条链的核苷酸序列。In yet another aspect, the invention provides a vector system comprising on one or more vectors encoding the invention. The nucleotide sequence of each chain of the bispecific polypeptide molecule.
在另一方面，本发明提供了宿主细胞，其包含本发明所述的核酸、载体或载体系统。In another aspect, the invention provides a host cell comprising a nucleic acid, vector or vector system of the invention.
任何细胞都可以用作本发明的核酸或载体的宿主细胞。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。为了产生本公开的多肽分子的目的，细胞优选为哺乳动物细胞。最优选地，宿主细胞为人细胞。Any cell can be used as a host cell for the nucleic acids or vectors of the invention. The cells may be eukaryotic cells, such as plants (without the potential to develop into plants), animals, fungi or algae, or may be prokaryotic cells, such as bacteria or protozoa. The cells may be cultured cells or primary cells, ie, isolated directly from an organism, such as a human. Cells can be adherent cells or suspension cells, that is, cells grown in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of producing the polypeptide molecules of the present disclosure, the cells are preferably mammalian cells. Most preferably, the host cells are human cells.
在一些实施方案中，所述细胞选自淋巴细胞(例如T细胞、NK细胞)、单核细胞(例如PBMC)和干细胞。例如，干细胞可以是淋巴祖细胞、诱导多能干细胞(iPSC)或造血干细胞(HSC)。在一些实施方案中，干细胞不包括通过破坏人类胚胎获得的胚胎干细胞，和/或不包括用于发育并形成动物个体的全能性干细胞。In some embodiments, the cells are selected from lymphocytes (eg, T cells, NK cells), monocytes (eg, PBMCs), and stem cells. For example, the stem cells may be lymphoid progenitor cells, induced pluripotent stem cells (iPSCs), or hematopoietic stem cells (HSCs). In some embodiments, stem cells do not include embryonic stem cells obtained by destroying human embryos, and/or do not include totipotent stem cells used to develop and form an animal individual.
在另一方面，本发明提供了缀合物，其包含本发明所述的双特异性多肽分子，以及与所述双特异性多肽分子缀合的化学部分。本发明还提供了缀合物，其包含本发明所述的TCR，以及与所述TCR缀合的化学部分。In another aspect, the invention provides conjugates comprising a bispecific polypeptide molecule of the invention, and a chemical moiety conjugated to the bispecific polypeptide molecule. The invention also provides conjugates comprising the TCR of the invention, and a chemical moiety conjugated to the TCR.
在一些实施方案中，所述化学部分选自治疗剂、免疫刺激分子和可检测标记。In some embodiments, the chemical moiety is selected from the group consisting of therapeutic agents, immunostimulatory molecules, and detectable labels.
在一些实施方案中，治疗剂包括但不限于免疫调节剂、放射性化合物、酶(例如穿孔素)、化学治疗剂(例如顺铂)或毒素。在一些实施方案中，治疗剂可以是例如美登素、格尔德霉素、微管蛋白抑制剂例如微管蛋白结合剂(例如奥瑞他汀类)或小沟结合剂例如加利车霉素(calicheamicin)。In some embodiments, therapeutic agents include, but are not limited to, immunomodulators, radioactive compounds, enzymes (eg, perforin), chemotherapeutic agents (eg, cisplatin), or toxins. In some embodiments, the therapeutic agent may be, for example, maytansine, geldanamycin, a tubulin inhibitor such as a tubulin binding agent (such as auristatins) or a minor groove binding agent such as calicheamicin (calicheamicin).
其他合适的治疗剂包括例如小分子细胞毒剂，即具有杀死哺乳动物细胞能力的分子量小于700道尔顿的化合物。这样的化合物还可包含能够具有细胞毒性作用的有毒金属。此外，应当理解，这些小分子细胞毒剂还包括药物前体，即在生理条件下分解或转化以释放细胞毒剂的化合物。此类药剂的实例包括顺铂、美登素衍生物、雷切霉素、加利车霉素、多西他赛、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、替莫唑胺、托泊替康、三甲双胍、奥瑞他汀E、长春生物碱和多柔比星；肽细胞毒素，即具有杀死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、DNA酶和RNA酶；放射性核素，即随着α或β粒子或γ射线的一种或多种的同时发射而衰变的元素的不稳定同位素，例如碘-131、铼-186、铟-111、钇-90、铋-210、铋-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。Other suitable therapeutic agents include, for example, small molecule cytotoxic agents, ie, compounds with a molecular weight of less than 700 daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that break down or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, racithromycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, Mitoxantrone, sorfimer porphyrin sodium II, temozolomide, topotecan, metformin, auristatin E, vinca alkaloids, and doxorubicin; peptide cytotoxins, i.e., proteins with the ability to kill mammalian cells or Fragments thereof, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides, i.e., with the simultaneous emission of one or more alpha or beta particles or gamma rays Unstable isotopes of decaying elements, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225, and astatine-213; chelating agents, which can be used to promote these radioactivities The binding of a nuclide to a molecule or its polymer.
在一些实施方案中，免疫刺激分子是激发免疫应答的免疫效应分子。例如，免疫刺激分子可以是细胞因子如IL-2和IFN-γ、趋化因子如IL-8、血小板因子4、黑色素瘤生长刺激蛋白、补体激活剂；病毒/细菌蛋白结构域，或病毒/细菌肽。在优选的实施方案中，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。In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule may be a cytokine such as IL-2 and IFN-γ, a chemokine such as IL-8, platelet factor 4, melanoma growth stimulating protein, complement activator; viral/bacterial protein domain, or viral/ Bacterial peptides. In a preferred embodiment, the immunostimulatory molecule is selected from the group consisting of cytokines, chemokines, platelet factors and complement initiators.
在一些实施方案中，可检测标记可以选自生物素、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、顺磁性金属离子或荧光、磷光，或化学发光分子。用于诊断目的的可检测部分包括例如荧光标记、放射性标记、酶、核酸探针和造影剂。In some embodiments, the detectable label can be selected from biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, or fluorescent, phosphorescent, or chemiluminescent molecules. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.
在另一方面，本发明提供了药物组合物，其包含本发明所述的双特异性多肽分子、核酸、载体、载体系统、宿主细胞、或缀合物，以及药学上可接受的赋形剂。In another aspect, the invention provides a pharmaceutical composition comprising the bispecific polypeptide molecule, nucleic acid, vector, vector system, host cell, or conjugate of the invention, and a pharmaceutically acceptable excipient. .
本发明的药物组合物特别适合施用于人；然而，其也适合施用于非人动物。所述组 合物及其组分(即活性剂和任选的赋形剂)优选为药物上可接受的，即在接受者中能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用。本发明的药物组合物可以是例如无菌的。The pharmaceutical compositions of the present invention are particularly suitable for administration to humans; however, they are also suitable for administration to non-human animals. the group The compounds and their components (i.e., active agents and optional excipients) are preferably pharmaceutically acceptable, i.e., capable of eliciting the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects. . Pharmaceutical compositions of the invention may, for example, be sterile.
赋形剂的实例包括但不限于填充剂、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药物组合物。用于本发明的药物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。Examples of excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, preservatives, antioxidants, flavoring agents, colorants, sweetening agents agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tonicity regulators. It is known to those skilled in the art to select suitable excipients for the preparation of pharmaceutical compositions of the invention. Exemplary carriers for use in pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the selection of a suitable excipient depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the composition.
根据所采用的活性剂，可将本发明的药物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。Depending on the active agent used, the pharmaceutical composition of the present invention can be prepared into various forms, such as solid, liquid, gaseous or freeze-dried forms, especially ointments, creams, transdermal patches, gels, in the form of a powder, tablet, solution, aerosol, granule, pill, suspension, emulsion, capsule, syrup, liquid, elixir, extract, tincture or liquid extract, or Particularly suitable for the form of application required. Processes for producing pharmaceuticals known in this invention are shown in Remington's Pharmaceutical Sciences, 22nd Edition (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar-coating, Grinding, emulsifying, encapsulating, embedding or freeze-drying processes.
在一些实施方案中，所述药物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。In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent, preferably the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs.
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。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 (sorfimer sodium photofrin II), temozolomide, topotecan, trimetreate glucuronate (trimetreate glucuronate), olefin auristatin E, vincristine, and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, Platelet factor 4; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; viral/bacterial protein domains and viral/bacterial peptides.
在又一方面，本发明提供了预防或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发明所述的双特异性多肽分子、TCR、核酸、载体、载体系统、宿主细胞、缀合物、或药物组合物，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。In yet another aspect, the invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the bispecific polypeptide molecule, TCR, nucleic acid, vector, vector of the invention A system, host cell, conjugate, or pharmaceutical composition, wherein the disease is selected from the group consisting of cancer, infectious diseases, autoimmune diseases, and inflammatory diseases.
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。在一些实施方案中，癌症为KRAS表达相关的癌症。在一些实施方案中，癌症为MAGE-A1表达相关的癌症。The cancer may be any cancer, such as cancer of the blood system, cancer of the central and peripheral nervous system, cancer of the lymphatic lineage, cancer of the myeloid lineage, cancer of mesenchymal origin, solid tumors, etc. Examples of cancers include, but are not limited to, acute lymphoblastic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct Cancer, joint cancer, neck cancer, gallbladder or pleural cancer, nose cancer, nasal cavity or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer Carcinoma, 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, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small bowel cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer and bladder cancer. In some embodiments, the cancer is a cancer associated with KRAS expression. In some embodiments, the cancer is a MAGE-A1 expression-associated cancer.
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。Infectious diseases are diseases caused by pathogenic infections, including communicable diseases and non-communicable diseases. cause The pathogens of infectious diseases include viruses, bacteria, mycoplasma, chlamydia, rickettsiae, prions, fungi, spirochetes and parasites. Examples of viruses include, but are not limited to, HPV, CMV, HBV, EBV, herpes viruses, human immunodeficiency virus (HIV), influenza viruses, and coronaviruses.
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。Common autoimmune and inflammatory diseases include but are not limited to rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, diabetes caused by autoimmune destruction of pancreatic islets, Sjogren's syndrome, Hashimoto's thyroid inflammatory bowel disease, Graves' thyroiditis, toxic diffuse goiter, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease (such as 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, autoimmunity of the central nervous system Diseases, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis, pulmonary hemorrhage-nephritis syndrome, autoimmune oophoritis, autoimmune orchitis, idiopathic leukopenia, primary adrenocortical atrophy, antiphospholipid antibody syndrome, post-infectious fibrosis, post-myocardial infarction fibrosis, liver fibrosis, and scar hyperplasia.
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。In some embodiments, the dosage administered to a subject may vary depending on the embodiment, the agent used, the method of administration, and the site and subject being treated. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine an effective amount for administration to a human or other subject to treat a medical condition. The precise amount required for the treatment to be effective may depend on many factors, such as the activity of the active agent and the route of administration.
本发明的多肽分子、缀合物或药物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、缀合物或药物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。The dose of the polypeptide molecule, conjugate or pharmaceutical composition of the invention can be administered to the mammal once or in a series of sub-doses over a suitable period of time, for example, daily, every half week, every week, every day as needed. Apply biweekly, semimonthly, bimonthly, semiannually, or annually. Dosage units containing an effective amount of a polypeptide molecule, conjugate or pharmaceutical composition may be administered in a single daily dose, or the total daily dose may be administered in two, three, four or more doses per day as desired. Give in divided doses.
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。The appropriate method of administration can be chosen by the physician. The route of administration may be parenteral, for example by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration can be by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, the polypeptide molecule, conjugate, or pharmaceutical composition is selected for parenteral delivery, inhalation, or delivery through the gastrointestinal tract, such as orally. The dosage and method of administration may vary according to the subject's weight, age, condition, etc., and may be appropriately selected.
在一些实施方案中，所述方法还包括向所述受试者施用第二治疗剂。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、缀合物或药物组合物。优选地，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。In some embodiments, the method further includes administering a second therapeutic agent to the subject. In certain embodiments, a polypeptide molecule, conjugate, or pharmaceutical composition of the invention is administered before, substantially simultaneously with, or after the second therapeutic agent. Preferably, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs. Preferred examples of second therapeutic agents are as described above.
在另一方面，本公开提供了检测(例如诊断)受试者中的疾病的方法，其中所述方法包括(i)使从受试者获得的样品与本发明的多肽分子、TCR或缀合物接触；和(ii)检测所述样品中靶抗原的存在，其中所述靶抗原的存在指示受试者患有相应疾病。In another aspect, the present disclosure provides a method of detecting (e.g., diagnosing) a disease in a subject, wherein the method comprises (i) conjugating a sample obtained from the subject with a polypeptide molecule, TCR or conjugate of the invention contact; and (ii) detecting the presence of a target antigen in the sample, wherein the presence of the target antigen indicates that the subject suffers from the corresponding disease.
靶抗原可以选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原。疾病可以选自癌症、感染性疾病、自身免疫病和炎性疾病。在一些实施方案中，靶抗原为KRAS。在一些实施方式中，所述抗原为MGAE-A1。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。Target antigens can be selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens. The disease may be selected from cancer, infectious diseases, autoimmune diseases and inflammatory diseases. In some embodiments, the target antigen is KRAS. In some embodiments, the antigen is MGAE-A1. The cancers, infectious diseases, autoimmune diseases and inflammatory diseases are as defined above.
在一些实施方案中，从受试者获得的样品可以是血液样品、尿液样品、组织样品或细胞样品。在某些实施方案中，所述方法在体外进行。在一些实施方案中，所述方法包括(i)使从受试者获得的样品与本发明的缀合物接触，其中该缀合物包含可检测标记；和(ii)通过检测可检测标记来检测所述样品中靶抗原的存在。可检测标记的实例包括但不限于生物素、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；优选酶或其催化活性片段、放射性核素、萤光、磷光或化学发光分子。In some embodiments, the sample obtained from the subject may be a blood sample, urine sample, tissue sample, or cell sample. In certain embodiments, the methods are performed in vitro. In some embodiments, the method includes (i) contacting a sample obtained from a subject with a conjugate of the invention, wherein the conjugate comprises a detectable label; and (ii) by detecting the detectable label. The sample is detected for the presence of the target antigen. Examples of detectable labels include, but are not limited to, biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, Nucleic acid probes, contrast agents, and fluorescent, phosphorescent or chemiluminescent molecules; preferably enzymes or catalytically active fragments thereof, radionuclides, fluorescent, phosphorescent or chemiluminescent molecules.
在又一方面，本公开提供了试剂盒，其包含本公开的多肽分子、TCR或缀合物，所述试剂盒用于检测待测样品中靶抗原的存在。In yet another aspect, the disclosure provides a kit comprising a polypeptide molecule, TCR or conjugate of the disclosure for detecting the presence of a target antigen in a sample to be tested.
在一些实施方式中，所述试剂盒为用于检测(例如诊断)受试者中的疾病的试剂盒，其包含本公开的多肽分子或缀合物。靶抗原可以选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原。疾病可以选自癌症、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。In some embodiments, the kit is a kit for detecting (eg, diagnosing) a disease in a subject, comprising a polypeptide molecule or conjugate of the disclosure. Target antigens can be selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens. The disease may be selected from cancer, infectious diseases, autoimmune diseases and inflammatory diseases. The cancers, infectious diseases, autoimmune diseases and inflammatory diseases are as defined above.
在一些实施方案中，所述缀合物包含可检测标记。可检测标记的实例包括但不限于生物素、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；优选酶或其催化活性片段、放射性核素、萤光、磷光或化学发光分子。在一些实施方案中，试剂盒还可以包含如何使用该试剂盒的说明书。In some embodiments, the conjugate includes a detectable label. Examples of detectable labels include, but are not limited to, biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent Or chemiluminescent molecules; preferably enzymes or catalytically active fragments thereof, radionuclides, fluorescent, phosphorescent or chemiluminescent molecules. In some embodiments, the kit may also include instructions on how to use the kit.
在另一方面，本公开提供了本发明双特异性多肽分子、TCR、核酸、载体、载体系统、宿主细胞、缀合物或药物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。In another aspect, the present disclosure provides bispecific polypeptide molecules, TCRs, nucleic acids, vectors, vector systems, host cells, conjugates or pharmaceutical compositions of the invention for use in the treatment or prevention of disease in a subject. Uses in medicines.
在又一方面，本公开提供了本发明的双特异性多肽分子、TCR、核酸、载体、载体系统、宿主细胞、缀合物或药物组合物，其用于治疗或预防受试者中的疾病。In yet another aspect, the disclosure provides a bispecific polypeptide molecule, TCR, nucleic acid, vector, vector system, host cell, conjugate or pharmaceutical composition of the invention for use in treating or preventing a disease in a subject .
在另一方面，本公开提供了本发明的双特异性多肽分子、TCR或缀合物在制备用于检测待测样品中靶抗原的存在的试剂盒中的用途。In another aspect, the disclosure provides the use of a bispecific polypeptide molecule, TCR or conjugate of the invention in the preparation of a kit for detecting the presence of a target antigen in a sample to be tested.
在一些实施方式中，本公开提供了本发明的双特异性多肽分子、TCR或缀合物在制备用于检测(例如诊断)受试者中的疾病的试剂盒中的用途。In some embodiments, the present disclosure provides the use of a bispecific polypeptide molecule, TCR, or conjugate of the invention in the preparation of a kit for detecting (eg, diagnosing) a disease in a subject.
在又一方面，本公开提供了本发明的双特异性多肽分子、TCR或缀合物，其用于检测待测样品中靶抗原的存在。In yet another aspect, the disclosure provides a bispecific polypeptide molecule, TCR or conjugate of the invention for use in detecting the presence of a target antigen in a sample to be tested.
在本公开的用途的一些实施方案中，靶抗原可以选自肿瘤相关抗原(TAA)、肿瘤特异性抗原(TSA)、病毒抗原和自身抗原。疾病可以选自癌症、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。In some embodiments of the uses of the present disclosure, the target antigen may be selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens, and autoantigens. The disease may be selected from cancer, infectious diseases, autoimmune diseases and inflammatory diseases. The cancers, infectious diseases, autoimmune diseases and inflammatory diseases are as defined above.
附图说明Description of the drawings
图1显示了本发明的format 22-2的一种具体形式的结构示意图、AlphaFold2图、免疫印迹结果图以及ELISPOT测定结果图。Figure 1 shows the structural schematic diagram, AlphaFold2 diagram, immunoblotting result diagram and ELISPOT assay result diagram of a specific form of format 22-2 of the present invention.
图2显示了本发明的format 64的一种具体形式的结构示意图、免疫印迹结果图以及ELISPOT测定结果图。Figure 2 shows a schematic structural diagram, an immunoblotting result diagram and an ELISPOT assay result diagram of a specific form of format 64 of the present invention.
图3显示了本发明的format 63的一种具体形式的结构示意图。Figure 3 shows a schematic structural diagram of a specific form of format 63 of the present invention.
图4显示了对照format 17的一种具体形式的结构示意图、蛋白染色和免疫印迹结果图、以及ELISPOT测定结果图。Figure 4 shows a schematic structural diagram of a specific form of control format 17, protein staining and immunoblotting results, and ELISPOT assay results.
图5显示了对照format 22的一种具体形式的结构示意图、蛋白染色和免疫印迹结果图、以及ELISPOT测定结果图。Figure 5 shows a schematic structural diagram of a specific form of control format 22, protein staining and immunoblotting results, and ELISPOT assay results.
图6显示了以KRAS作为靶肽时本发明的format 22-2的ELISPOT测定结果图。Figure 6 shows the ELISPOT measurement results of format 22-2 of the present invention when KRAS is used as the target peptide.
具体实施方式Detailed ways
通过下面的具体实施例进一步阐述本发明。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。The present invention is further illustrated by the following specific examples. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples were carried out in accordance with the standards in this field. Conventional conditions, for example, those described in Sambrook and Russeii et al., Molecular Cloning: Laboratory Manual (3rd Edition) (2001), CSHL Publishing, or as recommended by the manufacturer. Unless otherwise stated, experimental materials and reagents used in the following examples are commercially available.
实施例1基于TCR的多特异性多肽分子的构建Example 1 Construction of TCR-based multispecific polypeptide molecules
1.1靶向gp100的多特异性多肽分子的构建1.1 Construction of multi-specific peptide molecules targeting gp100
设计并构建了多种基于TCR的双特异性多肽分子format 22-2(如SEQ ID NO:1所示的第一链和如SEQ ID NO:2所示的第二链)、format 64(如SEQ ID NO:3所示的第一链和如SEQ ID NO:4所示的第二链)和format 63(如SEQ ID NO:5所示的第一链和如SEQ ID NO:6所示的第二链)，这些format的结构如图1-3所示。还通过AlphaFold2显示了这些format的三维结构图。Designed and constructed a variety of TCR-based bispecific peptide molecules format 22-2 (such as the first chain shown in SEQ ID NO: 1 and the second chain shown in SEQ ID NO: 2), format 64 (such as The first strand as shown in SEQ ID NO:3 and the second strand as shown in SEQ ID NO:4) and format 63 (the first strand as shown in SEQ ID NO:5 and the first strand as shown in SEQ ID NO:6 The second chain), the structures of these formats are shown in Figure 1-3. The three-dimensional structure diagrams of these formats are also displayed through AlphaFold2.
另外，为了验证本发明的format中使用的TRAC和TRBC相对于使用其他恒定区(例如抗体的CH3或铰链区-CH2-CH3)的优势，还构建了format 17和22作为对照。本发明的format 22-2的TRAC和TRBC替换为铰链区-CH2-CH3即为Format 17；本发明的format 22-2的TRAC和TRBC替换为CH3即为Format 22。Format 17的结构为：第一多肽链从N端到C端包含：TRAV-接头-VH-任选的接头-铰链区-CH2-CH3，并且第二多肽链从N端到C端包含：VL-接头-TRBV-任选的接头-铰链区-CH2-CH3。Format 22的结构为：第一多肽链从N端到C端包含：TRAV-接头-VH-任选的接头-CH3，并且第二多肽链从N端到C端包含：VL-接头-TRBV-任选的接头-CH3。Format 17和Format 22的结构示意图分别如图4和图5所示。In addition, in order to verify the advantages of TRAC and TRBC used in the format of the present invention over the use of other constant regions (such as CH3 or hinge region-CH2-CH3 of antibodies), formats 17 and 22 were also constructed as controls. The TRAC and TRBC of the format 22-2 of the present invention are replaced with the hinge region-CH2-CH3, which is Format 17; the TRAC and TRBC of the format 22-2 of the present invention are replaced with CH3, which is Format 22. The structure of Format 17 is: the first polypeptide chain contains from N-terminus to C-terminus: TRAV-linker-VH-optional linker-hinge region-CH2-CH3, and the second polypeptide chain contains from N-terminus to C-terminus :VL-linker-TRBV-optional linker-hinge region-CH2-CH3. The structure of Format 22 is: the first polypeptide chain from N-terminus to C-terminus contains: TRAV-linker-VH-optional linker-CH3, and the second polypeptide chain from N-terminus to C-terminus contains: VL-linker- TRBV-optional linker-CH3. The structural diagrams of Format 17 and Format 22 are shown in Figure 4 and Figure 5 respectively.
这些format中所用的氨基酸序列组件如下所示：The amino acid sequence components used in these formats are as follows:
TRAVTRAV
>gp100特异性TCR的TRAV
>TRAV of gp100-specific TCR
>gp100特异性TCR的TRAV，包含A104C突变
>TRAV of gp100-specific TCR, containing A104C mutation
TRACTRAC
>TRAC包含T48C、N113K、FFPSPESS缺失突变
>TRAC contains T48C, N113K, FFPSPESS deletion mutations
>TRAC包含T48C、N113K、PESS缺失突变
>TRAC contains T48C, N113K, and PESS deletion mutations
>TRAC包含T48C、N113K突变
>TRAC contains T48C, N113K mutations
>TRAC
>TRAC
TRBVTRBV
>gp100特异性TCR的TRBV
>gp100-specific TCR TRBV
>gp100特异性TCR的TRBV，包含N84C突变
>TRBV of gp100-specific TCR, containing N84C mutation
TRBCTRBC
>TRBC2,包含S57C、C187A、N210D突变
>TRBC2, contains S57C, C187A, N210D mutations
>TRBC2,包含S57C、C187A、N210D、FG环缺失突变
>TRBC2, contains S57C, C187A, N210D, and FG loop deletion mutations
>TRBC1,包含S57C、C187A、N210D突变
>TRBC1, contains S57C, C187A, N210D mutations
>TRBC1,包含S57C、C187A、N210D、FG环缺失突变
>TRBC1, contains S57C, C187A, N210D, and FG loop deletion mutations
>TRBC
>TRBC
VHVH
>VH1(抗CD3抗体huUCHT1,包含G44C突变)
>VH1 (anti-CD3 antibody huUCHT1, contains G44C mutation)
>VH1(抗CD3抗体huUCHT1，包含G44C、E89C突变)
>VH1 (anti-CD3 antibody huUCHT1, contains G44C, E89C mutations)
>VH2(抗CD28抗体，包含C50Y突变)
>VH2 (anti-CD28 antibody, contains C50Y mutation)
>VH2(抗CD28抗体，包含C50Y、D89C突变)
>VH2 (anti-CD28 antibody, containing C50Y, D89C mutations)
>VH-BB1(抗4-1BB抗体，单价)
>VH-BB1 (anti-4-1BB antibody, monovalent)
>VH-BB2(抗4-1BB抗体，双价)
>VH-BB2 (anti-4-1BB antibody, bivalent)
VLVL
>VL1(抗CD3抗体huUCHT1，包含E100C突变)
>VL1 (anti-CD3 antibody huUCHT1, contains E100C mutation)
>VL1(抗CD3抗体huUCHT1，包含E80C、E100C突变)
>VL1 (anti-CD3 antibody huUCHT1, contains E80C, E100C mutations)
>VL2(抗CD28抗体)
>VL2 (anti-CD28 antibody)
>VL2(抗CD28抗体，包含E80C突变)
>VL2 (anti-CD28 antibody, contains E80C mutation)
>VL-BB1(抗4-1BB抗体，单价)
>VL-BB1 (anti-4-1BB antibody, monovalent)
>VL-BB2(抗4-1BB抗体，双价)
>VL-BB2 (anti-4-1BB antibody, bivalent)
铰链-CH2-CH3Hinge-CH2-CH3
>含有二硫键的铰链-CH2-CH3 Hole(包含T366S、L368A和Y407V臼突变以及Y349C突变)
>Hinge-CH2-CH3 Hole containing disulfide bonds (contains T366S, L368A and Y407V acetal mutations and Y349C mutation)
>含有二硫键的铰链-CH2-CH3 Knob(包含T366W杵突变和S354C突变)
>Hinge-CH2-CH3 Knob containing disulfide bonds (contains T366W pestle mutation and S354C mutation)
>不含二硫键的铰链-CH2-CH3 Hole(包含T366S、L368A和Y407V臼突变以及Y349C突变)
>Hinge-CH2-CH3 Hole without disulfide bonds (contains T366S, L368A and Y407V acetabulum mutations and Y349C mutation)
>不含二硫键的铰链-CH2-CH3 Knob(包含T366W杵突变和S354C突变)
>Hinge-CH2-CH3 Knob without disulfide bonds (contains T366W pestle mutation and S354C mutation)
CH2-CH3CH2-CH3
CH2-CH3 Hole(包含T366S、L368A和Y407V臼突变以及Y349C突变)
CH2-CH3 Hole (contains T366S, L368A and Y407V hole mutations and Y349C mutation)
CH2-CH3 Knob(包含T366W杵突变和S354C突变)
CH2-CH3 Knob (contains T366W pestle mutation and S354C mutation)
CH3CH3
>CH3 Hole(包含T366S、L368A和Y407V臼突变以及Y349C突变)
>CH3 Hole (contains T366S, L368A and Y407V hole mutations and Y349C mutation)
>CH3 Knob(包含T366W杵突变和S354C突变)
>CH3 Knob (contains T366W mutation and S354C mutation)
Alb
Alb
接头Connector
S(SEQ ID NO:41)S(SEQ ID NO:41)
GGGGS(SEQ ID NO:42)GGGGS(SEQ ID NO:42)
GGGGSGGGGSGGGGSGGGGSGGGS(SEQ ID NO:43)GGGGSGGGGSGGGGSGGGGSGGGS(SEQ ID NO:43)
GGGGSGGGGSGGGGS(SEQ ID NO:44)GGGGSGGGGSGGGGS(SEQ ID NO:44)
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:45)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:45)
GGGGSGGGGS(SEQ ID NO:46)GGGGSGGGGS(SEQ ID NO:46)
GGSGGS(SEQ ID NO:47)GGSGGS(SEQ ID NO:47)
GGSGGSGGS(SEQ ID NO:48)GGSGGSGGS(SEQ ID NO:48)
GQPKAAP(SEQ ID NO:49)GQPKAAP(SEQ ID NO:49)
GGGSGGGG(SEQ ID NO:50)GGGSGGGG(SEQ ID NO:50)
RTSGPGDGGKGGPGKGPGGEGTKGTGPGG(SEQ ID NO:51)RTSGPGDGGKGGPGKGPGGEGTKGTGPGG(SEQ ID NO:51)
GGEGGGSEGGGS(SEQ ID NO:52)GGEGGGSEGGGS(SEQ ID NO:52)
TVLRT(SEQ ID NO:53) TVLRT(SEQ ID NO:53)
TVSSAS(SEQ ID NO:54)TVSSAS(SEQ ID NO:54)
GGEGG(SEQ ID NO:55)GGEGG(SEQ ID NO:55)
GSEGGGS(SEQ ID NO:56)GSEGGGS(SEQ ID NO:56)
RTSGPGDGGKGGPGKGPGGEGTKGTGPGG(SEQ ID NO:57)RTSGPGDGGKGGPGKGPGGEGTKGTGPGG(SEQ ID NO:57)
GKGPGGEGTKGTGPGG(SEQ ID NO:58)GKGPGGEGTKGTGPGG(SEQ ID NO:58)
TVLSSAS(SEQ ID NO:59)TVLSSAS(SEQ ID NO:59)
EDL(SEQ ID NO:60)EDL(SEQ ID NO:60)
ANI(SEQ ID NO:61)ANI(SEQ ID NO:61)
PNI(SEQ ID NO:88)PNI(SEQ ID NO:88)
对照format 17的两条多肽链的示例性氨基酸序列如下所示：Exemplary amino acid sequences of the two polypeptide chains of control format 17 are as follows:
第一链
first chain
第二链
second chain
对照format 22的两条多肽链的示例性氨基酸序列如下所示：Exemplary amino acid sequences of the two polypeptide chains of control format 22 are as follows:
第一链
first chain
第二链
second chain
在这些format的每一条多肽链的N端添加信号肽序列MGWSCIILFLVATATGVHS(SEQ ID NO:66)以增加多肽分子的分泌，并在format的任一条链的C端添加His6标签以用于纯化。本实施例中的format能够结合gp100抗原表位YLEPGPVTA(SEQ ID NO:67)或其突变形式(YLEPGPVTV,SEQ ID NO:68))与HLA-A*02的复合物。The signal peptide sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 66) is added to the N-terminus of each polypeptide chain of these formats to increase the secretion of the polypeptide molecules, and a His6 tag is added to the C-terminus of any chain of the format for purification. The format in this example can bind to the complex of gp100 epitope YLEPGPVTA (SEQ ID NO: 67) or its mutant form (YLEPGPVTV, SEQ ID NO: 68)) and HLA-A*02.
1.2靶向KRAS的多特异性多肽分子的构建1.2 Construction of multi-specific peptide molecules targeting KRAS
如实施例1.1所述构建靶向KRAS的多特异性多肽分子format。The multispecific polypeptide molecule format targeting KRAS was constructed as described in Example 1.1.
作为示例，根据这些序列构建了靶向KRAS的foramt 22-2分子KVA11-N03_B22-2_WT(其两条链的氨基酸序列分别如SEQ ID NO:99和100所示)、KVA11-N03_B22-2_Mut1(其两条链的氨基酸序列分别如SEQ ID NO:101和102所示)、KVA11-N03_B22-2_Mut2(其两条链的氨基酸序列分别如SEQ ID NO:103和104所示)、KVA11-N03_B22-2_Mut3(其两条链的氨基酸序列分别如SEQ ID NO:105和106所示)。As examples, the KRAS-targeting foramt 22-2 molecules KVA11-N03_B22-2_WT (the amino acid sequences of its two chains are shown in SEQ ID NO:99 and 100 respectively), KVA11-N03_B22-2_Mut1 (these The amino acid sequences of the two chains are shown in SEQ ID NO:101 and 102 respectively), KVA11-N03_B22-2_Mut2 (the amino acid sequences of the two chains are shown in SEQ ID NO:103 and 104 respectively), KVA11-N03_B22-2_Mut3 (The amino acid sequences of its two chains are shown in SEQ ID NO: 105 and 106 respectively).
本实施例中的format能够结合KRAS抗原表位VVGAVGVGK(SEQ ID NO:119)与HLA-A*11的复合物。The format in this example can bind to the complex of KRAS epitope VVGAVGVGK (SEQ ID NO: 119) and HLA-A*11.
1.3靶向MAGEA1的多特异性多肽分子的构建1.3 Construction of multispecific peptide molecules targeting MAGEA1
如实施例1.1所述构建靶向MAGEA1的多特异性多肽分子format，其format的结构与实施例1.1相同，区别仅在于用MAGEA1特异性TCR的TRAV和TRBV替换实施例1.1中的gp100特异性TCR的TRAV和TRBV。The multispecific polypeptide molecule format targeting MAGEA1 was constructed as described in Example 1.1. The structure of the format is the same as that in Example 1.1. The only difference is that the gp100-specific TCR in Example 1.1 was replaced with TRAV and TRBV of MAGEA1-specific TCR. TRAV and TRBV.
MAGEA1特异性TCR的TRAV和TRBV的氨基酸序列如下所示：The amino acid sequences of TRAV and TRBV of the MAGEA1-specific TCR are as follows:
TRAV
TRAV
TRBV
TRBV
本实施例中的format能够结合MAGEA1抗原表位KVLEYVIKV(SEQ ID NO:120)与HLA-A*02的复合物。The format in this example can bind to the complex of MAGEA1 epitope KVLEYVIKV (SEQ ID NO: 120) and HLA-A*02.
实施例2基于TCR的多特异性多肽分子的表达和纯化Example 2 Expression and Purification of TCR-Based Multispecific Polypeptide Molecules
1.载体构建和质粒提取1. Vector construction and plasmid extraction
将编码实施例1中的format的一条或多条多肽链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。The nucleotide sequence encoding one or more polypeptide chains of the format in Example 1 is directly cloned into the expression vector pTT5. The obtained vector clone is confirmed by sequencing and then amplified, cultured, and plasmid extracted.
2.转染2. Transfection
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SMS 293-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。Adjust the CHO cell density to 1X10 6 cells/ml, and the volume of cell fluid in each bottle is 40 mL. Then tighten the bottle mouth and place it on a shaker to continue culturing. Cultivate at 36.5°C, 175 rpm, and 5% CO2 for 2-4 hours. Then transfect with plasmid. Prepare transfection solution (1ml): Dilute 10μg DNA with about 800μL of 150mM sterilized NaCl solution, mix well and place it on the workbench for 5 minutes; add about 50μl of transfection reagent to the DNA diluent and mix well to obtain the final transfection solution. The total volume is 1mL. After leaving it on the workbench for 10 minutes, add the transfection solution dropwise to the cell culture medium. Shake evenly, tighten the bottle mouth and return it to the shaker (36.5°C, turn off 5% CO2, 175rpm). Add SMS 293-I feeding solution (0.7 mL/bottle) 20-24 hours after transfection, then add feeding solution every other day and culture for 6-10 days, and then collect the culture supernatant.
3.纯化3. Purification
用收集的培养上清液进行镍柱纯化。制备1ml压积镍柱，10ml灭菌水冲洗，10ml 1XBind Buffer进行平衡。将培养上清液在3000rpm下离心5min，然后450nm氯气过滤，上样。上样完成后，用10ml 1XBind Buffer洗柱，10ml 1XBind Buffer洗柱，10ml 1XWash Buffer洗柱子(收集洗脱液10管，流速在0.3-0.4ml/min左右)，1.2ml 1XElution Buffer洗脱蛋白，收集洗脱液即为纯化蛋白，分装后-80保存。The collected culture supernatant was used for nickel column purification. Prepare a 1ml packed nickel column, rinse with 10ml sterile water, and balance with 10ml 1XBind Buffer. The culture supernatant was centrifuged at 3000 rpm for 5 min, then filtered with chlorine gas at 450 nm, and loaded. After loading the sample, wash the column with 10ml 1XBind Buffer, wash the column with 10ml 1XBind Buffer, wash the column with 10ml 1XWash Buffer (collect 10 tubes of eluate, the flow rate is around 0.3-0.4ml/min), and elute the protein with 1.2ml 1XElution Buffer. Collect the eluate to obtain the purified protein, aliquot and store at -80°C.
4.蛋白染色和免疫印迹4. Protein Staining and Western Blotting
将纯化的蛋白经变性(R)或未变性(NR)处理后进行SDS-PAGE凝胶电泳和考马斯亮蓝染色，以评估蛋白表达纯度及聚集程度。另一组平行样品经凝胶电泳后进行western blot，用鼠抗His6作为一抗检测纯化蛋白的His6标签，以确认考马斯亮蓝染色的可靠性。结果表明，本发明提供的format可成功表达。The purified protein was denatured (R) or non-denatured (NR) and then subjected to SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining to evaluate the protein expression purity and degree of aggregation. Another set of parallel samples were subjected to gel electrophoresis and then subjected to western blotting. Mouse anti-His6 was used as the primary antibody to detect the His6 tag of the purified protein to confirm the reliability of Coomassie brilliant blue staining. The results show that the format provided by the invention can be successfully expressed.
示例性western blot结果如图1和2所示。根据收获的目的蛋白产量预测目的蛋白的浓度，根据SDS-PAGE考马斯亮蓝染色结果测定纯度。可见，本发明提供的format能够成功表达。Exemplary western blot results are shown in Figures 1 and 2. The concentration of the target protein is predicted based on the harvested target protein yield, and the purity is determined based on the SDS-PAGE Coomassie Brilliant Blue staining results. It can be seen that the format provided by the present invention can be successfully expressed.
实施例3 ELISPOT测定Example 3 ELISPOT assay
3.1培养上清液ELISPOT测定3.1 ELISPOT assay of culture supernatant
将本发明实施例2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。加完所有样品后，盖好板盖，放在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。 The culture supernatant collected in Example 2 of the present invention is subjected to gradient dilution (usually 2-fold dilution and 5-fold dilution are used). Then the corresponding target peptide and non-related peptide were added to DMSO to dissolve, and diluted with water to a usage concentration of 10-4M. T2 cells were used to load 10-6M of target peptide and irrelevant peptide respectively. Add 1640 complete medium containing 10% FBS to the ELISPOT plate and block for 30 min at room temperature. Discard the culture medium and add 5x105 cells/mL PBMC (100μL/well), 5x105 cells/mL peptide-loaded T2 cells (100μL/well), and different concentrations of format (2x dilution, 5x times dilution). For the negative control, add T2 cells and PBMC that are not loaded with polypeptides. The concentration of the candidate polypeptide molecules is consistent with the experimental group; for the positive control, add 10 μL of the anti-CD3-2 mAb that comes with the kit to PBMC as a positive control. After adding all samples, close the plate cover and place it on the After 20-24 hours of incubation in a 5% CO2 incubator, the secretion of IFN-γ was determined by ELISPOT detection to evaluate the immune cell activation induced by the format of the present invention through the target antigen peptide-MHC complex.
结果表明，本发明的format在有靶抗原的情况下能够激活免疫细胞，在加入无关靶肽和未加入多肽的阴性对照组均不能激活免疫细胞。The results show that the format of the present invention can activate immune cells in the presence of target antigen, but cannot activate immune cells when adding irrelevant target peptides or in the negative control group without adding polypeptides.
本发明format的示例性结果如图6所示，其中具有不同氨基酸序列的format 22-2的培养上清液在2倍和5倍稀释下均能激活免疫细胞。Exemplary results of the format of the present invention are shown in Figure 6, in which the culture supernatant of format 22-2 with different amino acid sequences can activate immune cells at both 2-fold and 5-fold dilution.
3.2纯化蛋白ELISPOT测定3.2 ELISPOT assay of purified protein
用含10％FBS的1640完全培养基将本发明实施例1纯化的format梯度稀释(10-7至10-13M)。将gp100多肽、KRAS多肽、MAGE-A1多肽分别加入DMSO中溶解，然后用水稀释至使用浓度10-4M。用T2细胞分别负载10-6M的gp100多肽、HPV E7多肽、MAGE-A1多肽。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(10-7、10-8、10-9、10-10、10-11、10-12M)。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。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。The format purified in Example 1 of the present invention was gradient diluted (10 -7 to 10 -13 M) with 1640 complete culture medium containing 10% FBS. Add gp100 polypeptide, KRAS polypeptide, and MAGE-A1 polypeptide to DMSO to dissolve respectively, and then dilute with water to a usage concentration of 10 -4 M. T2 cells were used to load 10 -6 M gp100 polypeptide, HPV E7 polypeptide, and MAGE-A1 polypeptide respectively. Add 1640 complete medium containing 10% FBS to the ELISPOT plate and block for 30 min at room temperature. Discard the culture medium and add 5x10 5 cells/mL PBMC (100 μL/well), 5x10 5 cells/mL peptide-loaded T2 cells (100 μL/well), and different concentrations of format (10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 M). For the negative control, add T2 cells and PBMC that are not loaded with polypeptides. The concentration of the candidate polypeptide molecules is consistent with the experimental group; for the positive control, add 10 μL of the anti-CD3-2 mAb that comes with the kit to PBMC as a positive control. After adding all samples, cover the plate, place it in a 37°C, 5% CO2 incubator for 20-24 hours, and then use ELISPOT detection to determine the secretion of IFN-γ to evaluate the format of the present invention through the target antigen peptide-MHC. Complex-induced immune cell activation.
结果表明，本发明的format能够在有靶抗原的情况下激活免疫细胞。The results show that the format of the present invention can activate immune cells in the presence of target antigen.
本发明format的示例性结果如图1和图2所示(附图标记“+”表示表示加入了gp100多肽的实验组，“-”表示没有加入gp100多肽的阴性对照组，“无关”表示加入了不相关多肽的对照组，“无靶”表示没有添加靶肽的对照组)。对照format 17和22的结果分别如图4和图5所示(附图标记“+”表示表示加入了gp100多肽的实验组，“-”表示没有加入gp100多肽的阴性对照组)。Exemplary results of the format of the present invention are shown in Figures 1 and 2 (the reference mark "+" indicates the experimental group in which gp100 polypeptide was added, "-" indicates the negative control group in which gp100 polypeptide was not added, and "irrelevant" indicates the addition of gp100 polypeptide. A control group with irrelevant peptides, "no target" means a control group without target peptide added). The results of control formats 17 and 22 are shown in Figures 4 and 5 respectively (the reference mark "+" indicates the experimental group in which gp100 polypeptide was added, and "-" indicates the negative control group in which gp100 polypeptide was not added).
结果显示，本发明的format 22-2和format 64分别在10-7-10-10M和10-7-10-9M浓度范围内能够实现对免疫细胞的有效激活，特别是format 22-2在10-10M的低浓度下就能有效激活免疫细胞(图1)，而对照format 17和22需要在10-8M的较高浓度下才能激活免疫细胞(图4和5)，表明本发明的format对免疫细胞的激活效果显著优于对照format 17和22。这提示了本发明的format中使用的TRAC和TRAC比使用抗体的CH3或铰链区-CH2-CH3在与特定的第一和第二抗原结合区组合时具有明显更好的效果。The results show that format 22-2 and format 64 of the present invention can achieve effective activation of immune cells in the concentration range of 10 -7 -10 -10 M and 10 -7 -10 -9 M respectively, especially format 22-2 Immune cells can be effectively activated at a low concentration of 10 -10 M (Figure 1), while control formats 17 and 22 require a higher concentration of 10 -8 M to activate immune cells (Figures 4 and 5), indicating that this The invented format has a significantly better activation effect on immune cells than the control formats 17 and 22. This suggests that TRAC and TRAC used in the format of the present invention have significantly better effects than using the CH3 or hinge region-CH2-CH3 of the antibody when combined with specific first and second antigen-binding regions.
实施例4靶细胞杀伤测定Example 4 Target Cell Killing Assay
用含10％FBS的1640完全培养基将本发明实施例1的format梯度稀释(10-7至10-13M)。将gp100多肽、KRAS多肽、MAGE-A1多肽分别加入DMSO中溶解，然后用水稀释至使用浓度10-4M。用T2细胞负载10-6M的靶标多肽。向96U型孔板中加入5x105个细胞/mL的PBMC(100μL/孔)，5x104个细胞/mL的负载多肽的T2细胞(100μL/孔)，和不同浓度的format(10-7、10-8、10-9、10-10、10-11、10-12M)。阴性对照加入不负载gp100多肽的T2细胞和PBMC。加完所有样品后，盖好板盖，放在37℃、5％CO2培养箱培养20-24小时后弃上清，加入100μL荧光素酶报告基因定量测定试剂盒的检测液通过检测靶细胞的荧光素酶活性以评估对靶细胞的杀伤。根据对靶细胞的杀伤数据，利用GraphPad软件，采用非线性回归计算各format对靶细胞杀伤的EC50。The format of Example 1 of the present invention was serially diluted (10 -7 to 10 -13 M) with 1640 complete culture medium containing 10% FBS. Add gp100 polypeptide, KRAS polypeptide, and MAGE-A1 polypeptide to DMSO to dissolve respectively, and then dilute with water to a usage concentration of 10 -4 M. T2 cells were loaded with 10 -6 M of target polypeptide. Add 5x10 5 cells/mL PBMC (100 μL/well), 5x10 4 cells/mL polypeptide-loaded T2 cells (100 μL/well), and different concentrations of format (10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 M). As a negative control, T2 cells and PBMC not loaded with gp100 polypeptide were added. After adding all samples, cover the plate and place it in a 37°C, 5% CO2 incubator for 20-24 hours. Discard the supernatant and add 100 μL of the detection solution of the luciferase reporter gene quantitative assay kit to detect the target cells. Luciferase activity to assess killing of target cells. Based on the target cell killing data, nonlinear regression was used to calculate the EC50 of each format for target cell killing using GraphPad software.
结果表明，本发明提供的format在10-7-10-10M浓度范围内或甚至更低的范围内可实现对靶细胞的杀伤。 The results show that the format provided by the present invention can kill target cells in the concentration range of 10 -7 -10 -10 M or even lower.
实施例5蛋白质的表达、重折叠和纯化Example 5 Expression, refolding and purification of proteins
将制备的含有TCRα-链和β-链的表达质粒各自转化入大肠杆菌菌株Rosetta(DE3)pLysS，诱导蛋白质表达，收获包涵体，用变性剂溶解包涵体，然后透析复性、纯化。获得了纯化的本发明提供的基于TCR的多特异性多肽分子。The prepared expression plasmids containing TCR α-chain and β-chain were each transformed into E. coli strain Rosetta (DE3) pLysS, protein expression was induced, inclusion bodies were harvested, inclusion bodies were dissolved with a denaturant, and then dialyzed, renatured, and purified. Purified TCR-based multispecific polypeptide molecules provided by the invention are obtained.
实施例6基于TCR的多特异性多肽分子与其配体的亲和力和动力学参数测定Example 6 Determination of affinity and kinetic parameters of TCR-based multispecific polypeptide molecules and their ligands
通过表面等离振子共振(SPR)生物传感器检测小型流动小室内传感器表面附近以响应单位(RU)表示的折射率变化，可用于检测受体配体相互作用和分析它们的亲和力以及动力学参数。本发明提供的基于TCR的多特异性多肽分子结合MHC-多肽复合物的KD小于或等于1μM和/或koff为1×10-3S-1或更慢，结合T细胞表面分子的KD小于或等于1μM和/或koff为1×10-3S-1或更慢。Surface plasmon resonance (SPR) biosensors detect refractive index changes expressed in response units (RU) near the sensor surface in small flow chambers, which can be used to detect receptor-ligand interactions and analyze their affinity and kinetic parameters. The TCR-based multispecific polypeptide molecules provided by the present invention have a KD that binds to MHC-polypeptide complexes less than or equal to 1 μM and/or a koff of 1×10-3S-1 or slower, and a KD that binds to T cell surface molecules is less than or equal to 1μM and/or koff is 1×10-3S-1 or slower.
实施例7体内抗肿瘤效力测定Example 7 In vivo anti-tumor efficacy assay
利用靶细胞异种移植模型，在免疫缺陷小鼠中研究抗肿瘤效力。测定本发明的基于TCR的多特异性多肽分子对移植瘤的杀伤，以评估其体内抑瘤作用。本发明提供的基于TCR的多特异性多肽分子具有体内抗肿瘤效力。Antitumor efficacy was studied in immunodeficient mice using a target cell xenograft model. The killing of transplanted tumors by the TCR-based multispecific polypeptide molecules of the present invention is determined to evaluate its tumor suppressive effect in vivo. The TCR-based multispecific polypeptide molecules provided by the present invention have anti-tumor efficacy in vivo.
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。This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated by reference into this application. If there is a conflict between any incorporated reference and this specification, this specification shall prevail. Furthermore, any specific embodiment of the invention that falls within the scope of the state of the art may be expressly excluded from any one or more claims. Because the embodiments described are considered to be known to those skilled in the art, they may be excluded, even if the exclusions are not explicitly listed in this application. Any specific embodiment of the invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.
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。 While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition, method, method step to the purpose, spirit and scope of the invention. All such modifications are intended to be within the scope of the claims.

Claims (31)

  1. 一种双特异性多肽分子，所述双特异性多肽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，A bispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on two polypeptide chains,
    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(TRAV)和β链可变区(TRBV)；wherein the first binding region comprises an alpha chain variable region (TRAV) and a beta chain variable region (TRBV) derived from a T cell receptor (TCR) bound to the first antigen-MHC complex;
    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；wherein 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;
    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；wherein the first antigen is selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens, and the second antigen is an immune cell surface molecule;
    其中所述双特异性多肽分子的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；    The two polypeptide chains of the bispecific polypeptide molecule respectively include:
    TRAV-VH and VL-TRBV;
    TRAV-VL and VH-TRBV;
    TRBV-VH and VL-TRAV; or
    TRBV-VL and 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And the adjacent variable regions in the two polypeptide chains are connected through a linker; and
    所述双特异性多肽分子还包含通过任选的接头连接在每条多肽链的C端的TCR恒定区或其片段。The bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof linked to the C-terminus of each polypeptide chain via an optional linker.
  2. 根据权利要求1的双特异性多肽分子，其中TCR恒定区选自TCRα链恒定区(TRAC)和TCRβ链恒定区(TRBC)，优选地，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；The bispecific polypeptide molecule according to claim 1, wherein the TCR constant region is selected from the group consisting of TCR alpha chain constant region (TRAC) and TCR beta chain constant region (TRBC). Preferably, one polypeptide chain of the bispecific polypeptide molecule comprises TRAC, The other polypeptide chain contains 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。Preferably, the TRAC and/or TRBC comprise 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 Position: position 48 of the constant region of wild-type TCRα chain and position 57 of the constant region of wild-type TCRβ chain.
  3. 根据权利要求2的双特异性多肽分子，其中所述双特异性多肽分子的两条多肽链分别包含：TRAV-VH和VL-TRBV。The bispecific polypeptide molecule according to claim 2, wherein the two polypeptide chains of the bispecific polypeptide molecule respectively comprise: TRAV-VH and VL-TRBV.
  4. 根据权利要求1-3中任一项的双特异性多肽分子，其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：The bispecific polypeptide molecule according to any one of claims 1 to 3, wherein the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:
    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The first polypeptide chain includes from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and
    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。The second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.
  5. 一种双特异性多肽分子，所述双特异性多肽分子在一条或多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，A bispecific polypeptide molecule comprising a first binding region that binds a first antigen and a second binding region that binds a second antigen on one or more polypeptide chains,
    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(TRAV)和β链可变区(TRBV)；wherein the first binding region comprises an alpha chain variable region (TRAV) and a beta chain variable region (TRBV) derived from a T cell receptor (TCR) bound to the first antigen-MHC complex;
    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；wherein 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;
    其中，所述TRAV、TRBV、VH和VL分布在所述一条或多条多肽链上，使得当所述一条或多条多肽链折叠时，所述TRAV和TRBV在空间上靠近以形成所述第一结合区， 且所述VH和VL在空间上靠近以形成第二结合区；Wherein, the TRAV, TRBV, VH and VL are distributed on the one or more polypeptide chains, so that when the one or more polypeptide chains are folded, the TRAV and TRBV are spatially close to form the first A bonding area, And the VH and VL are spatially close to form a second binding area;
    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，wherein the first antigen is selected from the group consisting of tumor-associated antigens (TAA), tumor-specific antigens (TSA), viral antigens and autoantigens, and the second antigen is an immune cell surface molecule,
    其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：Wherein the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:
    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The first polypeptide chain includes from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and
    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。The second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.
  6. 根据权利要求1-5中任一项的双特异性多肽分子，其中所述双特异性多肽分子还包含以下至少一种功能结构域：The bispecific polypeptide molecule according to any one of claims 1 to 5, wherein the bispecific polypeptide molecule further comprises at least one of the following functional domains:
    1)源于抗体的铰链区；1) Derived from the hinge region of the antibody;
    2)源于抗体的Fc结构域或其二聚化部分；2) Derived from the Fc domain of an antibody or its dimerization part;
    3)白蛋白(Alb)。3) Albumin (Alb).
  7. 根据权利要求6的双特异性多肽分子，其中所述功能结构域通过任选的接头连接在所述双特异性多肽分子的一条或两条多肽链的C末端。The bispecific polypeptide molecule according to claim 6, wherein the functional domain is connected to the C-terminus of one or both polypeptide chains of the bispecific polypeptide molecule through an optional linker.
  8. 根据权利要求6或7的双特异性多肽分子，其中所述功能结构域来源于白蛋白，所述白蛋白优选为血清白蛋白，更优选为人血清白蛋白。The bispecific polypeptide molecule according to claim 6 or 7, wherein the functional domain is derived from albumin, and the albumin is preferably serum albumin, more preferably human serum albumin.
  9. 根据权利要求8的双特异性多肽分子，其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：The bispecific polypeptide molecule according to claim 8, wherein the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:
    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The first polypeptide chain includes from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and
    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-任选的接头-Alb。The second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-Alb.
  10. 根据权利要求6或7的双特异性多肽分子，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；并且所述功能结构域通过任选的接头连接在所述双特异性多肽分子的两条多肽链的C末端。The bispecific polypeptide molecule according to claim 6 or 7, wherein the functional domain is derived from the hinge region of the antibody and/or the Fc domain of the antibody or a dimerization part thereof; and the functional domain is optionally passed through The linker is connected to the C-termini of the two polypeptide chains of the bispecific polypeptide molecule.
  11. 根据权利要求10的双特异性多肽分子，其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The bispecific polypeptide molecule according to claim 10, wherein the hinge region of the antibody is of human origin, for example, derived from the hinge domain of human IgGl, IgG2 or IgG4 or a part thereof; and/or
    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，并且优选地包含至少两个半胱氨酸残基突变，例如S354C和Y349C或L242C和K334C。The Fc domain or dimerization part thereof of the antibody is of human origin, for example, derived from the Fc domain or part thereof of human IgGl, IgG2 or IgG4, and preferably contains at least two cysteine residue mutations, e.g. S354C and Y349C or L242C and K334C.
  12. 根据权利要求10或11的双特异性多肽分子，其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：The bispecific polypeptide molecule according to claim 10 or 11, wherein the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:
    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-任选的接头-铰链区-CH2-CH3，并且The first polypeptide chain includes from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC-optional linker-hinge region-CH2-CH3, and
    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-任选的接头-铰链区-CH2-CH3。 The second polypeptide chain includes from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3.
  13. 根据权利要求1-12中任一项的双特异性多肽分子，其中所述多肽分子中的接头各自独立地选自由1-35个氨基酸组成的接头；The bispecific polypeptide molecule according to any one of claims 1-12, wherein the linkers in the polypeptide molecule are each independently selected from a linker consisting of 1-35 amino acids;
    优选地，所述接头各自独立地选自S、GGGS、GGGGS、GGGSGGGG、GGSGGS、GGSGGSGGS、GGGGSGGGGS、GGGGSGGGGSGGGGS、GGGGSGGGGSGGGGSGGGGSGGGS、GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS、GQPKAAP、TVLRT、TVSSAS、GGEGG、GSEGGGS、RTSGPGDGGKGGPGKGPGGEGTKGTGPGG、GKGPGGEGTKGTGPGG、TVLSSAS、EDLKN或其变体、EDLNK或其变体和ANIQK或其变体，或其任意组合；Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGSGGGS, GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS, GQPKAAP, TVLRT, TVSSAS, GGEGG, GSEGGGS, RTSGPGDGGKGGPGKGPGGEGTKGTGPGG, GKGPGG GEGTKGTGPGG, TVLSSAS, EDLKN or other Variants, EDLNK or its variants and ANIQK or its variants, or any combination thereof;
    优选地，EDLKN的变体为EDLKN经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或ANIQK的变体为ANIQK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列。Preferably, the variant of EDLKN is an amino acid sequence formed by the substitution, deletion or addition of one or several amino acids of EDLKN; and/or the variant of EDLNK is the amino acid sequence of EDLNK by substitution, deletion or addition of one or several amino acids. Sequence; and/or ANIQK variants are amino acid sequences formed by ANIQK substitution, deletion or addition of one or several amino acids.
  14. 根据权利要求1-13中任一项的双特异性多肽分子，其中所述多肽分子中的接头各自独立地选自由不超过12个氨基酸组成的接头；The bispecific polypeptide molecule according to any one of claims 1-13, wherein the linkers in the polypeptide molecule are each independently selected from a linker consisting of no more than 12 amino acids;
    优选地，所述接头各自独立地选自S、GGGS、GGGGS、GGGSGGGG、GGSGGS、GGSGGSGGS、GGGGSGGGGS、EDLKN或其变体、EDLNK或其变体和ANIQK或其变体；Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, EDLKN or variants thereof, EDLNK or variants thereof and ANIQK or variants thereof;
    优选地，EDLKN的变体为EDLKN经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或EDLNK的变体为EDLNK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列；和/或ANIQK的变体为ANIQK经过取代、缺失或添加一个或几个氨基酸所形成的氨基酸序列。Preferably, the variant of EDLKN is an amino acid sequence formed by the substitution, deletion or addition of one or several amino acids of EDLKN; and/or the variant of EDLNK is the amino acid sequence of EDLNK by substitution, deletion or addition of one or several amino acids. Sequence; and/or ANIQK variants are amino acid sequences formed by ANIQK substitution, deletion or addition of one or several amino acids.
  15. 根据权利要求1-14中任一项的双特异性多肽分子，其中所述VH和VL包含至少一个半胱氨酸突变以在VH和VL之间形成二硫键，优选地，所述半胱氨酸突变在以下位置处：VH的第44位和VL的第100位。The bispecific polypeptide molecule according to any one of claims 1 to 14, wherein said VH and VL comprise at least one cysteine mutation to form a disulfide bond between VH and VL, preferably said cysteine The amino acid mutations are at the following positions: position 44 of VH and position 100 of VL.
  16. 根据权利要求1-15中任一项的双特异性多肽分子，其中所述多肽分子还包含能够增强其亲和力或生物学活性、增强其稳定性、延长其半衰期、降低其免疫原性或减少其翻译后修饰的一个或多个氨基酸突变、插入或缺失；例如，所述氨基酸突变包括能够增强对FcRn的结合的突变和/或效应子功能沉默突变。The bispecific polypeptide molecule according to any one of claims 1 to 15, wherein the polypeptide molecule further contains a compound capable of enhancing its affinity or biological activity, enhancing its stability, extending its half-life, reducing its immunogenicity or reducing its One or more amino acid mutations, insertions or deletions that modify post-translationally; for example, such amino acid mutations include mutations that enhance binding to FcRn and/or effector function silencing mutations.
  17. 根据权利要求1-16中任一项的双特异性多肽分子，其中所述多肽分子在一条或两条多肽链的N端还包含信号肽序列，优选在每一条链的N端包含信号肽序列，例如衍生自白蛋白或免疫球蛋白的信号肽序列，优选MGWSCIILFLVATATGVHS。The bispecific polypeptide molecule according to any one of claims 1 to 16, wherein the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or both polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain. , for example, a signal peptide sequence derived from albumin or immunoglobulin, preferably MGWSCIILFLVATATGVHS.
  18. 根据权利要1-17中任一项的双特异性多肽分子，其中所述第一抗原为选自以下的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；或者 The bispecific polypeptide molecule according to any one of claims 1-17, wherein the first antigen is 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 endogenous Reverse transcriptase antigen; or
    其中所述第一抗原为选自以下的TSA：KRAS、TP53、PIK3CA、CTNNB1、EGFR、BRAF和GNAS；或者wherein the first antigen is a TSA selected from: KRAS, TP53, PIK3CA, CTNNB1, EGFR, BRAF and GNAS; or
    其中所述第一抗原为选自以下的病毒抗原：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；或者Wherein the first antigen is a viral antigen selected from the following: 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; or
    所述第一抗原为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。The first antigen is an autoantigen selected from the following: AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUCl, 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-γ, CHRNA1, MUSK, LRP4, AQP4, MOG, GRIN1, COL4A3, PLA2R, GM-SCF, PR3 and MPO.
  19. 根据权利要求1-18中任一项的双特异性多肽分子，其中所述第二抗原选自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、CD80、BTLA、TIM3、TIGIT和LAG-3。The bispecific polypeptide molecule according to any one of claims 1 to 18, wherein the second antigen is selected from the group consisting of CD3 (eg CD3γ, CD3δ and CD3ε chains), 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, CD80, BTLA, TIM3, TIGIT and LAG-3.
  20. 根据权利要求1-19中任一项的双特异性多肽分子，其中所述第一抗原选自The bispecific polypeptide molecule according to any one of claims 1-19, wherein said first antigen is selected from
    gp100、MAGEA1、KRAS和HPVE7，和/或所述第二抗原选自CD3、CD28或4-1BB(CD137)。gp100, MAGEA1, KRAS and HPVE7, and/or the second antigen is selected from CD3, CD28 or 4-1BB (CD137).
  21. 根据权利要求1-20中任一项的双特异性多肽分子，其中所述多肽分子包含:The bispecific polypeptide molecule according to any one of claims 1-20, wherein said polypeptide molecule comprises:
    具有如SEQ ID NO:1所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:2所示的氨基酸序列的第二多肽链；A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 1, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 2;
    具有如SEQ ID NO:3所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:4所示的氨基酸序列的第二多肽链；A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO:3, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO:4;
    具有如SEQ ID NO:5所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:6所示的氨基酸序列的第二多肽链；A first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 5, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 6;
    具有如SEQ ID NO:99所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:100所示的氨基酸序列的第二多肽链；A first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 99, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 100;
    具有如SEQ ID NO:101所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:102所示的氨基酸序列的第二多肽链；A first polypeptide chain having an amino acid sequence shown in SEQ ID NO: 101, and a second polypeptide chain having an amino acid sequence shown in SEQ ID NO: 102;
    具有如SEQ ID NO:103所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:104所示的氨基酸序列的第二多肽链；或A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 103, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 104; or
    具有如SEQ ID NO:105所示的氨基酸序列的第一多肽链，和具有如SEQ ID NO:106所示的氨基酸序列的第二多肽链。A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 105, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 106.
  22. 核酸，其包含编码权利要求1-21中任一项所述的双特异性多肽分子的每一条链的核苷酸序列。 A nucleic acid comprising a nucleotide sequence encoding each strand of the bispecific polypeptide molecule of any one of claims 1-21.
  23. 载体，其包含权利要求22所述的核酸。A vector comprising the nucleic acid of claim 22.
  24. 载体系统，其在一个或多个载体上包含编码权利要求1-21中任一项所述的双特异性多肽分子的每一条链的核苷酸序列。A vector system comprising, on one or more vectors, a nucleotide sequence encoding each chain of the bispecific polypeptide molecule of any one of claims 1-21.
  25. 宿主细胞，其包含权利要求22所述的核酸、权利要求23所述的载体或权利要求24所述的载体系统。A host cell comprising the nucleic acid of claim 22, the vector of claim 23, or the vector system of claim 24.
  26. 缀合物，其包含权利要求1-21中任一项所述的双特异性多肽分子，以及与所述双特异性多肽分子缀合的化学部分。A conjugate comprising the bispecific polypeptide molecule of any one of claims 1-21 and a chemical moiety conjugated to the bispecific polypeptide molecule.
  27. 根据权利要求26的缀合物，其中所述化学部分选自治疗剂、免疫刺激分子和可检测标记；The conjugate according to claim 26, wherein said chemical moiety is selected from the group consisting of therapeutic agents, immunostimulatory molecules and detectable labels;
    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。Preferably, the therapeutic agent is selected from immunomodulators, radioactive compounds, enzymes, chemotherapeutic agents and toxins.
    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；Preferably, the immunostimulatory molecule is selected from the group consisting of cytokines, chemokines, platelet factors and complement initiators;
    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。Preferably, the detectable label is selected from the group consisting of biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescence, Phosphorescent or chemiluminescent molecules.
  28. 药物组合物，其包含权利要求1-21中任一项所述的双特异性多肽分子、权利要求22所述的核酸、权利要求23所述的载体、权利要求24所述的载体系统、权利要求25所述的宿主细胞、权利要求26或27所述的缀合物，以及药学上可接受的赋形剂。A pharmaceutical composition comprising the bispecific polypeptide molecule of any one of claims 1-21, the nucleic acid of claim 22, the vector of claim 23, the vector system of claim 24, the The host cell of claim 25, the conjugate of claim 26 or 27, and a pharmaceutically acceptable excipient.
  29. 根据权利要求28的药物组合物，其中所述药物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。The pharmaceutical composition according to claim 28, wherein the pharmaceutical composition further comprises a second therapeutic agent. Preferably, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs.
  30. 预防或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1-21中任一项所述的双特异性多肽分子、权利要求22所述的核酸、权利要求23所述的载体、权利要求24所述的载体系统、权利要求25所述的宿主细胞、权利要求26或27所述的缀合物、或权利要求28或29所述的药物组合物，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。A method of preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the bispecific polypeptide molecule of any one of claims 1-21, the nucleic acid of claim 22, The vector of claim 23, the vector system of claim 24, the host cell of claim 25, the conjugate of claim 26 or 27, or the pharmaceutical composition of claim 28 or 29, wherein said disease is selected from the group consisting of cancer, infectious diseases, autoimmune diseases and inflammatory diseases.
  31. 根据权利要求30的方法，其还包括向所述受试者施用第二治疗剂，优选地，所述第二治疗剂选自抗体、化疗剂和小分子药物。 The method according to claim 30, further comprising administering to the subject a second therapeutic agent, preferably, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs.
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