Multi-specific polypeptide molecule based on T cell receptor and application thereof

Abstract

The invention provides multi-specific polypeptide molecules based on T cell receptors, and specifically discloses tri-specific polypeptide molecules and bi-specific polypeptide molecules. The invention also provides nucleic acids encoding the polypeptide molecules of the invention, vectors or vector systems comprising the nucleic acids of the invention, host cells comprising the polypeptide molecules of the invention, conjugates and pharmaceutical compositions comprising the polypeptide molecules of the invention, and methods of using the polypeptide molecules of the invention to prevent or treat diseases in subjects.

Description

Multispecific polypeptide molecules based on T cell receptors and uses thereof

[0001] This international patent application claims the priority benefit of Chinese patent application CN202210730436.0 filed on June 24, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to multispecific polypeptide molecules based on T cell receptors and uses thereof, in particular, uses in the treatment of cancer, infectious diseases, autoimmune diseases and / or inflammatory diseases. Background Art

[0003] T cells are an important component of the vertebrate adaptive immune system, playing a crucial role in viral infection, cancer, and autoimmunity. TCR recognition of the major histocompatibility complex (MHC)-antigen peptide complex (pMHC) is a crucial step in T cell-mediated immune responses. The TCR non-covalently associates with the CD3 signaling apparatus, composed of γ, δ, ε, and ζ subunits, to form the TCR–CD3 complex. Binding of the pMHC to the TCR activates LCK-induced phosphorylation of the intracellular immunoreceptor tyrosine-based activation motif (ITAM) in the TCR CD3ζ subunit. CD3ζ phosphorylation then triggers a cascade of phosphorylation signaling, involving ζ chain-associated protein kinase 70 (ZAP70) and the activating T cell linker (LAT) in T cells, leading to the recruitment of multiple downstream adaptors and signaling molecules and the formation of the LAT signalosome. The assembled LAT signalosome activates multiple signaling pathways involving transcription factors such as activator protein 1 (AP-1), nuclear factor-κB (NF-κB), and nuclear factor of activated T cells (NFAT). Activated transcription factors can lead to subsequent T cell activation, proliferation, cytokine production, and effector function.

[0004] Bispecific antibodies (BsAbs) are antibody molecules that simultaneously target two antigens or two different antigenic epitopes of one antigen. Compared with conventional 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 formats have been produced. Multispecific antibodies are used for various purposes, including (1) receptor activation (2) blocking (3) internalization (4) aggregation, (5) binding of membrane-associated proteins, or (6) directed targeting of cytotoxic effector cells.

[0005] T cell-redirecting bsAbs (also known as T cell engagers, TcEs) are an important form of cytotoxic effector cell redirection and are the central pillar of current cancer immunotherapy. This bsAb can recognize targets on the surface of tumor cells and molecules on the surface of T cells (in most cases, this molecule is CD3). In this way, the bsAb can couple tumor cells and cytotoxic T cells together to activate the T cell TCR downstream signaling pathway and kill tumor cells through T cell cytotoxicity. Blincyto (blinatumomab) is a CD19 / CD3 bispecific antibody that resulted in complete remission in 69% of patients with relapsed / refractory B-precursor acute lymphoblastic leukemia (ALL). A large number of new T cell-redirecting antibody formats have emerged, such as BITE, BITE-Fc, DART-Fc, TriTAC, etc.

[0006] bsAbs, which rely on antibodies to recognize target cell surface molecules, only recognize tumor cell surface antigens and are unable to recognize nearly 90% of intracellular proteins. TCRs, on the other hand, can recognize both intracellular and cell surface tumor-specific antigens, as well as antigenic peptides that have been processed and presented on cell surface MHC molecules, thus offering a broader recognition range. In TCR-based T cell-redirecting bispecific antibodies, the target cell recognition region is replaced by the TCR, leveraging the broad recognition properties of the TCR to increase target selectivity.

[0007] Summary of the Invention

[0008] To enhance the in vivo effectiveness of TCEs, in addition to increasing the affinity of the TCR for pMHC, it is also possible to increase the potency of the TCE against target cell targets and the potency of the TCE against molecules on the surface of effector cells, thereby enhancing T cell cytotoxicity and proliferation. Furthermore, to maintain the ability of TCEs to enter tumors while maintaining an appropriate half-life in vivo, the molecular size of TCEs needs to be kept within a certain range. Accordingly, the present invention provides TCR-based multispecific polypeptide molecules, such as trispecific or bispecific TCR-based polypeptide molecules.

[0009] the term

[0010] Unless otherwise specified or defined, all terms used herein have the common meanings in the art known to those skilled in the art. For example, the terms used herein are as defined in Janeway CA Jr, Travers P, Walport M et al., Immunobiology, Fifth Edition, New York: Garland Science (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Definitions as described in H., ed. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0011] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Similarly, the terms "comprising," "including," and "having" can be used interchangeably.

[0012] Unless otherwise stated or defined, the term "comprise" and variations thereof such as "comprises" and "comprising" should 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 a preferred embodiment of the term "comprising." If a group is defined hereinafter as comprising or containing at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists only of these embodiments.

[0013] As used herein, the term "T cell receptor" or "TCR" includes native TCRs as well as TCR variants, fragments, and constructs. The term thus includes heterodimers, as well as multimers and single-chain constructs comprising a TCR alpha chain and a TCR beta chain; optionally comprising other domains and / or portions, so long as the TCR retains its ability to recognize an antigen target.

[0014] In its native form, the TCR exists as a complex of several proteins on the surface of the T cell. The T cell receptor consists of two (separate) protein chains, which are produced by independent T cell receptor α and β (TCRα and TCRβ) genes and are called α chain and β chain. Each chain of the TCR has an N-terminal immunoglobulin-like (Ig)-variable (V) region / domain, an Ig-constant (C) region / domain, a transmembrane / cell membrane spanning region that anchors the chain in the plasma membrane, and a short cytoplasmic tail at the C-terminus.

[0015] Antigen specificity is conferred by the variable regions of the α and β chains (TRAV and TRBV). Both variable regions of the TCR α and β chains contain three hypervariable or complementarity-determining regions (CDR1α / β, CDR2α / β, and CDR3α / β) surrounded by framework (FR) regions. CDR3 is the primary determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 primarily interact with the MHC molecules that present the antigenic peptide.

[0016] TCR recognizes antigenic peptides that are bound to major histocompatibility complex (MHC) molecules at the surface of antigen-presenting cells ("presented / displayed on MHC molecules"). Antigenic peptides presented on MHC molecules are also referred to herein as "complexes of epitopes and MHC molecules," "epitope-MHC complexes," "antigen-MHC complexes," or "target antigen peptide-MHC complexes." There are two different classes of MHC molecules: MHC I and MHC II, which present peptides from different cell compartments. MHC class I molecules are expressed on the surface of all nucleated cells in the human body and display peptides or protein fragments from intracellular compartments to cytotoxic T cells. In humans, MHC is also known as 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 exerts biological effector functions.

[0017] As used herein, the term "TCR constant region" includes the TCR alpha chain constant region (TRAC) and beta chain constant region (TRBC), which may be human constant regions or derived from another species, such as mouse.

[0018] It has been reported that the addition of disulfide bonds in the constant region can promote the correct pairing of TCRα and β chains (Kuball J et al. Blood. 2007 Mar 15; 109(6): 2331-8). Therefore, the present invention also contemplates the addition of one or more cysteine ​​modifications in the constant region to form a disulfide bond between the TCRα chain and the TCRβ chain.

[0019] The sequence of the wild-type TCR constant region can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant domain sequence of the TCR molecule α chain is "TRAC*01", and the constant domain sequence of the TCR molecule β chain is "TRBC1*01" or "TRBC2*01".

[0020] The positions of the amino acid sequences of wild-type TCRs in the present invention are numbered according to the nomenclature of the International Immunogenetics Information System (IMGT). For example, if an amino acid in the TCRα chain variable region (TRAV) is numbered as position 104 in IMGT, it will be described as the 104th amino acid of TRAV in the present invention; if an amino acid in the TCRβ chain variable region (TRBV) is numbered as position 84 in IMGT, it will be described as the 84th amino acid of TRBV in the present invention; and so on. For example, if an amino acid in the TCRα chain constant region (TRAC) is numbered as position 48 in IMGT, it will be described as the 48th amino acid of TRAC in this article; if an amino acid in the TCRβ chain constant region (TRBC) is numbered as position 57 in IMGT, it will be described as the 57th amino acid of TRBC in this article; and so on. In the present invention, if the sequence position numbering of other amino acids is specially specified, it shall be in accordance with the special instructions.

[0021] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Such molecules typically comprise 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) (abbreviated herein 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) (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains of an antibody contain a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin 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 first component in the classical pathway of complement activation).

[0022] The heavy chain of an immunoglobulin can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region contains the VH and CH1 domains and combines with the light chain to form the Fab (antigen binding fragment). The Fc region, also known as the 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 the CH2 and CH3 domains. In the native structure of immunoglobulins, the Fc regions of the two heavy chains dimerize to form a dimerized portion. The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to cognate Fc receptors on effector cells.

[0023] The hinge region of IgG class antibodies refers to the short amino acid sequence region between the heavy chain CH1 and CH2 parts, which is relatively flexible in the native state of the antibody. The hinge region found in IgG, IgA and IgD immunoglobulin classes acts as a flexible spacer, allowing the Fab part to move freely in space relative to the Fc region. The hinge domain is structurally diverse, with different sequences and lengths between immunoglobulin classes and subclasses. According to crystallographic studies, the immunoglobulin hinge region can be further divided into three regions in structure and function: the upper hinge, the core hinge and the lower hinge (Shin et al., Immunological Reviews 130:87, 1992). The upper hinge includes the amino acid residues from the carboxyl terminus of CH1 to the first residue in the hinge that restricts movement, typically the first cysteine ​​residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region is related to the flexibility of the antibody fragment. The core hinge region contains an inter-heavy chain disulfide bond. 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: 177), which, when dimerized through disulfide bonds, generates a cyclic octapeptide that is believed to act as a pivot, conferring flexibility. The conformational changes allowed by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the antibody Fc portion.

[0024] 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 regions coordinate the CDRs for specific binding to an antigenic epitope. The CDRs contain the amino acid residues primarily responsible for antigen binding in an antibody. From amino-terminus to carboxyl-terminus, both VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0025] The amino acid assignments for each VL and VH domain follow any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)); the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 878-883, 1989); a composite of the Chothia Kabat CDRs, in which CDR-H1 is a composite of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (World Wide Web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use the Kabat defined CDRs.

[0026] The term "antibody" as used herein should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) comprising at least two antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0027] 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 to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0028] Examples of antigen-binding fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a Fab' fragment, which is essentially a Fab but has a portion of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment (Ward et al. (1989) Nature 557(5)). 341: 544-546), which consists of a VH domain; (viii) a single complementarity determining region (CDR); and (ix) a nanobody, which is a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that enables them to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (ScFv); see, e.g., Bird et al. (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 encompassed by the term "antigen-binding fragment" of an antibody. In addition, the term also includes "linear antibodies" containing a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides and modified versions of any of the foregoing segments that retain antigen-binding activity form an antigen-binding region.

[0029] As used herein, the term "antigen" refers to any substance capable of inducing an immune response in the body. Specifically, it refers to a substance that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, and produce immune response products (sensitized lymphocytes or antibodies), and that can specifically bind to corresponding products in vivo or in vitro.

[0030] Herein, the antigen may be a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, a self-antigen, and an immune cell surface molecule.

[0031] As used herein, the term "tumor-associated antigen (TAA)" refers to an antigen that is differentially expressed in tumor cells (eg, has significantly increased expression on tumor cells) compared to normal cells.

[0032] As used herein, the term "tumor-specific antigen (TSA)" refers to an antigen that is unique to tumor cells and not present in normal tissues and cells.

[0033] As used herein, the term "viral antigen" refers to a substance in a virus that can induce an immune response in the body.

[0034] As used herein, the term "autoantigen" refers to an antigenic substance that causes changes in the structure and composition of one's own tissue cells caused by biological, physical, and chemical factors, thereby causing the body's immune system to produce an immune response to these self-tissue cell components. The release of hidden antigens can produce an immune response. Changes in one's own components may produce autoantigens. For example, denatured IgG can stimulate the body to produce anti-denatured IgG antibodies (rheumatoid factor), causing rheumatoid arthritis. After clinical use of certain drugs, the surface antigenicity of blood cells can be changed, causing autoimmune hemolytic anemia or granulocytopenia. The cross-reaction caused by common antigens can produce an immune response. Certain bacteria, viruses and certain tissue cells of the normal human body have similar antigenic determinants. The autoantibodies and sensitized lymphocytes produced for these bacteria and virus antigenic determinants can cross-react with one's own tissue cells, causing autoimmune diseases.

[0035] "Immune cell surface molecules" may include, for example, immune cell surface antigens (eg, T cell antigens) and co-stimulatory molecules.

[0036] The site on the antigen that is bound by TCR or antibody is called "epitope". The epitope can be formed by continuous amino acids or non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. The epitope formed by continuous amino acids (also referred to as linear epitope) is usually retained after exposure to denaturing solvents, while the epitope formed by tertiary folding (also referred to as conformational epitope) is usually lost in the treatment of denaturing solvents. The epitope usually comprises at least 3, more generally at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the minimum binding site of TCR or antibody and is therefore the specific target of TCR or antibody or its antigen-binding fragment.

[0037] As used herein, the term "albumin" encompasses any natural albumin of any vertebrate origin, including mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). Albumin also encompasses full-length, unprocessed albumin preproproteins and any form of albumin produced by processing in cells. Albumin also encompasses naturally occurring variants of albumin, such as splice variants or allelic variants. The term also encompasses any recombinant form of albumin. Albumin sequences are known in the art. For example, information about the human serum albumin gene (including genomic DNA sequences) can be found 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, 17 disulfide bonds in the molecule, and no 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. After 2 hours, about 10% of exogenously infused albumin enters the extravascular tissue space, and equilibrium is reached after 20 days. The half-life of albumin in plasma is 15-19 days.

[0038] >gi|4502027|ref|NP_000468.1|Serum albumin preproprotein [Homo sapiens]

[0039] (SEQ ID NO: 200; underline indicates signal peptide, bold indicates propeptide, italics indicates mature human serum albumin).

[0040] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acids) have identical residues at identical positions when aligned. For example, "an 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 described as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y.

[0041] Computer programs are typically 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., 1984).

[0042] In addition, when determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of a similar chemical structure, which has little or substantially no effect on the function, activity or other biological properties of the 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) the type and / or combination of these substitutions can be selected according to the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and the further references cited therein.

[0043] Such conservative substitutions are preferably substitutions in which an amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar 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, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0044] 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.

[0045] As used herein, the term "cysteine-containing peptide segment" refers to a polypeptide segment containing cysteine ​​residues that is capable of forming a disulfide bond between two polypeptide chains comprising the peptide segment, and has a length of 3-10 amino acids.

[0046] The term "vector" as used herein is a nucleic acid molecule used as a vehicle for transferring (exogenous) genetic material into a host cell where it can, for example, be replicated and / or expressed.

[0047] As used herein, the term "host cell" refers to any type of cell into which an expression vector has been introduced so as to be capable of expressing foreign genetic material.

[0048] The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof, and / or that the carrier or adjuvant is approved or acceptable for inclusion in pharmaceutical compositions for parenteral administration to humans.

[0049] As used herein, the terms "treat", "treat", and the like refer to the administration of an agent or the performance of a procedure in order to obtain an effect. These effects may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of affecting a partial or complete cure of a disease and / or disease symptoms. As used herein, "treatment" may include the treatment of a disease or condition (e.g., cancer, infectious diseases, autoimmune diseases, or inflammatory diseases) in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease or disease symptoms in a subject susceptible to the disease but not yet diagnosed as having the disease (e.g., including diseases that may be associated with or caused by the primary disease); (b) inhibiting the disease, i.e., preventing its development; (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any reference to success in treating, ameliorating, or preventing cancer, including any objective or subjective parameter, such as elimination; alleviation; reducing symptoms or making the disease condition more tolerable for the patient; slowing the rate of deterioration or decline; or reducing the end point of deterioration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes administering a multispecific polypeptide molecule, pharmaceutical composition, or conjugate disclosed herein to prevent, delay, alleviate, arrest, or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptom, or disease side effect in a subject.

[0050] As used herein, the term "effective amount" means an amount of a therapeutic agent that, when administered to a subject for the treatment or prevention of a disease, is sufficient to achieve 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. A "therapeutically effective amount" refers to an effective amount for therapeutic treatment. A "prophylactically effective amount" refers to an effective amount for preventive treatment.

[0051] As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock, and laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0052] Preferred embodiments of the present invention

[0053] The features and advantages of the present invention and its additional features and advantages will be more clearly understood below through the detailed description of the following embodiments in conjunction with the accompanying drawings. The embodiments described herein with reference to the accompanying drawings are explanatory and illustrative and are used for a general understanding of the present invention. The embodiments should not be interpreted as limiting the scope of the present invention. The same or similar elements and elements having the same or similar functions are represented by the same reference numerals throughout the description.

[0054] In one aspect, the present invention provides a trispecific polypeptide molecule, wherein the trispecific polypeptide molecule comprises a first binding region that binds a first antigen, a second binding region that binds a second antigen, and a third binding region that binds a third antigen on one or more polypeptide chains,

[0055] wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

[0056] wherein the second binding region comprises a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen; and

[0057] wherein the third binding region comprises a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody that binds to the third antigen;

[0058] wherein said TRAV1, TRBV1, VH1, VL1, VH2 and VL2 are distributed on said one or more polypeptide chains such that when said one or more polypeptide chains are folded, said TRAV1 and TRBV1 are spatially close to form a first binding region, said VH1 and VL1 are spatially close to form a second binding region, and said VH2 and VL2 are spatially close to form a third binding region;

[0059] The first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens and self-antigens, the second antigen is a first immune cell surface molecule, and the third antigen is a second immune cell surface molecule; the first immune cell surface molecule and the second immune cell surface molecule are different.

[0060] In some embodiments, the TRAV1, TRBV1, VH1, VL1, VH2 and VL2 are distributed on the one or more polypeptide chains and adjacent variable regions on the same chain are separated by a linker sequence.

[0061] In some embodiments, the polypeptide molecule further comprises at least one pair of TRAV2 and TRBV2, which form a fourth binding region that binds to a fourth antigen; wherein the fourth antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the fourth antigen is the same as or different from the first antigen. In a preferred embodiment, the polypeptide molecule comprises an additional copy of TRAV1 and TRBV1, which form a fourth binding region that binds to the first antigen.

[0062] In some embodiments, the polypeptide molecule further comprises one or more of the following structures:

[0063] (1) a CH3 domain, wherein the CH3 domain is derived from the CH3 domain of an antibody, and the CH3 of any one polypeptide chain or both polypeptide chains comprises at least one mutation that promotes the formation of heterodimers of the polypeptide molecules;

[0064] (2) a TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0065] (3) hinge region, CH2 and CH3, wherein the hinge region is derived from the hinge domain of an antibody, the CH2 and CH3 are derived from the Fc domain of an antibody, and the CH3 in any one polypeptide chain or both polypeptide chains contains at least one mutation that can promote the formation of heterodimers of the polypeptide molecules;

[0066] (4) CL and CH1, wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain;

[0067] (5) Cysteine-containing peptide segments, wherein the cysteine-containing peptide segments are capable of forming a disulfide bond between two polypeptide chains and are 3-10 amino acids in length, preferably 4-8 amino acids in length, such as 4 or 6 amino acids.

[0068] In some embodiments, the polypeptide molecule comprises the first binding region, the second binding region, the third binding region, and, when present, the fourth binding region, on one polypeptide chain.

[0069] In some embodiments, the polypeptide molecule comprises the following structure from N-terminus to C-terminus:

[0070] TRBV1-linker-VH1-linker-VH2-linker-TRAV2-linker-TRBV2-linker-VL2-linker-VL1-linker-TRAV1 (format 13).

[0071] In some embodiments, the polypeptide molecule comprises a derivative form of format 13, in which the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2) constituting the binding region can be interchanged and the order of the binding regions formed therefrom can be interchanged, as long as the ability to bind to the corresponding antigen is retained. For example, VH1 and VL1 in format 13 can be interchanged, and / or VH2 and VL2 can be interchanged, and / or TRAV1 and TRBV1 can be interchanged, and / or TRAV2 and TRBV2 can be interchanged; for example, the order of the first binding region, the second binding region, the third binding region, and the fourth binding region can also be interchanged. Specifically, the derivative form of format 13 includes, but is not limited to, the following structures from N-terminus to C-terminus:

[0072] TRAV2-linker-VH1-linker-VH2-linker-TRBV1-linker-TRAV1-linker-VL2-linker-VL1-linker-TRBV2 (format 13-1); or

[0073] TRBV1-linker-VH1-linker-VL2-linker-TRAV2-linker-TRBV2-linker-VH2-linker-VL1-linker-TRAV1 (format 13-2); or

[0074] TRAV2-linker-VH1-linker-VL2-linker-TRBV1-linker-TRAV1-linker-VH2-linker-VL1-linker-TRBV2 (format 13-3); or

[0075] VH1-Linker-TRBV1-Linker-VH2-Linker-TRAV2-Linker-TRBV2-Linker-VL2-Linker-TRAV1-Linker-VL1 (format 13-4); or

[0076] VH1-Linker-TRAV2-Linker-VH2-Linker-TRBV1-Linker-TRAV1-Linker-VL2-Linker-TRBV2-Linker-VL1 (format 13-5); or

[0077] VH1-Linker-TRBV1-Linker-VL2-Linker-TRAV2-Linker-TRBV2-Linker-VH2-Linker-TRAV1-Linker-VL1 (format 13-6); or

[0078] VH1-Linker-TRAV2-Linker-VL2-Linker-TRBV1-Linker-TRAV1-Linker-VH2-Linker-TRBV2-Linker-VL1 (format 13-7); or

[0079] VH1-Linker-TRBV1-Linker-TRAV2-Linker-VH2-Linker-VL2-Linker-TRBV2-Linker-TRAV1-Linker-VL1 (format 13-8); or

[0080] VH1-Linker-TRBV1-Linker-TRAV2-Linker-VL2-Linker-VH2-Linker-TRBV2-Linker-TRAV1-Linker-VL1 (format 13-9); or

[0081] VH1-Linker-TRAV2-Linker-TRBV1-Linker-VH2-Linker-VL2-Linker-TRAV1-Linker-TRBV2-Linker-VL1 (format 13-10); or

[0082] VH1-Linker-TRAV2-Linker-TRBV1-Linker-VL2-Linker-VH2-Linker-TRAV1-Linker-TRBV2-Linker-VL1 (format 13-11); or

[0083] VL1-Linker-TRAV2-Linker-VH2-Linker-TRAV1-Linker-TRBV1-Linker-VL2-Linker-TRBV2-Linker-VH1 (format 13-12); or

[0084] VL1-linker-TRAV2-linker-TRBV1-linker-VL2-linker-VH2-linker-TRAV1-linker-TRBV2-linker-VH1 (format 13-13).

[0085] In some embodiments, the polypeptide molecule comprises a derivative form of format 13, in which the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2) constituting the binding region can be interchanged, and TRAV1 and TRBV1, or TRAV2 and TRBV2, can be replaced by CH3 / CH3, CH1 / CL, or TRAC / TRBC of an antibody, as long as the ability to bind to the corresponding antigen is retained. For example, TRAV1 and TRBV1, or TRAV2 and TRBV2 in format 13 can be replaced by CH3 / CH3 of an antibody. For example, derivative forms of format 13 include, but are not limited to, the following structures from N-terminus to C-terminus:

[0086] CH3-Linker-VH1-Linker-VH2-Linker TRAV-Linker-TRBV-Linker-VL2-Linker-VL1-Optional Linker-CH3 (format 13-14); or

[0087] CH3-Linker-VH1-Linker-VH2-Linker TRBV-Linker-TRAV-Linker-VL2-Linker-VL1-Optional Linker-CH3 (format 13-15); or

[0088] CH3-Linker-VH1-Linker-VL2-Linker TRAV-Linker-TRBV-Linker-VH2-Linker-VL1-Optional Linker-CH3 (format 13-16); or

[0089] CH3-Linker-VH1-Linker-VL2-Linker TRBV-Linker-TRAV-Linker-VH2-Linker-VL1-Optional Linker-CH3 (format 13-17); or

[0090] VH1-optional linker-CH3-linker-VH2-linker TRAV-linker-TRBV-linker-VL2-optional linker-CH3-linker-VL1 (format 13-18); or

[0091] VH1-optional linker-CH3-linker-VH2-linker TRBV-linker-TRAV-linker-VL2-optional linker-CH3-linker-VL1 (format 13-19); or

[0092] VH1-optional linker-CH3-linker-VL2-linker TRAV-linker-TRBV-linker-VH2-optional linker-CH3-linker-VL1 (format 13-20); or

[0093] VH1-optional linker-CH3-linker-VL2-linker TRBV-linker-TRAV-linker-VH2-optional linker-CH3-linker-VL1 (format 13-21); or

[0094] VH1-Linker-TRBV-Linker-VH2-Optional Linker-CH3-Optional Linker-CH3-Linker-VL2-Linker-TRAV-Linker-VL1 (format 13-22); or

[0095] VH1-Linker-TRAV-Linker-VH2-Optional Linker-CH3-Optional Linker-CH3-Linker-VL2-Linker-TRBV-Linker-VL1 (format 13-23); or

[0096] VH1-Linker-TRBV-Linker-VL2-Optional Linker-CH3-Optional Linker-CH3-Linker-VH2-Linker-TRAV-Linker-VL1 (format 13-24); or

[0097] VH1-Linker-TRAV-Linker-VL2-Optional Linker-CH3-Optional Linker-CH3-Linker-VH2-Linker-TRBV-Linker-VL1 (format 13-25); or

[0098] VH1-optional linker-CH3-linker-TRAV-linker VH2-linker-VL2-linker-TRBV-optional linker-CH3-linker-VL1 (format 13-26); or

[0099] VH1-optional linker-CH3-linker-TRAV-linker VL2-linker-VH2-linker-TRBV-optional linker-CH3-linker-VL1 (format 13-27); or

[0100] VH1-optional linker-CH3-linker-TRBV-linker VH2-linker-VL2-linker-TRAV-optional linker-CH3-linker-VL1 (format 13-28); or

[0101] VH1-optional linker-CH3-linker-TRBV-linkerVL2-linker-VH2-linker-TRAV-optional linker-CH3-linker-VL1 (format 13-29); or

[0102] VL1-optional linker-CH3-linker-VH2-linker TRAV-linker-TRBV-linker-VL2-optional linker-CH3-linker-VH1 (format 13-30); or

[0103] VL1-Linker-TRAV-Linker-VH2-Optional Linker-CH3-Optional Linker-CH3-Linker-VL2-Linker-TRBV-Linker-VH1 (format 13-31); or

[0104] VL1-optional linker-CH3-linker-TRBV-linker VL2-linker-VH2-linker-TRAV-optional linker-CH3-linker-VH1 (format 13-32).

[0105] In some embodiments, the polypeptide molecule further comprises a CH3 domain, wherein the CH3 domain is derived from a CH3 domain of an antibody and comprises at least one mutation capable of inhibiting the polypeptide molecule from forming a homodimer.

[0106] In some embodiments, the polypeptide molecule comprises the following structure from N-terminus to C-terminus:

[0107] VL1-Linker-TRBV1-Linker-TRAV1-Linker-VH1-Optional Linker-CH3-Linker-VL2-Linker-TRBV2-Linker-TRAV2-Linker-VH2-Optional Linker-CH3 (format 43).

[0108] In some embodiments, the polypeptide molecule comprises a derivative of format 43. In these derivatives, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, and CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in derivatives of format 43, the two CH3 domains can be replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. Specifically, derivatives of format 43 include, but are not limited to, derivatives of format 43 formed by concatenating derivatives of format 42 into a single chain with linkers, which are not specifically listed here.

[0109] In some embodiments, the CH3s each independently comprise a mutation at one or more positions selected from amino acid positions 366, 368, 405, and 407, and the two CH3s do not comprise the same mutation. In a preferred embodiment, one of the CH3s comprises a T366W mutation and the other comprises T366S, L368A, and Y407V mutations.

[0110] In some embodiments, the polypeptide molecule comprises the first binding region, the second binding region, the third binding region, and when present, the fourth binding region on two polypeptide chains.

[0111] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the domains constituting the binding region (e.g., VH1, VL1, VH2, VL2, TRAV1, TRBV1, and optionally TRAV2, TRBV2) are evenly distributed on the two polypeptide chains.

[0112] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0113] The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1 (format 7).

[0114] In some embodiments, the polypeptide molecule comprises a derivative form of format 7, in which the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1) comprising the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. For example, VH1 and VL1 in format 7 can be interchanged, and / or VH2 and VL2 can be interchanged, and / or TRAV1 and TRBV1 can be interchanged. For example, the order of the first binding region, the second binding region, and the third binding region can also be interchanged. Derivative forms of format 7 include, but are not limited to, the following structures:

[0115] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL2-linker-TRAV1 (format 7-1); or

[0116] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRBV1 (format 7-2); or

[0117] The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VL1-linker-TRAV1, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL2-linker-TRBV1 (format 7-3; or

[0118] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VL2 (format 7-4); or

[0119] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-VH2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-linker-VL2 (format 7-5); or

[0120] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV1-linker-VH2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-VL1-linker-VL2 (format 7-6); or

[0121] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRAV1-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH1-linker-VL2 (format 7-7).

[0122] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH1-linker-VH2-linker TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-VL2-linker-VL1-linker-TRAV1 (format 11).

[0123] In some embodiments, the polypeptide molecule comprises a derivative form of format 11, in which the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2) constituting the binding region can be interchanged and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. For example, VH1 and VL1 in format 11 can be interchanged, and / or VH2 and VL2 can be interchanged, and / or TRAV1 and TRBV1, and / or TRAV2 and TRBV2 can be interchanged. For example, the order of the first binding region, the second binding region, the third binding region, and the fourth binding region can also be interchanged. Derivative forms of format 11 include, but are not limited to, the following structures:

[0124] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VH1-linker-VH2-linker-TRBV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL2-linker-VL1-linker-TRBV2 (format 11-1); or

[0125] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-VH1-Linker-VL2-Linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-Linker-VH2-Linker-VL1-Linker-TRAV1 (format 11-2); or

[0126] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VH1-linker-VL2-linker-TRBV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH2-linker-VL1-linker-TRBV2 (format 11-3); or

[0127] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2-linker-TRAV2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VL2-linker-TRAV1-linker-VL1 (format 11-4); or

[0128] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV2-linker-VH2-linker-TRBV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VL2-linker-TRBV2-linker-VL1 (format 11-5); or

[0129] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VL2-linker-TRAV2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VH2-linker-TRAV1-linker-VL1 (format 11-6); or

[0130] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV2-linker-VL2-linker-TRBV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH2-linker-TRBV2-linker-VL1 (format 11-7); or

[0131] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV2-linker-VH2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV1-linker-VL1 (format 11-8); or

[0132] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-TRAV2-linker-VL2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV2-linker-TRAV1-linker-VL1 (format 11-9); or

[0133] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV2-linker-TRBV1-linker-VH2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV1-linker-TRBV2-linker-VL1 (format 11-10); or

[0134] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRAV2-linker-TRBV1-linker-VL2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV1-linker-TRBV2-linker-VL1 (format 11-11); or

[0135] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV2-linker-VH2-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-VL2-linker-TRBV2-linker-VH1 (format 11-12); or

[0136] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRAV2-linker-TRBV1-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRAV1-linker-TRBV2-linker-VH1 (format 11-13).

[0137] In some embodiments, the polypeptide molecule comprises a derivative form of format 11, in which TRAV1 and TRBV1 or TRAV2 and TRBV2 are replaced by the CH3 / CH3, CH1 / CL, or TRAC / TRBC of an antibody, and the domains can be interchanged as long as the ability to bind to the corresponding antigen is retained. For example, TRAV1 and TRBV1 or TRAV2 and TRBV2 in format 11 can be replaced by the CH3 / CH3 of an antibody. Derivative forms of format 11 include, but are not limited to, the following structures:

[0138] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VH1-linker-VH2-linker TRAV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VL2-linker-VL1-optional linker-CH3 (format 11-14); or

[0139] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VH1-linker-VH2-linker TRBV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VL2-linker-VL1-optional linker-CH3 (format 11-15); or

[0140] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VH1-linker-VL2-linker TRAV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VH2-linker-VL1-optional linker-CH3 (format 11-16); or

[0141] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VH1-linker-VL2-linker TRBV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH2-linker-VL1-optional linker-CH3 (format 11-17); or

[0142] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-optional linker-CH3-linker-VH2-linker TRAV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VL2-optional linker-CH3-linker-VL1 (format 11-18); or

[0143] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-optional linker-CH3-linker-VH2-linker TRBV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VL2-optional linker-CH3-linker-VL1 (format 11-19); or

[0144] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-optional linker-CH3-linker-VL2-linker TRAV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VH2-optional linker-CH3-linker-VL1 (format 11-20); or

[0145] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-optional linker-CH3-linker-VL2-linker TRBV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH2-optional linker-CH3-linker-VL1 (format 11-21); or

[0146] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV-linker-VH2-optional linker CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VL2-linker-TRAV-linker-VL1 (format 11-22); or

[0147] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRAV-linker-VH2-optional linker CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VL2-linker-TRBV-linker-VL1 (format 11-23); or

[0148] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV-linker-VL2-optional linker CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VH2-linker-TRAV-linker-VL1 (format 11-24); or

[0149] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRAV-linker-VL2-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VH2-linker-TRBV-linker-VL1 (format 11-25); or

[0150] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-optional linker-CH3-linker-TRAV-linker VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV-optional linker-CH3-linker-VL1 (format 11-26); or

[0151] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-optional linker-CH3-linker-TRAV-linker VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRBV-optional linker-CH3-linker-VL1 (format 11-27); or

[0152] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-optional linker-CH3-linker-TRBV-linker VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRAV-optional linker-CH3-linker-VL1 (format 11-28); or

[0153] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-optional linker-CH3-linker-TRBV-linker VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRAV-optional linker-CH3-linker-VL1 (format 11-29); or

[0154] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-optional linker-CH3-linker-VH2-linker TRAV, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VL2-optional linker-CH3-linker-VH1 (format 11-30); or

[0155] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRAV-linker-VH2-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-VL2-linker-TRBV-linker-VH1 (format 11-31); or

[0156] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-optional linker-CH3-linker-TRBV-linker VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRAV-optional linker-CH3-linker-VH1 (format 11-32); or

[0157] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH1-linker-CH3-linker TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-CH3-linker-VL1-linker-TRAV1 (format 11-33).

[0158] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein VH1 and VL1, or VH2 and VL2 are operably linked together and connected to the C-terminus of one of the polypeptide chains via the N-terminus.

[0159] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-linker-VH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV2-linker-VL1-linker-VL2-linker-VH2 (format 12).

[0160] In some embodiments, the polypeptide molecule comprises a derivative form of format 12, in which the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2) constituting the binding region can be interchanged and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. For example, VH1 and VL1 in format 12 can be interchanged, and / or VH2 and VL2 can be interchanged, and / or TRAV1 and TRBV1, and / or TRAV2 and TRBV2 can be interchanged. For example, the order of the first binding region, the second binding region, the third binding region, and the fourth binding region can also be interchanged. Derivative forms of format 12 include, but are not limited to, the following structures:

[0161] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV2-linker-VH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VL1-linker-VH2-linker-VL2 (format 12-1); or

[0162] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-linker-VL1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV2-linker-VH1-linker-VH2-linker-VL2 (format 12-2); or

[0163] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-TRAV2-Linker-VL1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-TRBV2-Linker-VH1-Linker-VH2-Linker-VL2 (format 12-3); or

[0164] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VL1-linker-TRAV2-linker-VH2-linker-VL2 (format 12-4); or

[0165] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-VH1-Linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-VL1-Linker-TRBV2-Linker-VH2-Linker-VL2 (format 12-5); or

[0166] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-VL1-Linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-VH1-Linker-TRAV2-Linker-VH2-Linker-VL2 (format 12-6); or

[0167] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-VL1-Linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-VH1-Linker-TRBV2-Linker-VH2-Linker-VL2 (format 12-7); or

[0168] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-VL1-Linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-VH1-Linker-TRAV2-Linker-VH2-Linker-VL2 (format 12-8); or

[0169] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-CH3-Linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-Linker-CH3-Linker-TRAV1-Linker-VH2-Linker-VL2 (format 12-9); or

[0170] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRAC-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRBC-linker-TRAV1-linker-VH2-linker-VL2 (format 12-10).

[0171] In some embodiments, the polypeptide molecule further comprises a TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC).

[0172] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and wherein VH1 and VL1, and / or VH2 and VL2 are operably linked together and connected to the C-terminus of one of the polypeptide chains via the N-terminus, or connected to the N-terminus of one of the polypeptide chains via the C-terminus.

[0173] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0174] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL2-linker-VH2 (format 15).

[0175] In some embodiments, the polypeptide molecule comprises a derivative of format 15. In this derivative, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAC, and TRBC) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative of format 15, TRAC and TRBC are replaced by the CH3 / CH3 or CH1 / CL of the antibody. Preferably, the derivative of format 15 may also include a cysteine-containing peptide segment. For example, derivatives of format 15 include, but are not limited to, the following structures:

[0176] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC-linker-VL2-linker-VH2 (format 15-1); or

[0177] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-TRAV1-optional linker-TRAC (format 15-2); or

[0178] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CH3-linker-VL2-linker-VH2 (format 15-3); or

[0179] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-CH3-linker-VL2-linker-VH2 (format 15-4); or

[0180] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-TRAV1-optional linker-CH3 (format 15-5); or

[0181] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CL-linker-VL2-linker-VH2 (format 15-6); or

[0182] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRAV1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-CL-linker-VL2-linker-VH2 (format 15-7); or

[0183] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-TRAV1-optional linker-CL (format 15-8); or

[0184] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL2-linker-TRAV1-linker-optional linker-CL-optional linker-cysteine-containing peptide segment (format 15-9); or

[0185] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH2-linker-VL2 (format 15-10).

[0186] In some embodiments, the polypeptide molecule further comprises a CH3 domain on both polypeptide chains, wherein the CH3 domain is derived from the CH3 domain of an antibody, and the CH3 in any one or both polypeptide chains comprises at least one mutation that can promote the polypeptide molecule to form a heterodimer.

[0187] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the domains contained in at least one of the binding regions formed by VH1 and VL1, VH2 and VL2, TRAV1 and TRBV1, and optionally TRAV2 and TRBV2 are distributed on different polypeptide chains.

[0188] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0189] The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-CH3 (format 30).

[0190] In some embodiments, the polypeptide molecule comprises a derivative of format 30. In this derivative, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in a derivative of format 30, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, a derivative of format 30 may also include a cysteine-containing peptide. For example, derivatives of format 30 include, but are not limited to, the following structures:

[0191] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VL2-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2-optional linker-CH3 (format 30-1); or

[0192] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL2-linker-TRAV1-optional linker-TRAC (format 30-2); or

[0193] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VL2-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2-optional linker-TRAC (format 30-3); or the first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL2-linker-TRAV1-optional linker-CL (format 30-4); or

[0194] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VL2-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2-optional linker-CL (format 30-5); or

[0195] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 30-6); or

[0196] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VL2-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 30-7); or

[0197] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL2-linker-TRAV1-optional linker-CH3 (format 30-8); or

[0198] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VL2-linker-VH1-optional linker-CH3 (format 30-9); or

[0199] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL2-linker-TRAV1-optional linker-TRBC (format 30-10); or

[0200] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VL2-linker-VH1-optional linker-TRBC (format 30-11); or

[0201] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL2-linker-TRAV1-optional linker-CL (format 30-12); or

[0202] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-TRBV1-Linker-VL1-Optional Linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-Linker-VL2-Linker-VH1-Optional Linker-CL (format 30-13); or

[0203] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VL2-linker-TRAV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 30-14); or

[0204] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VL2-linker-VH1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 30-15) or

[0205] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-TRAC (format 30-16); or

[0206] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-TRBC-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-TRAC-optional linker-cysteine-containing peptide segment (format 30-17).

[0207] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and the domains contained in at least one of the binding regions formed by VH1 and VL1, VH2 and VL2, TRAV1 and TRBV1, and optionally TRAV2 and TRBV2 are operably linked together and connected to the C-terminus of one of the polypeptide chains via the N-terminus, or connected to the N-terminus of one of the polypeptide chains via the C-terminus.

[0208] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH3-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-CH3 (format 32).

[0209] In some embodiments, the polypeptide molecule comprises a derivative of format 32. In this derivative, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in a derivative of format 32, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, a derivative of format 32 may also include a cysteine-containing peptide. For example, derivatives of format 32 include, but are not limited to, the following structures:

[0210] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-TRBC-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-TRAC (format 32-1); or

[0211] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-Linker-VL1-Optional Linker-CH1-Linker-TRBV1-Linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-Linker-VH2-Optional Linker-CL (format 32-2); or

[0212] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-CH3-linker-TRBV1-linker-TRAV1 (format 32-3); or

[0213] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-TRAC-linker-TRBV1-linker-TRAV1 (format 32-4); or

[0214] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-CL-linker-TRBV1-linker-TRAV1 (format 32-5); or

[0215] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3-linker-VH2-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH3 (format 32-6); or

[0216] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-TRBC-linker-VH2-linker-VL2 (format 32-7); or

[0217] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CL, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH1-linker-VH2-linker-VL2 (format 32-8); or

[0218] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH3-linker-VH2-linker-VL2 (format 32-9); or

[0219] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-TRAC-linker-VH2-linker-VL2 (format 32-10); or

[0220] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CL-linker-VH2-linker-VL2 (format 32-11); or

[0221] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-linker-VL1-linker-TRBV1-optional linker-CH3 (format 32-12); or

[0222] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-linker-VL1-linker-TRBV1-optional linker-TRBC (format 32-13); or

[0223] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-linker-VL1-linker-TRBV1-optional linker-CL (format 32-14); or

[0224] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-VL1-linker-TRBV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 32-15); or

[0225] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-TRAV1-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH3 (format 32-16); or

[0226] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-TRBC (format 32-17); or

[0227] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-TRAV1-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CL (format 32-18); or

[0228] The first polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-linker-TRAV1-linker-VH1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 32-19); or

[0229] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-TRAV1-Linker-VH2-Linker-VH1-Optional Linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-Linker-VL2-Optional Linker-CH3 (format 32-20); or

[0230] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-TRAV1-Linker-VH2-Linker-VH1-Optional Linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-Linker-VL2-Optional Linker-TRAC (format 32-21); or

[0231] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-TRAV1-Linker-VH2-Linker-VH1-Optional Linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-Linker-VL2-Optional Linker-CL (format 32-22); or

[0232] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-VH2-linker-VH1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 32-23); or

[0233] The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-VL1-linker-VL2-optional linker-CH3 (format 32-24); or

[0234] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-VL1-linker-VL2-optional linker-TRAC (format 32-25); or

[0235] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-VL1-linker-VL2-optional linker-CL (format 32-26); or

[0236] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-VL1-linker-VL2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 32-27); or

[0237] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3-linker-TRBV2-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH3 (format 32-28); or

[0238] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-optional linker-CH3-linker-VH2-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV1-optional linker-CH3 (format 32-29); or

[0239] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL1-linker-TRBV1-optional linker-CH3 (format 32-30); or

[0240] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRBC-linker-TRBV2-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV2-optional linker-TRAC (format 32-31); or

[0241] The first polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL1-linker-TRAV1-linker-TRBV2-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-CH3 (format 32-45).

[0242] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-optional linker-CH3-linker-VH2-linker-VL2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3 (format 35).

[0243] In some embodiments, the polypeptide molecule comprises a derivative of format 35. In this derivative, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in a derivative of format 35, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, a derivative of format 35 may also include a cysteine-containing peptide. For example, derivatives of format 35 include, but are not limited to, the following structures:

[0244] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-TRBC-linker-VH2-linker-VL2 (format 35-1); or

[0245] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CL-linker-VH2-linker-VL2 (format 35-2); or

[0246] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3-linker-VH2-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL1-optional linker-CH3 (format 35-3); or

[0247] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-TRAC-linker-VH2-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL1-optional linker-TRBC (format 35-4); or

[0248] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH1-linker-VH2-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL1-optional linker-CL (format 35-5).

[0249] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and the domains contained in at least one of the binding regions formed by VH1 and VL1, VH2 and VL2, TRAV1 and TRBV1, and TRAV2 and TRBV2 are operably linked together and connected to the C-terminus of one of the polypeptide chains via the N-terminus, or connected to the N-terminus of one of the polypeptide chains via the C-terminus.

[0250] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH3-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-CH3-linker-TRBV2-linker-TRAV2 (format 37).

[0251] In some embodiments, the polypeptide molecule comprises a derivative of format 37. In this derivative, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative of format 37, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, the derivative of format 37 may also include a cysteine-containing peptide segment. For example, derivatives of format 37 include, but are not limited to, the following structures:

[0252] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VH2-optional linker-TRAC-linker-TRBV1-linker-TRBA1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VL1-optional linker-TRBC-linker-TRBV2-linker-TRAV2 (format 37-1); or

[0253] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VH2-optional linker-CH1-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VL1-optional linker-CL-linker-TRBV2-linker-TRAV2 (format 37-2); or

[0254] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3-linker-TRBV2-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH3-linker-VL2-linker-VH2 (format 37-3)

[0255] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRAC-linker-VL2-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-TRBC-linker-TRBV2-linker-TRAV2 (format 37-4); or

[0256] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH1-linker-VL2-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CL-linker-TRBV2-linker-TRAV2 (format 37-5); or

[0257] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3-linker-VH1-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH3-linker-TRAV2-linker-TRBV2 (format 37-6); or

[0258] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRAC-linker-VH1-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-TRBC-linker-TRAV2-linker-TRBV2 (format 37-7); or

[0259] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH1-linker-VH1-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CL-linker-TRAV2-linker-TRBV2 (format 37-8); or

[0260] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-linker-CH3-linker-VL2-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-linker-CH3-linker-TRAV2-linker-TRBV2 (format37-9).

[0261] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VH1-linker-VL1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VL2-linker-VH2-optional linker-CH3 (format 39).

[0262] In some embodiments, the polypeptide molecule comprises a derivative form of format 39. In this derivative form, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 39, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, the derivative form of format 39 may also include a cysteine-containing peptide segment. For example, derivative forms of format 39 include, but are not limited to, the following structures:

[0263] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VH1-linker-VL1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VL2-linker-VH2-optional linker-TRBC (format 39-1).

[0264] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-VH1-linker-VL1-optional linker-CH3-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VL2-linker-VH2-optional linker-CH3-linker-TRAV2 (format 40).

[0265] In some embodiments, the polypeptide molecule comprises a derivative form of format 40. In this derivative form, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 40, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, the derivative form of format 40 may also include a cysteine-containing peptide segment. For example, derivative forms of format 40 include, but are not limited to, the following structures:

[0266] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VL1-linker-VH1-optional linker-CH3-linker-TRAV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-VL2-linker-VH2-optional linker-CH3-linker-TRAV2 (format 40-1).

[0267] In some embodiments, format 40-1 comprises the following amino acid sequence:

[0268] >TRBV-G4S2-VL3-G4S3-VH3-CH3 Hole-G4S3-TRAV

[0269] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and the domains contained in at least one of the binding regions formed by VH1 and VL1, VH2 and VL2, TRAV1 and TRBV1, and TRAV2 and TRBV2 are operably linked together.

[0270] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the two polypeptide chains of the bispecific polypeptide molecule respectively comprise:

[0271] VL1-linker-TRAV1-linker-TRBV1-linker-VH1 and VL2-linker-TRAV2-linker-TRBV2-linker-VH2;

[0272] VL1-linker-TRBV1-linker-TRAV1-linker-VH1 and VL2-linker-TRBV2-linker-TRAV2-linker-VH2;

[0273] VH1-linker-TRAV1-linker-TRBV1-linker-VL1 and VH2-linker-TRAV2-linker-TRBV2-linker-VL2;

[0274] VH1-linker-TRBV1-linker-TRAV1-linker-VL1 and VH2-linker-TRBV2-linker-TRAV2-linker-VL2;

[0275] VL1-linker-TRAV1-linker-TRBV1-linker-VH1 and VH2-linker-TRAV2-linker-TRBV2-linker-VL2;

[0276] VL1-linker-TRBV1-linker-TRAV1-linker-VH1 and VH2-linker-TRBV2-linker-TRAV2-linker-VL2;

[0277] VH1-Linker-TRAV1-Linker-TRBV1-Linker-VL1 and VL2-Linker-TRAV2-Linker-TRBV2-Linker-VH2; or

[0278] VH1-linker-TRBV1-linker-TRAV1-linker-VL1 and VL2-linker-TRBV2-linker-TRAV2-linker-VH2.

[0279] In some embodiments, the bispecific polypeptide molecule further comprises a domain selected from the following: a TCR constant region or fragment thereof, CH3, or CH1 / CL, connected to the C-terminus of each polypeptide chain via an optional linker. In preferred embodiments, the bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker. In more preferred embodiments, the two polypeptide chains of the bispecific polypeptide molecule comprise: VL1-Linker-TRAV1-Linker-TRBV1-Linker-VH1 and VL2-Linker-TRAV2-Linker-TRBV2-Linker-VH2, respectively; and the bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker. In some embodiments, the TCR constant region is selected from the TCR α chain constant region (TRAC) and the TCR β chain constant region (TRBC). In preferred embodiments, one polypeptide chain of the bispecific polypeptide molecule comprises TRAC and the other polypeptide chain comprises TRBC. In some embodiments, 1-5 amino acids at the N-terminus of TRAC may be substituted. In some embodiments, 1-5 amino acids at the N-terminus of TRBC may be substituted.

[0280] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-CH3 (format 42).

[0281] In some embodiments, the polypeptide molecule comprises a derivative form of format 42. In this derivative form, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 42, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, the derivative form of format 42 may also include a cysteine-containing peptide segment. For example, derivative forms of format 42 include, but are not limited to, the following structures:

[0282] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-TRBC (format 42-1); or

[0283] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-TRBC (format 42-20); or

[0284] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-CL (format 42-2); or

[0285] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 42-3); or

[0286] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VH2-linker-VL2-linker-TRAV2-optional linker-CH3 (format 42-4); or

[0287] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VH2-linker-VL2-linker-TRAV2-optional linker-TRBC (format 42-5); or

[0288] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VH2-linker-VL2-linker-TRAV2-optional linker-CL (format 42-6); or

[0289] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VL2-linker-VH2-linker-TRBV2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 42-7); or

[0290] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-VH1-Linker-VL1-Linker-TRBV1-Optional Linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-Linker-VH2-Linker-VL2-Linker-TRAV2-Optional Linker-CH3 (format 42-8); or

[0291] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-VH1-Linker-VL1-Linker-TRBV1-Optional Linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-Linker-VH2-Linker-VL2-Linker-TRAV2-Optional Linker-TRBC (format 42-9); or

[0292] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-VH1-Linker-VL1-Linker-TRBV1-Optional Linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-Linker-VH2-Linker-VL2-Linker-TRAV2-Optional Linker-CL (format 42-10); or

[0293] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VH1-linker-TRBV1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VL2-linker-VH2-linker-TRBV2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 42-11).

[0294] In some embodiments, derivatives of format 42 lack TRAV1 and TRBV1, or TRAV2 and TRBV2, relative to format 42. The domains may be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in derivatives of format 42, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, derivatives of format 42 may also include cysteine-containing peptides. For example, derivatives of format 42 include, but are not limited to, the following structures:

[0295] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV-linker-TRAV-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-optional linker-CH3 (format 42-12); or

[0296] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV-linker-TRAV-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-optional linker-TRBC (format 42-13); or

[0297] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV-linker-TRAV-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-optional linker-CL (format 42-14); or

[0298] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV-linker-TRBV-linker-VH1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 42-15); or

[0299] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH1-linker-VL1-linker-TRBV-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-optional linker-CH3 (format 42-16); or

[0300] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH1-linker-VL1-linker-TRBV-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-optional linker-TRBC (format 42-17); or

[0301] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VH1-linker-VL1-linker-TRBV-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-optional linker-CL (format 42-18); or

[0302] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV-linker-VL1-linker-VH1-linker-TRBV-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-VH2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 42-19).

[0303] In a preferred embodiment, 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: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-TRBC (format 42-20).

[0304] In other preferred 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: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-TRAC.

[0305] In other preferred 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: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-TRBC.

[0306] In other preferred 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: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-TRAC.

[0307] In other preferred 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: VH1-linker-TRAV1-linker-TRBV1-linker-VL1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV2-linker-TRBV2-linker-VL2-optional linker-TRBC.

[0308] In other preferred 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: VH1-linker-TRAV1-linker-TRBV1-linker-VL1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV2-linker-TRBV2-linker-VL2-optional linker-TRAC.

[0309] In other preferred 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: VH1-linker-TRBV1-linker-TRAV1-linker-VL1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV2-linker-TRAV2-linker-VL2-optional linker-TRBC.

[0310] In other preferred 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: VH1-linker-TRBV1-linker-TRAV1-linker-VL1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV2-linker-TRAV2-linker-VL2-optional linker-TRAC.

[0311] In other preferred 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: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV2-linker-TRBV2-linker-VL2-optional linker-TRBC.

[0312] In other preferred 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: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV2-linker-TRBV2-linker-VL2-optional linker-TRAC.

[0313] In other preferred 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: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV2-linker-TRAV2-linker-VL2-optional linker-TRBC.

[0314] In other preferred 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: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRBV2-linker-TRAV2-linker-VL2-optional linker-TRAC.

[0315] In other preferred 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: VH1-linker-TRAV1-linker-TRBV1-linker-VL1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-TRBC.

[0316] In other preferred 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: VH1-linker-TRAV1-linker-TRBV1-linker-VL1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-TRAC.

[0317] In other preferred 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: VH1-linker-TRBV1-linker-TRAV1-linker-VL1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-TRBC.

[0318] In other preferred 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: VH1-linker-TRBV1-linker-TRAV1-linker-VL1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-TRAC.

[0319] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH2-linker-VL2-optional linker-CH3-linker-TRBV, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV-optional linker-CH3-linker-VH1 (format 54).

[0320] In some embodiments, the polypeptide molecule comprises a derivative form of format 54. In this derivative form, the positions of the various domains (VH1, VL1, VH2, VL2, TRAV, TRBV, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 54, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, the derivative form of format 54 may also include a cysteine-containing peptide segment. For example, derivative forms of format 54 include, but are not limited to, the following structures:

[0321] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH2-linker-VL2-optional linker-TRBC-linker-TRBV, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV-optional linker-TRAC-linker-VH1 (format 54-1); or

[0322] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH2-linker-VL2-optional linker-CH1-linker-TRBV, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV-optional linker-CL-linker-VH1 (format 54-2); or

[0323] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV-linker-TRBV-optional linker-CH3-linker-VL2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-optional linker-CH3-linker-VH1 (format 54-3); or

[0324] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV-linker-TRBV-optional linker-TRBC-linker-VL2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-optional linker-TRAC-linker-VH1 (format 54-4); or

[0325] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV-linker-TRBV-optional linker-CH1-linker-VL2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH2-optional linker-CL-linker-VH1 (format 54-5); or

[0326] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV-Linker-VH1-Linker-VL1-Optional Linker-CH3-Linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-Optional Linker-CH3-Linker-TRAV (format 54-6); or

[0327] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VH1-linker-VL1-optional linker-TRBC-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-optional linker-TRAC-linker-TRAV (format 54-7); or

[0328] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV-linker-VH1-linker-VL1-optional linker-CH1-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH2-optional linker-CL-linker-TRAV (format 54-8).

[0329] In some embodiments, the polypeptide molecule further comprises a hinge region, CH2 and CH3 on the two polypeptide chains, wherein the hinge region is derived from the hinge domain of an antibody, the CH2 and CH3 are derived from the Fc domain of an antibody, and the CH3 in any one polypeptide chain or both polypeptide chains comprises at least one mutation that can promote the polypeptide molecule to form a heterodimer.

[0330] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0331] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-hinge region-CH2-CH3; and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-hinge region-CH2-CH3 (format 44).

[0332] In some embodiments, the polypeptide molecule comprises a derivative of format 44, in which the domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2) that comprise the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. For example, derivatives of format 44 include, but are not limited to, the following structures:

[0333] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VH2-linker-VL2-linker-TRAV2-optional linker-hinge region-CH2-CH3 (format 44-1).

[0334] In some embodiments, derivatives of format 44 lack TRAV1 and TRBV1, or TRAV2 and TRBV2, relative to format 44, and the domains can be interchanged as long as the ability to bind to the corresponding antigen is retained. For example, derivatives of format 44 include, but are not limited to, the following structures:

[0335] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV-linker-TRAV-linker-VH1-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-VH2-optional linker-hinge region-CH2-CH3 (format 44-2).

[0336] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0337] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-hinge region-CH2-CH3-linker-VH2-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-hinge region-CH2-CH3-linker-TRBV2-linker-TRAV2 (format 20).

[0338] In some embodiments, the polypeptide molecule comprises a derivative of format 20. In this derivative, the domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2) can be interchanged and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative of format 20, the two hinge domains - CH2-CH3 - can be replaced by the CH1 / CL or CH3 / CH3 of an antibody, or the TRAC / TRBC of a TCR. Preferably, this derivative of format 20 also includes a cysteine-containing peptide segment.

[0339] In some embodiments, the polypeptide molecule further comprises a cysteine-containing peptide segment on the two polypeptide chains that is capable of forming a disulfide bond between the two polypeptide chains, wherein the cysteine-containing peptide segment is 3-10 amino acids in length, preferably 4-8 amino acids in length, such as 4 or 6 amino acids in length, and

[0340] (1) a CH3 domain, wherein the CH3 domain is derived from the CH3 domain of an antibody, and the CH3 of any one polypeptide chain or both polypeptide chains comprises at least one mutation that promotes the formation of heterodimers of the polypeptide molecule; or

[0341] (2) a hinge region, CH2, and CH3, wherein the hinge region is derived from the hinge domain of an antibody, the CH2 and CH3 are derived from the Fc domain of an antibody, and the CH3 in any one polypeptide chain or both polypeptide chains contains at least one mutation that can promote the formation of heterodimers of the polypeptide molecules.

[0342] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0343] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3-linker-VL2-linker

[0344] -TRBV2-optional linker-cysteine-containing peptide, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH3-linker-TRAV2-linker-VH2-optional linker-cysteine-containing peptide (format 36).

[0345] In some embodiments, the polypeptide molecule comprises a derivative of format 36, in which the positions of the domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in a derivative of format 36, the two CH3 domains can be replaced by the CL / CH1 of an antibody or the TRAC / TRBC of a TCR.

[0346] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3-linker-VH2-linker-VH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-CH3-linker-VL1-linker-VL2-optional linker-cysteine-containing peptide segment (format 38).

[0347] In some embodiments, the polypeptide molecule comprises a derivative form of format 38, in which the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in a derivative form of format 38, the two CH3 domains can be replaced by the CL / CH1 of an antibody or the TRAC / TRBC of a TCR. For example, derivative forms of format 38 include, but are not limited to, the following structures:

[0348] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-TRAC-linker-VH1-linker-VH2-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-TRBC-linker-VL2-linker-VH2-optional linker-cysteine-containing peptide segment (format 38-1); or

[0349] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH1-linker-VH1-linker-VH2-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-CL-linker-VL2-linker-VH2-optional linker-cysteine-containing peptide segment (format 38-2); or

[0350] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3-linker-TRBV2-linker-VL2-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-optional linker-CH3-linker-VH2-linker-TRAV2-optional linker-cysteine-containing peptide segment (format 38-3); or

[0351] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-TRAC-linker-TRBV2-linker-VL2-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-optional linker-TRBC-linker-VH2-linker-TRAV2-optional linker-cysteine-containing peptide segment (format 38-4); or

[0352] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH1-linker-TRBV2-linker-VL2-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL1-optional linker-CL-linker-VH2-linker-TRAV2-optional linker-cysteine-containing peptide segment (format 38-5).

[0353] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-hinge region-CH2-CH3-linker-VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-hinge region-CH2-CH3-linker-TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment (format 21).

[0354] In some embodiments, the polypeptide molecule comprises a derivative of format 21. In this derivative, the domains comprising the binding region (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2) can be interchanged, and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative of format 21, the two hinge regions (CH2-CH3) can be replaced by the CL / CH1 or CH3 / CH3 structure of an antibody, or the TRAC / TRBC structure of a TCR.

[0355] In some embodiments, the polypeptide molecule further comprises a TCR constant region or a fragment thereof and CL and CH1, wherein the TCR constant region, for example, comprises a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC), the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain.

[0356] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0357] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-CL-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-CH1; and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-optional linker-CL-linker-TRBV1-optional linker-TRBC-linker-VH2-optional linker-CH1 (format 25).

[0358] In some embodiments, the polypeptide molecule comprises a derivative form of format 25, in which the positions of the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, CL, CH1, TRAC, TRBC) can be interchanged and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 25, each of the two pairs of CL / CH1 is independently replaced by the CH3 / CH3 of the antibody or the TRAC / TRBC of the TCR. In some embodiments, in the derivative form of format 25, TRAC and TRBC are replaced by the CH3 / CH3 or CL / CH1 of the antibody. Preferably, the derivative form of format 25 may also include a cysteine-containing peptide segment. For example, derivative forms of format 25 include, but are not limited to, the following structures:

[0359] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-CL-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-optional linker CH3-linker-TRBV1-optional linker-TRBC-linker-VH2-optional linker-CH3 (format 25-1); or

[0360] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-TRBC-linker-TRAV1-optional linker-TRAC-linker-VH1-optional linker-TRAC; and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-optional linker TRBC-linker-TRBV1-optional linker-TRBC-linker-VH2-optional linker-TRAC (format 25-2).

[0361] In some embodiments, the polypeptide molecule comprises one or more characteristics selected from the group consisting of:

[0362] (a) the TRAC and / or TRBC comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, preferably, the cysteine ​​mutation is at position 48 of the wild-type TCR α chain constant region and position 57 of the wild-type TCR β chain constant region;

[0363] (b) the CH3 each independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407 and the two CH3s do not comprise the same mutation, preferably, one of the CH3s comprises a T366W mutation and the other comprises T366S, L368A and Y407V mutations;

[0364] (c) said CH2 and CH3 are derived from the Fc domain of human IgG1, IgG2 or IgG4 or a portion thereof, and preferably contain at least two additional cysteine ​​residue mutations, such as S354C and Y349C or L242C and K334C;

[0365] (d) the hinge region is derived from the hinge domain of human IgG1, IgG2 or IgG4, or a portion thereof;

[0366] (e) The cysteine-containing peptide is derived from an antibody hinge domain, such as the C-terminus of a kappa or lambda chain or the hinge region of IgG1; preferably, the cysteine-containing peptide is selected from SSGGC (SEQ ID NO: 259), LGGC (SEQ ID NO: 260), FNRGEC (SEQ ID NO: 261), VAPTEC (SEQ ID NO: 262) and VEPKSC (SEQ ID NO: 263).

[0367] In some embodiments, TRAC and TRBC can be human or murine. TRAC and TRBC can be wild-type or derivatives thereof. For example, a derivative form of TRAC can include one or more of T48C, N113K, PESS deletion mutation, or FFPSPESS deletion mutation relative to the wild-type sequence. For example, a derivative form of TRBC can include one or more of S57C, C187A, N210D, or FG loop deletion mutation relative to the wild-type sequence.

[0368] In some embodiments, CL can comprise a terminal NEC deletion mutation relative to the wild-type sequence. In some embodiments, CH1 can comprise a terminal VPRDC deletion mutation relative to the wild-type sequence.

[0369] In some embodiments, the polypeptide molecule comprises the first binding region, the second binding region, the third binding region, and when present, the fourth binding region on four polypeptide chains.

[0370] In some embodiments, the polypeptide molecule further comprises one or more of the following structures:

[0371] (1) a CH3 domain, wherein the CH3 domain is derived from the CH3 domain of an antibody, and the CH3 of any one polypeptide chain or both polypeptide chains comprises at least one mutation that promotes the formation of heterotetramers of the polypeptide molecules;

[0372] (2) a TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC);

[0373] (3) a hinge region, CH2, and CH3, wherein the hinge region is derived from an antibody hinge domain, the CH2 and CH3 are derived from an antibody Fc domain, and the CH3 in any one or both polypeptide chains contains at least one mutation that promotes the formation of heterotetramers by the polypeptide molecule;

[0374] (4) CL and CH1, wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain;

[0375] (5) Cysteine-containing peptide segments, wherein the cysteine-containing peptide segments are capable of forming a disulfide bond between two polypeptide chains and are 3-10 amino acids in length, preferably 4-8 amino acids in length, such as 4 or 6 amino acids.

[0376] In some embodiments, the polypeptide molecule comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein:

[0377] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1, the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3, the third polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV2, and the fourth polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-CH3 (format 41).

[0378] In some embodiments, the polypeptide molecule comprises a derivative of format 41. In this derivative, the positions of the domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative of format 41, the two CH3 domains can be replaced by the CL / CH1 of an antibody or the TRAC / TRBC of a TCR. Preferably, this derivative of format 41 also includes a cysteine-containing peptide.

[0379] In some embodiments, the polypeptide molecule comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein:

[0380] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-hinge region-CH2-CH3, the third polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment-optional linker-hinge region-CH2-CH3 (format 45).

[0381] In some embodiments, the polypeptide molecule comprises a derivative form of format 45. In this derivative form, the various domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2) that comprise the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 45, the two hinge regions -CH2-CH3 can be replaced by CL / CH1 or CH3 / CH3 of an antibody, or TRAC / TRBC of a TCR. Preferably, the derivative form of format 45 can also include a cysteine-containing peptide segment. For example, derivative forms of format 45 include, but are not limited to, the following structures:

[0382] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-hinge region-CH2-CH3, the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment-optional linker-hinge region-CH2-CH3 (format 45-1); or

[0383] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-CH3, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment-optional linker-CH3, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment (format 45-2); or

[0384] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-TRAC, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment-optional linker-TRBC, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment (format 45-3); or

[0385] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-CH1, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment-optional linker-CL, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment (format 45-4); or

[0386] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-CH1-optional linker-cysteine-containing peptide segment, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the third polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment-optional linker-CL-optional linker-cysteine-containing peptide segment, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment (format 45-5); or

[0387] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-CH3, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the third polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment-optional linker-CH3, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment (format 45-6); or

[0388] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-TRAC, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the third polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV2-optional linker-cysteine-containing peptide segment-optional linker-TRBC, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VL2-optional linker-cysteine-containing peptide segment (format 45-7); or

[0389] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-CH1, the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the third polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-TRAV2-optional linker-cysteine-containing peptide segment-optional linker-CL, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VL2-optional linker-cysteine-containing peptide segment (format 45-8); or

[0390] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-CH1-optional linker-cysteine-containing peptide segment, the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the third polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRAV2-optional linker-cysteine-containing peptide segment-optional linker-CL-optional linker-cysteine-containing peptide segment, and the fourth polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-VL2-optional linker-cysteine-containing peptide segment (format 45-9).

[0391] In some embodiments, the polypeptide molecule comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein:

[0392] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL2-optional linker-CL, the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-CH1-optional linker-hinge region-CH2-CH3, the third polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VL1-optional linker-TRAC, and the fourth polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV2-optional linker-TRBC-optional linker-hinge region-CH2-CH3 (format 46).

[0393] In some embodiments, the polypeptide molecule comprises a derivative form of format 46. In this derivative form, the positions of the domains (VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CL, CH1, TRAC, and TRBC) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 46, CL / CH1 can be replaced with CH3 / CH3 of an antibody or TRAC / TRBC of a TCR. In some embodiments, in the derivative form of format 46, TRAC / TRBC can be replaced with CL / CH1 or CH3 / CH3 of an antibody or TCR. Preferably, the derivative form of format 46 also includes a cysteine-containing peptide segment.

[0394] In a preferred embodiment of the trispecific polypeptide molecule of the present invention, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0395] The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VL2 (format 7-4); or

[0396] The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL2-linker-VH2 (format 15); or the first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH2-linker-VL2 (format 15-10); or

[0397] The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-CH3 (format 30); or

[0398] The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-TRAC (foramt 30-16); or

[0399] The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL2-linker-TRAV1-optional linker-TRAC (foramt 30-2); or

[0400] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRAC-linker-VL2-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-TRBC-linker-TRBV2-linker-TRAV2 (foramt 37-4); or

[0401] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-TRBC (foramt 42-1); or

[0402] The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-TRBC (foramt 42-20).

[0403] In some embodiments, the polypeptide molecule comprises one or more characteristics selected from the group consisting of:

[0404] (a) the polypeptide molecule comprises a TCR constant region or a fragment thereof, wherein the TCR constant region comprises a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC), wherein the TRAC and / or TRBC comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, preferably, the cysteine ​​mutation is at the following positions: position 48 of the wild-type TCR α chain constant region and position 57 of the wild-type TCR β chain constant region;

[0405] (b) the polypeptide molecule comprises a CH3 domain, wherein the CH3s each independently comprise a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407 and the two CH3s do not comprise the same mutation, preferably, one of the CH3s comprises a T366W mutation and the other comprises T366S, L368A and Y407V mutations;

[0406] (c) the polypeptide molecule comprises CH2 and CH3, wherein the CH2 and CH3 are derived from the Fc domain of human IgG1, IgG2 or IgG4 or a portion thereof, and preferably comprise at least two additional cysteine ​​residue mutations, such as S354C and Y349C or L242C and K334C;

[0407] (d) the polypeptide molecule comprises a hinge region, wherein the hinge region is derived from the hinge domain of human IgG1, IgG2 or IgG4 or a portion thereof;

[0408] (e) the polypeptide molecule comprises a cysteine-containing peptide segment, wherein the cysteine-containing peptide segment is derived from an antibody hinge domain, such as the C-terminus of a kappa or lambda chain or the hinge region of IgG1; preferably, the cysteine-containing peptide segment is selected from SSGGC, LGGC, FNRGEC, VAPTEC and VEPKSC;

[0409] (f) The polypeptide molecule comprises CL and CH1, wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain.

[0410] In the embodiments of the trispecific polypeptide molecules of the present invention, the domains of the polypeptide molecules (e.g., VH1, VL1, VH2, VL2, TRAV1, TRBV1, TRAV2, TRBV2, CL, CH1, CH3, TRAC, TRBC) can be interchanged as long as the ability to bind to the corresponding antigen is retained.

[0411] In some embodiments, VH1 and VL1 comprise at least one cysteine ​​mutation to form a disulfide bond between VH1 and VL1, and the cysteine ​​is introduced into FR4 in the case of VL and into FR2 in the case of VH. In some embodiments, VH2 and VL2 comprise at least one cysteine ​​mutation to form a disulfide bond between VH2 and VL2, and the cysteine ​​is introduced into FR4 in the case of VL and into FR2 in the case of VH.

[0412] In some embodiments, VH1 and VL1 comprise at least one cysteine ​​mutation to form a disulfide bond between VH1 and VL1, preferably, the cysteine ​​mutation is at the following positions: position 44 of VH1 and position 100 of VL1.

[0413] In some embodiments, TRAV1 and / or TRAV2 can be native TRAV or a functional derivative thereof, as long as the functional derivative retains the ability to bind to the target antigen. For example, TRAV1 and / or TRAV2 can be a truncated derivative of native TRAV, such as a derivative with 8 amino acids truncated at the N-terminus or C-terminus.

[0414] In some embodiments, TRBV1 and / or TRBV2 may be native TRBV or a functionally derived form thereof, as long as the functionally derived form retains the ability to bind to the target antigen.

[0415] In some embodiments, the polypeptide molecule further comprises one or more amino acid mutations, insertions or deletions 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 post-translational modification (e.g., glycosylation modification), such as 1, 2, 3, 4, 5 or more amino acid mutations, insertions or deletions. The 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: Ala, Gly. In some embodiments, the amino acid mutations, insertions or deletions are located at the following amino acid positions: the beginning of TRAV, TRBV, VL, VH, the boundary amino acids of TRAV / TRAC, the boundary amino acids of TRBV / TRBC, the boundary amino acids of VL / CL, the boundary amino acids of VH / CH1, the hinge region and its adjacent amino acids, the boundary amino acids of CH2 / CH3, the C-terminus of CH3, and 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 that enhance binding to FcRn include, for example, one or more of the following mutations: L234A, L235A, M252Y, S254T, T256E, M428L, N434S, T250R, M428L, N434A in the CH2 and CH3 domains, preferably L234A L235A, M252Y S254T T256E, M428L N434S and T250R M428L N434A. In some embodiments, the effector function silent 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 silent mutation is generated by replacing at least one residue at positions 233, 234, 235, 236, and 331 with the corresponding residue derived from IgG2 or IgG4. In some embodiments, amino acid mutations that can reduce post-translational modification of the polypeptide molecule include, for example, N113K in TRAC and N210D in TRBC.

[0416] In embodiments of the trispecific polypeptide molecules of the present invention, when the polypeptide molecules comprise more than one linker, the linkers may be identical or different. In some embodiments, the linkers in the polypeptide molecules are each independently a linker consisting of 1-35 amino acids.

[0417] In some embodiments, the linkers in the polypeptide molecule are each independently selected from S, GGGS (SEQ ID NO: 178), GGGGS (SEQ ID NO: 179), GGGSGGGG (SEQ ID NO: 180), GGSGGS (SEQ ID NO: 181), GGSGGSGGS (SEQ ID NO: 182), GGGGSGGGGS (SEQ ID NO: 183), GGGGSGGGGSGGGGS (SEQ ID NO: 184), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 185), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 186), GQPKAAP (SEQ ID NO: 187), TVLRT (SEQ ID NO: 188), TVSSAS (SEQ ID NO: 189), GGEGG (SEQ ID NO: 190), GSEGGGS (SEQ ID NO: 191). NO: 191), GGEGGGSEGGGS (SEQ ID NO: 192), RTSGPGDGGKGGPGKGPGGEGTKGTGPGG (SEQ ID NO: 193), GDGGKGGPGKGPGGEGTKGTGPGG (SEQ ID NO: 194), GKGPGGEGTKGTGPGG (SEQ ID NO: 195), TVLSSAS (SEQ ID NO: 196), EDLKN (SEQ ID NO: 197) or a variant thereof, EDLNK (SEQ ID NO: 198) or a variant thereof, and ANIQK (SEQ ID NO: 199) or a variant thereof, or any combination thereof.

[0418] In some embodiments, the variant of EDLKN is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to ANIQK.

[0419] Preferably, the variant of EDLKN is an amino acid sequence formed by replacing one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by replacing one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by replacing one or several amino acids of ANIQK.

[0420] Preferably, the variant of EDLKN is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLKN; and / or the variant of EDLNK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLNK; and / or the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from ANIQK.

[0421] Preferably, the variant of EDLKN is EDL, DL, L, ED, E, D; and / or the variant of ANIQK is ANI, NI, I, AN, N, A, PNI, P, PN.

[0422] 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 an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to EDLNK (preferably at the N-terminus); and / or the variant of ANIQK is an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to ANIQK (preferably at the N-terminus).

[0423] In some embodiments, the polypeptide molecule comprises one or more domains selected from the group consisting of:

[0424] CH3 domain, TCR constant region or fragment thereof, hinge region, CH2, CH3, CL, CH1, cysteine-containing peptide segment and combinations thereof, when the N-terminus of one or more domains is connected to the C-terminus of its upstream sequence through a linker, the linkers are each independently selected from a linker consisting of no more than 12 amino acids.

[0425] Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, EDLKN or a variant thereof, EDLNK or a variant thereof and ANIQK or a variant thereof.

[0426] In some embodiments, the variant of EDLKN is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to ANIQK.

[0427] Preferably, the variant of EDLKN is an amino acid sequence formed by replacing one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by replacing one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by replacing one or several amino acids of ANIQK.

[0428] Preferably, the variant of EDLKN is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLKN; and / or the variant of EDLNK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLNK; and / or the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from ANIQK.

[0429] Preferably, the variant of EDLKN is EDL, DL, L, ED, E, D; and / or the variant of ANIQK is ANI, NI, I, AN, N, A, PNI, P, PN.

[0430] 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 an amino acid sequence consisting of 6-12 amino acids formed by adding one or several amino acids to EDLNK (preferably at the N-terminus); and / or the variant of ANIQK is an amino acid sequence consisting of 6-12 amino acids formed by adding one or several amino acids to ANIQK (preferably at the N-terminus).

[0431] In some embodiments, the polypeptide molecule comprises TRAC or a fragment thereof, which is not connected to TRAV via a linker. Preferably, 1-5 amino acids at the N-terminus of TRAC may be substituted.

[0432] In some embodiments, the polypeptide molecule comprises TRBC or a fragment thereof, which is not connected to TRBV via a linker. Preferably, 1-5 amino acids at the N-terminus of TRBC may be substituted.

[0433] Unless otherwise indicated, the term "cis-linked" refers to the linkage between the TCR constant region and the variable region in the natural state.

[0434] In some embodiments, the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or more polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain, such as a signal peptide sequence derived from albumin or immunoglobulin, preferably MGWSCIILFLVATATGVHS.

[0435] In some embodiments, the first antigen and the fourth antigen are each independently a TAA selected from the following: melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A and an endogenous reverse transcriptase antigen.

[0436] In some embodiments, the first antigen and the fourth antigen are each independently a TSA selected from the group consisting of KRAS (e.g., G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (e.g., R175H, R173H, R273C, R248W, R248Q, R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (e.g., E542K, E545K, H1047R), CTNNB1 (e.g., S45P, T41A), EGFR (e.g., L858R, T790M), BRAF (e.g., V600E), and GNAS (e.g., R201C, R201H).

[0437] In some embodiments, the first antigen and the fourth antigen are each independently a viral antigen selected from the group consisting of HPV E6 or E7 antigens, CMV antigens, HBV antigens, EBV antigens, herpes virus antigens, human immunodeficiency virus (HIV) antigens, influenza virus antigens, and coronavirus antigens.

[0438] In some embodiments, the first antigen and the fourth antigen are each independently an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Ins ulin, 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.

[0439] In some embodiments, the second antigen and the third antigen are each independently selected from CD3 (e.g., CD3γ, CD3δ, and CD3ε chain), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, , FcεRI, TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD244), HVEM, LAG3, DAP 10. DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT, B7-H3, CTLA-4, PD-1, CD80, BTLA, TIM3, TIGIT and LAG-3.

[0440] In some embodiments, the first antigen and the fourth antigen are each independently a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen; and the second antigen and the third antigen are each independently selected from the group consisting of CD3 (e.g., CD3γ, C D3δ and CD3ε chains), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD 41b, 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(C D244), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT and B7-H3.

[0441] In some embodiments, the first antigen and the fourth antigen are each independently a TSA selected from the group consisting of KRAS (e.g., G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (e.g., R175H, R173H, R273C, R248W, R248Q, R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (e.g., E542K, E545K, H1047R), CTNNB1 (e.g., S45P, T41A), EGFR (e.g., L858R, T790M), BRAF (e.g., V600E), and GNAS (e.g., R201C, R201H); and the second antigen and the third antigen are each independently selected from the group consisting of CD3 (e.g., CD3γ, CD3δ), and CD3ε chain), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41 b. 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(C D244), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT and B7-H3.

[0442] In some embodiments, the first antigen and the fourth antigen are each independently a viral antigen selected from the group consisting of HPV E6 or E7 antigens, CMV antigens, HBV antigens, EBV antigens, herpes virus antigens, human immunodeficiency virus (HIV) antigens, influenza virus antigens, and coronavirus antigens; and the second antigen and the third antigen are each independently 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.

[0443] In some embodiments, the first antigen and the fourth antigen are each independently an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin, 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 and the third antigen are each independently selected from CTLA-4, PD-1, CD80, BTLA, TIM3, TIGIT and LAG-3.

[0444] In some embodiments, the first antigen and the fourth antigen are each independently selected from gp100, MAGEA1, KRAS, and HPVE7.

[0445] In some embodiments, the second antigen and the third antigen are different and each independently is CD3, CD28, or 4-1BB (CD137). In some embodiments, the second antigen is CD3. In some embodiments, the third antigen is CD28. In some embodiments, the second antigen or the third antigen is 4-1BB (CD137). In some embodiments, the second antigen is CD3 and the third antigen is CD28. In some embodiments, the second antigen is CD3 or CD28, and the third antigen is 4-1BB (CD137). In some embodiments, the second antigen is 4-1BB (CD137) and the third antigen is CD3 or CD28. In some embodiments, the second antigen is CD3, and the second binding region comprises VH1 and VL1 derived from an anti-CD3 antibody selected from the group consisting of OKT3, UCHT-1, BMA031, and 12F6. In some embodiments, the third antigen is CD3, and the third binding region comprises VH2 and VL2 derived from an anti-CD3 antibody selected from the group consisting of OKT3, UCHT-1, BMA031, and 12F6.

[0446] In some embodiments, the polypeptide molecule comprises:

[0447] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 11, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 12;

[0448] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 17, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 18;

[0449] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 19, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 20, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 20;

[0450] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:23, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:23, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:24, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:24;

[0451] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:27, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:27, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:28, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:28;

[0452] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:35, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:35, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:36, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:36;

[0453] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:43, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:43, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:44, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:44;

[0454] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:45, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:45, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:46, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:46;

[0455] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:49, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:49, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:50, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:50;

[0456] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:51, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:51, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:52, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:52;

[0457] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:53, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:53, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:54, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:54;

[0458] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:55, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:55, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:56, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:56;

[0459] a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:57, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:57, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:58, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:58; or

[0460] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:59, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:59, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:60, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:60; or

[0461] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 127, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 127, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 128; or

[0462] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 133, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 133, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 134, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 134; or

[0463] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 135, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 135, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 136, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 136; or

[0464] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 137, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 137, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 138, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 138; or

[0465] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 139, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 139, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 140, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 140; or

[0466] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 151, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 151, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 152, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 152; or

[0467] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 157, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 157, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 158, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 158; or

[0468] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 159, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 159, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 160, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 160; or

[0469] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 161, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 161, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 162, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 162; or

[0470] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 163, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 163, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 164, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 164; or

[0471] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 165, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 165, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 166, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 166; or

[0472] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 171, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 171, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 172, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 172; or

[0473] A first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 175, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 175, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 176, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 176.

[0474] In some embodiments, the polypeptide molecule comprises:

[0475] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:61, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:61; a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:62, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:62; a third polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:63, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:63; and a fourth polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:64, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:64.

[0476] In another aspect, the present 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.

[0477] wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

[0478] 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;

[0479] wherein the TRAV1, TRBV1, VH and VL are distributed on the one or more polypeptide chains such that when the one or more polypeptide chains are folded, the TRAV1 and TRBV1 are spatially close to form the first binding region, and the VH and VL are spatially close to form the second binding region;

[0480] The first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and a self-antigen, and the second antigen is an immune cell surface molecule.

[0481] In some embodiments, the TRAV1, TRBV1, VH and VL are distributed on the one or more polypeptide chains and adjacent variable regions on the same chain are separated by a linker sequence.

[0482] In some embodiments, the polypeptide molecule further comprises at least one pair of TRAV2 and TRBV2, which form a third binding region that binds to a third antigen; wherein the third antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the third antigen is the same as or different from the first antigen. In a preferred embodiment, the polypeptide molecule comprises an additional copy of TRAV1 and TRBV1, which form a third binding region that binds to the first antigen.

[0483] In some embodiments, the polypeptide molecule comprises an additional copy of the VH and VL, which form a fourth binding region that binds to the second antigen.

[0484] In some embodiments, the polypeptide molecule comprises the first binding region, the second binding region, when present, the third binding region, and when present, the fourth binding region on one polypeptide chain.

[0485] In some embodiments, the polypeptide molecule does not comprise the constant region of a TCR and does not comprise the hinge domain and Fc domain of an antibody.

[0486] In some embodiments, the polypeptide molecule comprises the following structure from N-terminus to C-terminus:

[0487] TRAV1-linker-TRBV1-linker-VH-linker-VL (format 5).

[0488] In some embodiments, the polypeptide molecule comprises a derivative form of format 5, in which the domains (VH, VL, TRAV1, TRBV1) that make up the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained.

[0489] In some embodiments, the polypeptide molecule comprises the following structure from N-terminus to C-terminus:

[0490] VL-linker-TRAV1-linker-TRBV1-linker-VH (format 6).

[0491] In some embodiments, the polypeptide molecule comprises a derivative form of format 6, in which the domains (VH, VL, TRAV1, TRBV1) that make up the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained.

[0492] In some embodiments, the polypeptide molecule comprises the following structure from N-terminus to C-terminus: TRAV1-linker-VH-linker-TRAV2-linker-TRBV2-linker-VL-linker-TRBV1 (format 10).

[0493] In some embodiments, the polypeptide molecule comprises a derivative of format 10. In this derivative, the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2) that comprise the binding region can be interchanged, and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. Preferably, the derivative of format 10 further comprises the CH3 / CH3, CH1 / CL, or TRAC / TRBC of an antibody. For example, format 10 further comprises CH3 / CH3. For example, derivatives of format 10, from N-terminus to C-terminus, include, but are not limited to, the following structures:

[0494] TRBV1-Linker-VH-Optional Linker-CH3-Linker-TRAV2-Linker-TRBV2-Optional Linker-CH3-Linker-VL-Linker-TRAV1 (format 10-1); or

[0495] TRBV2-optional linker-CH3-linker-VL-linker-TRAV1-linker-TRBV1-linker-VH-optional linker-CH3-linker-TRAV2 (format 10-2); or

[0496] TRAV1-Linker-VH-Optional Linker-CH3-Linker-TRBV2-Linker-TRAV2-Optional Linker-CH3-Linker-VL-Linker-TRBV1 (format 10-3); or

[0497] TRAV2-optional linker-CH3-linker-VL-linker-TRBV1-linker-TRAV1-linker-VH-optional linker-CH3-linker-TRBV2 (format 10-4); or

[0498] TRBV1-Linker-VL-Optional Linker-CH3-Linker TRAV2-Linker-TRBV2-Optional Linker-CH3-Linker-VH-Linker-TRAV1 (format 10-5); or

[0499] TRBV2-optional linker-CH3-linker-VH-linker-TRAV1-linker-TRBV1-linker-VL-optional linker-CH3-linker-TRAV2 (format 10-6); or

[0500] TRAV1-Linker-VL-Optional Linker-CH3-Linker TRBV2-Linker-TRAV2-Optional Linker-CH3-Linker-VH-Linker-TRBV1 (format 10-7); or

[0501] TRAV2-optional linker-CH3-linker-VH-linker-TRBV1-linker-TRAV1-linker-VL-optional linker-CH3-linker TRBV2 (format 10-8); or

[0502] VH-Linker-TRBV1-Optional Linker-CH3-Linker-TRAV2-Linker-TRBV2-Optional Linker-CH3-Linker-TRAV1-Linker-VL (format 10-9); or

[0503] TRBV2-optional linker-CH3-linker-TRAV1-linker-VL-linker VH-linker-TRBV1-optional linker-CH3-linker-TRAV2 (format 10-10); or

[0504] VH-Linker-TRAV1-Optional Linker-CH3-Linker-TRBV2-Linker-TRAV2-Optional Linker-CH3-Linker-TRBV1-Linker-VL (format 10-11); or

[0505] TRAV2-optional linker-CH3-linker-TRBV1-linker-VL-linker-VH-linker-TRAV1-optional linker-CH3-linker-TRBV2 (format 10-12); or

[0506] CH3-Linker-TRBV1-Linker-VH-Linker-TRAV2-TRBV2-Linker-VL-Linker-TRAV1-Optional Linker-CH3 (format 10-13); or

[0507] TRBV2-Linker-VL-Linker-TRAV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRBV1-Linker-VH-Linker-TRAV2 (format 10-14); or

[0508] CH3-Linker-TRAV1-Linker-VH-Linker-TRBV2-Linker-TRAV2-Linker-VL-Linker-TRBV1-Optional Linker-CH3 (format 10-15); or

[0509] TRAV2-Linker-VL-Linker-TRBV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRAV1-Linker-VH-Linker-TRBV2 (format 10-16); or

[0510] CH3-Linker-TRBV1-Linker-VL-Linker-TRAV2-Linker-TRBV2-Linker-VH-Linker-TRAV1-Optional Linker-CH3 (format 10-17); or

[0511] TRBV2-Linker-VH-Linker-TRAV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRBV1-Linker-VL-Linker-TRAV2 (format 10-18); or

[0512] CH3-Linker-TRAV1-Linker-VL-Linker-TRBV2-Linker-TRAV2-Linker-VH-Linker-TRBV1-Optional Linker-CH3 (format 10-19); or

[0513] TRAV2-Linker-VH-Linker-TRBV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRAV1-Linker-VL-Linker-TRBV2 (format 10-20); or

[0514] CH3-Linker-TRBV1-Linker-TRAV2-Linker-VH-Linker-VL-Linker-TRBV2-Linker-TRAV1-Optional Linker-CH3 (format 10-21); or

[0515] VL-Linker-TRBV2-Linker-TRAV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRBV1-Linker-TRAV2-Linker-VH (format 10-22); or

[0516] CH3-Linker-TRBV1-Linker-TRAV2-Linker-VL-Linker-VH-Linker-TRBV2-Linker-TRAV1-Optional Linker-CH3 (format 10-23); or

[0517] VH-Linker-TRBV2-Linker-TRAV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRBV1-Linker-TRAV2-Linker-VL (format 10-24); or

[0518] CH3-Linker-TRAV1-Linker-TRBV2-Linker VH-Linker-VL-Linker-TRAV2-Linker-TRBV1-Optional Linker-CH3 (format 10-25); or

[0519] VL-Linker-TRAV2-Linker-TRBV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRAV1-Linker-TRBV2-Linker VH (format 10-26); or

[0520] CH3-Linker-TRAV1-Linker-TRBV2-Linker VL-Linker-VH-Linker-TRAV2-Linker-TRBV1-Optional Linker-CH3 (format 10-27); or

[0521] VH-Linker-TRAV2-Linker-TRBV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRAV1-Linker-TRBV2-Linker VL (format 10-28); or

[0522] VL-Linker-TRAV1-Optional Linker-CH3-Linker-TRAV2-Linker-TRBV2-Optional Linker-CH3-Linker-TRBV1-Linker-VH (format 10-29); or

[0523] TRBV2-optional linker-CH3-linker-TRBV1-linker-VH-linker-VL-linker-TRAV1-optional linker-CH3-linker-TRAV2 (format 10-30); or

[0524] CH3-Linker-TRAV1-Linker-VH-Linker-TRAV2-Linker-TRBV2-Linker-VL-Linker-TRBV1-Optional Linker-CH3 (format 10-31); or

[0525] TRBV2-Linker-VL-Linker-TRBV1-Optional Linker-CH3-Optional Linker-CH3-Linker-TRAV1-Linker-VH-Linker-TRAV2 (format 10-32).

[0526] In some embodiments, the polypeptide molecule comprises the following structure from N-terminus to C-terminus: TRAV1-linker-TRAC-linker-TRBV2-linker-TRAV2-linker-TRBC-linker-TRBV1-linker-VH1-linker-VL1 (format 12-11).

[0527] In some embodiments, the polypeptide molecule comprises the first binding region, the second binding region, when present, the third binding region, and when present, the fourth binding region on two polypeptide chains.

[0528] In some embodiments, the polypeptide molecule does not comprise the constant region of a TCR and does not comprise the hinge domain and Fc domain of an antibody.

[0529] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0530] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1 (format 1).

[0531] In some embodiments, the polypeptide molecule comprises a derivative form of format 1, in which the domains (VH, VL, TRAV1, TRBV1) that make up the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained.

[0532] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0533] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV1 (format 2).

[0534] In some embodiments, the polypeptide molecule comprises a derivative form of format 2, in which the domains (VH, VL, TRAV1, TRBV1) that make up the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained.

[0535] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0536] The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL (format 3).

[0537] In some embodiments, the polypeptide molecule comprises a derivative form of format 3, in which the domains (VH, VL, TRAV1, TRBV1) that make up the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained.

[0538] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0539] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-linker-VH, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV2-linker-VL (format 8).

[0540] In some embodiments, the polypeptide molecule comprises a derivative form of format 8, in which the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2) that make up the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained.

[0541] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VL-linker-TRBV2 (format 9).

[0542] In some embodiments, the polypeptide molecule comprises a derivative of format 9. In this derivative, the domains comprising the binding region (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2) can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. Preferably, the derivative of format 9 further comprises the CH3 / CH3, CH1 / CL of an antibody, or the TRAC / TRBC of a TCR. For example, format 9 further comprises CH3 / CH3. For example, derivatives of format 9 include, but are not limited to, the following structures:

[0543] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH-optional linker-CH3-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-optional linker-CH3-linker-VL-linker-TRAV1 (format 9-1); or

[0544] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH3-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-CH3-linker-VL-linker-TRBV1 (format 9-2); or

[0545] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VL-optional linker-CH3-linker TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-optional linker-CH3-linker-VH-linker-TRAV1 (format 9-3); or

[0546] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL-optional linker-CH3-linker TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-CH3-linker-VH-linker-TRBV1 (format 9-4); or

[0547] The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV1-optional linker-CH3-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-optional linker-CH3-linker-TRAV1-linker-VL (format 9-5); or

[0548] The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRAV1-optional linker-CH3-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-CH3-linker-TRBV1-linker-VL (format 9-6); or

[0549] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRBV1-linker-VH-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-VL-linker-TRAV1-optional linker-CH3 (format 9-7); or

[0550] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRAV1-linker-VH-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VL-linker-TRBV1-optional linker-CH3 (format 9-8); or

[0551] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRBV1-linker-VL-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-VH-linker-TRAV1-optional linker-CH3 (format 9-9); or

[0552] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRAV1-linker-VL-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VH-linker-TRBV1-optional linker-CH3 (format 9-10); or

[0553] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRBV1-linker-TRAV2-linker-VH, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV2-linker-TRAV1-optional linker-CH3 (format 9-11); or

[0554] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRBV1-linker-TRAV2-linker-VL, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV2-linker-TRAV1-optional linker-CH3 (format 9-12); or

[0555] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRAV1-linker-TRBV2-linker VH, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV2-linker-TRBV1-optional linker-CH3 (format 9-13); or

[0556] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRAV1-linker-TRBV2-linker VL, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRAV2-linker-TRBV1-optional linker-CH3 (format 9-14); or

[0557] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV1-optional linker-CH3-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-optional linker-CH3-linker-TRBV1-linker-VH (format 9-15); or

[0558] The first polypeptide chain comprises from N-terminus to C-terminus: CH3-linker-TRAV1-linker-VH-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-VL-linker-TRBV1-optional linker-CH3 (format 9-16).

[0559] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL-optional linker-cysteine-containing peptide segment (format 4); wherein the cysteine-containing peptide segment is capable of forming a disulfide bond between the first polypeptide chain and the second polypeptide chain, and has a length of 3-10 amino acids, preferably a length of 4-8 amino acids, for example 4 or 6 amino acids.

[0560] In some embodiments, the polypeptide molecule comprises a derivative form of format 4, in which the domains (VH, VL, TRAV1, TRBV1) that make up the binding region can be interchanged and the order of the binding region formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained.

[0561] In some embodiments, the cysteine-containing peptide is derived from an antibody hinge domain, such as the C-terminus of a Kappa or Lambda chain or the hinge region of IgG1; preferably, the cysteine-containing peptide is selected from SSGGC, LGGC, FNRGEC, VAPTEC, and VEPKSC.

[0562] In some embodiments, the polypeptide molecule does not comprise the hinge domain and CH2 domain of an antibody, and the two polypeptide chains comprise a CH3 domain derived from an antibody; wherein the CH3 domains in the two polypeptide chains comprise at least one mutation that can promote the polypeptide molecule to form a heterodimer.

[0563] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the domains contained in the binding region formed by VH and VL as well as TRAV1 and TRBV1 are distributed on different polypeptide chains.

[0564] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the two polypeptide chains of the bispecific polypeptide molecule respectively comprise:

[0565] TRAV-VH and VL-TRBV;

[0566] TRAV-VL and VH-TRBV;

[0567] TRBV-VH and VL-TRAV; or

[0568] TRBV-VL and VH-TRAV;

[0569] Furthermore, adjacent variable regions in the two polypeptide chains are connected via a linker.

[0570] In some embodiments, the bispecific polypeptide molecule further comprises a domain selected from the group consisting of a TCR constant region or fragment thereof, CH3, and CH1 / CL, connected to the C-terminus of each polypeptide chain via an optional linker. In preferred embodiments, the bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker. In more preferred embodiments, the two polypeptide chains of the bispecific polypeptide molecule comprise TRAV-VH and VL-TRBV, respectively, with adjacent variable regions in the two polypeptide chains connected by a linker; and the bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker. In some embodiments, the TCR constant region is selected from the group consisting of a TCR α chain constant region (TRAC) and a TCR β chain constant region (TRBC). In preferred embodiments, one polypeptide chain of the bispecific polypeptide molecule comprises TRAC and the other polypeptide chain comprises TRBC. In some embodiments, 1-5 amino acids at the N-terminus of TRAC may be substituted. In some embodiments, 1-5 amino acids at the N-terminus of TRBC may be substituted.

[0571] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0572] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-CH3 (format 22).

[0573] In some embodiments, the polypeptide molecule comprises a derivative form of format 22. In this derivative form, the positions of the various domains (VH, VL, TRAV1, TRBV1, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in a derivative form of format 22, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. Preferably, the derivative form of format 22 may also include a cysteine-containing peptide segment. For example, derivative forms of format 22 include, but are not limited to, the following structures:

[0574] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV1-optional linker-CH3 (format 22-1); or

[0575] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-TRBC (format 22-2); or

[0576] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV1-optional linker-TRBC (format 22-3); or

[0577] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-CL (format 22-4); or

[0578] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL-optional linker-CL, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV1-optional linker-CH1 (format 22-5); or

[0579] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 22-6); or

[0580] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL-optional linker-CL-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV1-optional linker-CH1-optional linker-cysteine-containing peptide segment (format 22-7).

[0581] In a preferred embodiment, the bispecific polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC (format 22-2).

[0582] In other preferred 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC.

[0583] In other preferred 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC.

[0584] In other preferred 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC.

[0585] In other preferred 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-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC.

[0586] In other preferred 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC.

[0587] In other preferred 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC.

[0588] In other preferred 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC.

[0589] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CL-linker-VH-optional linker-CH3-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-CH1-linker-VL-optional linker-CH3-linker-TRAV2-optional linker-CL (format 23); wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain.

[0590] In some embodiments, the polypeptide molecule comprises a derivative form of format 23. In this derivative form, the positions of the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CL, CH1, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative form of format 23, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. In some embodiments, in this derivative form of format 23, the CH1 / CL can be replaced by the CH3 / CH3 of an antibody or the TRAC / TRBC of a TCR. Preferably, this derivative form of format 23 also includes a cysteine-containing peptide.

[0591] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH-optional linker-CH3-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-TRBC-linker-VL-optional linker-CH3-linker-TRAV2-optional linker-TRAC (format 24); wherein the TRAC is derived from the TCRα chain constant region, and the TRBC is derived from the TCRβ chain constant region.

[0592] In some embodiments, the polypeptide molecule comprises a derivative form of format 24, in which the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, TRAC, TRBC, CH3) can be interchanged and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 24, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. In some embodiments, in the derivative form of format 24, TRAC / TRBC can be replaced by the CH1 / CL or CH3 / CH3 of an antibody. Preferably, the derivative form of format 24 can also contain a cysteine-containing peptide segment. For example, derivative forms of format 24 include, but are not limited to, the following structures:

[0593] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH-optional linker-TRAC-linker-TRAV2-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-TRBC-linker-VL-optional linker-TRBC-linker-TRBV1-optional linker-TRBC (format 24-2).

[0594] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH-optional linker-TRAC-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-TRBC-linker-VL-optional linker-TRBC-linker-TRAV2-optional linker-TRAC (format 24-1).

[0595] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VH-optional linker-CH3 (format 27).

[0596] In some embodiments, the polypeptide molecule comprises a derivative form of format 27, in which the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 27, TRAV1 and TRBV1, or TRAV2 and TRBV2 are replaced by another copy of VH and VL. In some embodiments, in the derivative form of format 27, two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, the derivative form of format 27 may also include a cysteine-containing peptide segment. For example, derivative forms of format 27 include, but are not limited to, the following structures:

[0597] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-TRBV2-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV2-optional linker-CH3 (format 27-1); or

[0598] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-VH-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV1-optional linker-CH3 (format 27-2); or

[0599] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-VH-optional linker-CH3 (format 27-3); or

[0600] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VH-optional linker-TRBC (format 27-4); or

[0601] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-TRBV2-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV2-optional linker-TRBC (format 27-5); or

[0602] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-VH-linker-TRBV1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV1-optional linker-TRBC (format 27-6); or

[0603] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-VH-optional linker-TRBC (format 27-7); or

[0604] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VH-optional linker-CL (format 27-8); or

[0605] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-TRBV2-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV2-optional linker-CL (format 27-9); or

[0606] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-VH-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV1-optional linker-CL (format 27-10); or

[0607] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-VH-optional linker-CL (format 27-11); or

[0608] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VH-optional linker-CL-optional linker-cysteine-containing peptide segment (format 27-12); or

[0609] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-TRBV2-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-VL-linker-TRAV2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 27-13); or

[0610] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-VH-linker-TRBV1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-VL-linker-TRAV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 27-14); or

[0611] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-VH-optional linker-CL-optional linker-cysteine-containing peptide segment (format 27-15).

[0612] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-VL-optional linker-CH3-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-VH-optional linker-CH3-linker-TRAV2 (format 28).

[0613] In some embodiments, the polypeptide molecule comprises a derivative of format 28. In this derivative, the positions of the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative, the two CH3 domains can be replaced by the CL / CH1 of an antibody or the TRAC / TRBC of a TCR. Preferably, the derivative of format 28 also includes a cysteine-containing peptide.

[0614] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-CH3 (format 29).

[0615] In some embodiments, the polypeptide molecule comprises a derivative of format 29. In this derivative, the positions of the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative of format 29, the two CH3 domains can be replaced by the CL / CH1 of an antibody or the TRAC / TRBC of a TCR. Preferably, this derivative of format 29 also includes a cysteine-containing peptide.

[0616] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VL-optional linker-CH3-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-VH-optional linker-CH3-linker-TRAV1 (format 31).

[0617] In some embodiments, the polypeptide molecule comprises a derivative of format 31. In this derivative, the positions of the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be swapped, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative, the two CH3 domains can be replaced by the CL / CH1 of an antibody or the TRAC / TRBC of a TCR. Preferably, derivatives of format 31 can also include cysteine-containing peptides.

[0618] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises, from N-terminus to C-terminus: VL-optional linker-CH3-linker-TRAV2-linker-TRBV2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-optional linker-CH3-linker-TRAV1-linker-TRBV1 (format 31-1).

[0619] In some embodiments, the polypeptide molecule comprises a derivative of format 31-1. In these derivatives, the positions of the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in derivatives of format 31-1, the two CH3 domains can be replaced by the CL / CH1 of an antibody or the TRAC / TRBC of a TCR. Preferably, derivatives of format 31-1 can also include cysteine-containing peptides.

[0620] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-CH3-linker-VH-linker-VL (format 33).

[0621] In some embodiments, the polypeptide molecule comprises a derivative form of format 33. In this derivative form, the positions of the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 33, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, the derivative form of format 33 may also include a cysteine-containing peptide segment. For example, derivative forms of format 33 include, but are not limited to, the following structures:

[0622] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-TRBC-linker-VH-linker-VL (format 33-1); or

[0623] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-CL-linker-VH-linker-VL (format 33-2).

[0624] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-optional linker-CH3-linker-TRAV2-linker-TRBV2 (format 34).

[0625] In some embodiments, the polypeptide molecule comprises a derivative of format 34. In this derivative, the positions of the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in a derivative of format 34, the two CH3s are replaced by CL / CH1 of an antibody or TRAC / TRBC of a TCR. Preferably, a derivative of format 34 may also include a cysteine-containing peptide. For example, derivatives of format 34 include, but are not limited to, the following structures:

[0626] The first polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-VL-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-TRBC-linker-TRAV2-linker-TRBV2 (format 34-1); or

[0627] The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CL-linker-TRAV2-linker-TRBV2 (format 34-2); or

[0628] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3-linker-TRAV2-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-optional linker-CH3 (format 34-3); or

[0629] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-TRAC-linker-TRAV2-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-optional linker-TRBC (format 34-4); or

[0630] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH1-linker-TRAV2-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-optional linker-CL (format 34-5).

[0631] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0632] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV2-optional linker-CH3-linker-TRBV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-CH3-linker-TRAV2-linker-VH-linker-VL (format 47).

[0633] In some embodiments, the polypeptide molecule comprises a derivative form of format 47. In this derivative form, the positions of the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 47, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. Preferably, the derivative form of format 47 may also include a cysteine-containing peptide segment. For example, derivative forms of format 47 include, but are not limited to, the following structures:

[0634] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-CH3-linker-TRAV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-CH3-linker-TRBV2-linker-VH-linker-VL (format 47-1); or

[0635] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-CH3-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-CH3-linker-TRBV2-linker-VH-linker-VL (format 47-2); or

[0636] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-CH3-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-CH3-linker-TRAV2-linker-VH-linker-VL (format 47-3).

[0637] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, and the domains contained in at least one of the binding regions formed by VH and VL, TRAV1 and TRBV1, and TRAV2 and TRBV2 are operably linked together and connected to the C-terminus of one of the polypeptide chains through the N-terminus, or connected to the N-terminus of one of the polypeptide chains through the C-terminus.

[0638] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0639] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH3-linker-TRBV2-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-CH3 (format 49).

[0640] In some embodiments, the polypeptide molecule comprises a derivative form of format 49. In this derivative form, the positions of the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 49, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. Preferably, the derivative form of format 49 may also include a cysteine-containing peptide segment. For example, derivative forms of format 49 include, but are not limited to, the following structures:

[0641] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRBC-linker-TRBV2-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-TRAC (format 49-1); or

[0642] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH1-linker-TRBV2-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-CL (format 49-2); or

[0643] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-CH3-linker-TRBV2-linker-TRAV2 (format 49-3); or

[0644] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-TRAC-linker-TRBV2-linker-TRAV2 (format 49-4); or

[0645] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-CL-linker-TRBV2-linker-TRAV2 (format 49-5); or

[0646] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-optional linker-CH3-linker-VH-linker-VL, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV1-optional linker-CH3 (format 49-6); or

[0647] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV1-optional linker-TRAC-linker-VH-linker-VL (format 49-7); or

[0648] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV1-optional linker-CL-linker-VH-linker-VL (format 49-8).

[0649] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-CH3 (format 49-9); or

[0650] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-TRBC (format 49-10); or

[0651] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-CL (format 49-11); or

[0652] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 49-12); or

[0653] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-TRAV2-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV2-optional linker-CH3 (format 49-13); or

[0654] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-TRAV2-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV2-optional linker-TRBC (format 49-14); or

[0655] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-TRAV2-linker-VH-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV2-optional linker-CL (format 49-15); or

[0656] The first polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-linker-TRAV1-linker-TRAV2-linker-VH-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 49-16); or

[0657] The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV1-linker-TRBV2-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV1-optional linker-CH3 (format 49-17); or

[0658] The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV1-linker-TRBV2-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV1-optional linker-TRAC (format 49-18); or

[0659] The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV1-linker-TRBV2-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV1-optional linker-CL (format 49-19); or

[0660] The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-linker-TRAV1-linker-TRBV2-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 49-20).

[0661] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0662] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-linker-TRAV1-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-CH3 (format 50).

[0663] In some embodiments, the polypeptide molecule comprises a derivative form of format 50. In this derivative form, the positions of the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in the derivative form of format 50, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. Preferably, the derivative form of format 50 may also include a cysteine-containing peptide segment. For example, derivative forms of format 50 include, but are not limited to, the following structures:

[0664] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV1-linker-TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-TRBC (format 50-1); or

[0665] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV1-linker-TRAV1-linker-VH-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-CL (format 50-2); or

[0666] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV1-linker-TRAV1-linker-VH-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 50-3); or

[0667] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-linker-VL-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-CH3 (format 50-4); or

[0668] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-linker-VL-linker-TRBV1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-TRBC (format 50-5); or

[0669] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-linker-VL-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-CL (format 50-6); or

[0670] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH-linker-VL-linker-TRBV1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-CL-optional linker-cysteine-containing peptide segment (format 50-7); or

[0671] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV1-linker-TRBV1-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-CH3 (format 50-8).

[0672] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0673] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV1-linker-TRBV1-optional linker-CH3-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-CH3-linker-VH (format 52).

[0674] In some embodiments, the polypeptide molecule comprises a derivative form of format 52. In this derivative form, the positions of the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in a derivative form of format 52, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. Preferably, the derivative form of format 52 may also include a cysteine-containing peptide segment. For example, derivative forms of format 52 include, but are not limited to, the following structures:

[0675] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV1-linker-TRBV1-optional linker-TRBC-linker-TRBV2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-optional linker-TRAC-linker-VH (format 52-1); or

[0676] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV1-linker-TRBV1-optional linker-CH1-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-CL-linker-VH (format 52-2).

[0677] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0678] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH-linker-VL-optional linker-CH3-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-CH3-linker-TRAV1 (format 53).

[0679] In some embodiments, the polypeptide molecule comprises a derivative form of format 53. In this derivative form, the positions of the various domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 53, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR. Preferably, the derivative form of format 53 may also include a cysteine-containing peptide segment. For example, derivative forms of format 53 include, but are not limited to, the following structures:

[0680] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH-linker-VL-optional linker-TRBC-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-TRAC-linker-TRAV1 (format 53-1); or

[0681] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH-linker-VL-optional linker-CH1-linker-TRBV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-optional linker-CL-linker-TRAV1 (format 53-2); or

[0682] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-TRAV2-Linker-TRBV2-Optional Linker-CH3-Linker-VL, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-Optional Linker-CH3-Linker-TRAV1 (format 53-3); or

[0683] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV2-linker-TRBV2-optional linker-TRBC-linker-VL, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-optional linker-TRAC-linker-TRAV1 (format 53-4); or

[0684] The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-TRAV2-Linker-TRBV2-Optional Linker-CH1-Linker-VL, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-Optional Linker-CL-Linker-TRAV1 (format 53-5); or

[0685] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-Linker-VL1-Linker-VH1-Linker-CH3-Linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-Linker-CH3-Linker-TRBV1 (format 53-6).

[0686] In some embodiments, each of the CH3s independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405, and 407, and the two CH3s do not comprise the same mutation. Preferably, one of the CH3s comprises a T366W mutation and the other comprises a T366S, L368A, and Y407V mutation. In some embodiments, the TRAC and / or TRBC comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC; preferably, the cysteine ​​mutation is at position 48 of the wild-type TCR α chain constant region and position 57 of the wild-type TCR β chain constant region.

[0687] In some embodiments, the polypeptide molecule does not comprise the hinge domain and Fc domain of an antibody, and comprises the α chain constant region (TRAC) and the β chain constant region (TRBC) of a TCR.

[0688] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0689] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-VH-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-TRAC (format 14).

[0690] In some embodiments, the polypeptide molecule comprises a derivative form of format 14. In this derivative form, the positions of the various domains (VH, VL, TRAV1, TRBV1, TRAC, TRBC) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in the derivative form of format 14, TRAC and TRBC can be replaced by the CH3 / CH3 or CH1 / CL of the antibody. Preferably, the derivative form of format 14 can also include a cysteine-containing peptide segment. For example, derivative forms of format 14 include, but are not limited to, the following structures:

[0691] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-VH-linker-TRAV1-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-TRBC (format 14-1); or

[0692] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-VH-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-CH3 (format 14-2); or

[0693] The first polypeptide chain comprises from N-terminus to C-terminus: VL-linker-VH-linker-TRAV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-optional linker-CH3 (format 14-3); or

[0694] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-VH-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CL (format 14-4); or

[0695] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-VH-linker-TRAV1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-CL (format 14-5); or

[0696] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-VH-linker-TRBV1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 14-6); or

[0697] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-VH-linker-TRAV1-optional linker-CH1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV1-optional linker-CL-optional linker-cysteine-containing peptide segment (format 14-7).

[0698] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0699] The first polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV1-optional linker-TRAC-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV2-optional linker-TRBC-linker-TRAV2-optional linker-TRAC (format 26).

[0700] In some embodiments, the polypeptide molecule comprises a derivative of format 26. In this derivative, the positions of the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, TRAC, and TRBC) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative of format 26, TRAC and TRBC can be replaced by the CH3 / CH3 or CH1 / CL of the antibody. Preferably, this derivative of format 26 also includes a cysteine-containing peptide.

[0701] In some embodiments, the polypeptide molecule does not comprise the hinge domain and Fc domain of an antibody, and comprises the α chain constant region (TRAC) and β chain constant region (TRBC) of a TCR, and the TRAC and TRBC are respectively connected to the C-termini of the two polypeptide chains of the polypeptide molecule through an optional linker.

[0702] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0703] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-TRBC (format 22-2); or

[0704] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-TRBC (format 49-10); or

[0705] The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH-optional linker-TRAC-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-TRBC-linker-VL-optional linker-TRBC-linker-TRAV2-optional linker-TRAC (format 24-1).

[0706] In some embodiments, the TRAC and / or TRBC comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, preferably, the cysteine ​​mutation is at the following positions: position 48 of the wild-type TCR α chain constant region and position 57 of the wild-type TCR β chain constant region.

[0707] In some embodiments, the polypeptide molecule does not contain a constant region of a TCR, and the two polypeptide chains contain a hinge domain and CH2 and CH3 domains derived from an antibody; wherein the CH3 domains in the two polypeptide chains contain at least one mutation that can promote the polypeptide molecule to form a heterodimer.

[0708] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0709] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-hinge domain-CH2-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-VH-optional linker-hinge domain-CH2-CH3 (format 16).

[0710] In some embodiments, the polypeptide molecule comprises a derivative of format 16. In this derivative, the positions of the domains (VH, VL, TRAV1, TRBV1) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative of format 16, the two hinge domains - CH2-CH3 - can be replaced by the CH1 / CL or CH3 / CH3 of an antibody, or the TRAC / TRBC of a TCR. Preferably, this derivative of format 16 can also contain a cysteine-containing peptide segment.

[0711] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0712] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-hinge domain-CH2-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-hinge domain-CH2-CH3 (format 17).

[0713] In some embodiments, the polypeptide molecule comprises a derivative of format 17. In this derivative, the positions of the domains (VH, VL, TRAV1, TRBV1) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative of format 17, the two hinge domains - CH2-CH3 - can be replaced by the CH1 / CL or CH3 / CH3 of an antibody, or the TRAC / TRBC of a TCR. Preferably, this derivative of format 17 can also contain a cysteine-containing peptide segment.

[0714] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0715] The first polypeptide chain comprises, from N-terminus to C-terminus: hinge domain-CH2-CH3-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: hinge domain-CH2-CH3-linker-VH-linker-VL (format 18).

[0716] In some embodiments, the polypeptide molecule comprises a derivative of format 18. In this derivative, the positions of the domains (VH, VL, TRAV1, TRBV1) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative of format 18, the two hinge domains - CH2-CH3 - can be replaced by the CH1 / CL or CH3 / CH3 of an antibody, or the TRAC / TRBC of a TCR. Preferably, this derivative of format 18 can also include a cysteine-containing peptide segment.

[0717] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0718] The first polypeptide chain comprises from N-terminus to C-terminus: hinge domain-CH2-CH3-linker-TRAV1-linker-VH, and the second polypeptide chain comprises from N-terminus to C-terminus: hinge domain-CH2-CH3-linker-VL-linker-TRBV1 (format 19).

[0719] In some embodiments, the polypeptide molecule comprises a derivative of format 19. In these derivatives, the positions of the domains (VH, VL, TRAV1, TRBV1) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in derivatives of format 19, the two hinge domains - CH2-CH3 - can be replaced by the CH1 / CL or CH3 / CH3 of an antibody, or the TRAC / TRBC of a TCR. Preferably, derivatives of format 19 may also include cysteine-containing peptides.

[0720] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0721] The first polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV1-linker-TRAV1-linker-VH-optional linker-hinge domain-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-linker-TRAV2-optional linker-hinge domain-CH2-CH3 (format 51).

[0722] In some embodiments, the polypeptide molecule comprises a derivative of format 51. In this derivative, the positions of the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind the corresponding antigen is retained. In some embodiments, in this derivative of format 51, the two hinge domains (CH2-CH3) can be replaced by the CH1 / CL or CH3 / CH3 structures of an antibody, or the TRAC / TRBC structures of a TCR. Preferably, this derivative of format 51 may also include a cysteine-containing peptide segment.

[0723] In some embodiments, the polypeptide molecule does not comprise the hinge domain and CH2 domain of an antibody.

[0724] In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0725] The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-optional linker-CL-linker-TRAV2-optional linker-TRAC-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-optional linker-CH3-linker-TRBV2-optional linker-TRBC-linker-VL-optional linker-CH3 (format 48).

[0726] In some embodiments, the polypeptide molecule comprises a derivative form of format 48. In this derivative form, the positions of the domains (VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, TRAC, TRBC, CL, CH1, and CH3) can be interchanged, and the order of the binding regions formed by these domains can be interchanged, as long as the ability to bind to the corresponding antigen is retained. In some embodiments, in this derivative form of format 48, the two CH3 domains are replaced by the CH1 / CL of an antibody or the TRAC / TRBC of a TCR; and / or the TRAC and TRBC are replaced by the CH1 / CL or CH3 / CH3 of an antibody; and / or the CH1 and CL are replaced by the CH3 / CH3 of an antibody or the TRAC / TRBC of a TCR. Preferably, the derivative form of format 48 may also include a cysteine-containing peptide.

[0727] It will be appreciated that the above describes the main structure of the format of the present invention and the concepts derived from the main structure, and provides exemplary derivative forms of some formats. Those skilled in the art, upon understanding the concepts of the present invention, should consider other derivative forms of the format derived from the concepts to be within the scope of protection of the present invention.

[0728] In some embodiments, the CH3 each independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405, and 407, and the two CH3s do not comprise the same mutation. Preferably, one of the CH3s comprises a T366W mutation and the other comprises a T366S, L368A, and Y407V mutation. In some embodiments, the CH2 and CH3 are derived from the Fc domain of human IgG1, IgG2, or IgG4, or a portion thereof, and preferably comprise at least two additional cysteine ​​residue mutations, such as S354C and Y349C or L242C and K334C. In some embodiments, the hinge region is derived from the hinge domain of human IgG1, IgG2, or IgG4, or a portion thereof.

[0729] In embodiments of the bispecific polypeptide molecules of the present invention, TRAC and TRBC can be human or murine. TRAC and TRBC can be wild-type or variants thereof. For example, a TRAC variant can comprise one or more of T48C, N113K, PESS deletion mutation, or FFPSPESS deletion mutation relative to the wild-type sequence. For example, a TRBC variant can comprise one or more of S57C, C187A, N210D, or FG loop deletion mutation relative to the wild-type sequence.

[0730] In some embodiments, CL can comprise a terminal NEC deletion mutation relative to the wild-type sequence. In some embodiments, CH1 can comprise a terminal VPRDC deletion mutation relative to the wild-type sequence.

[0731] In the embodiments of the bispecific polypeptide molecules of the present invention, the domains of the polypeptide molecules (e.g., VH, VL, TRAV1, TRBV1, TRAV2, TRBV2, CL, CH1, CH3, TRAC, TRBC) can be interchanged as long as the ability to bind to the corresponding antigen is retained.

[0732] In some embodiments, VH and VL contain 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 into FR2 in the case of VH. In a preferred embodiment, the cysteine ​​mutation is at the following positions: position 44 of VH and position 100 of VL.

[0733] In some embodiments, the TRAV1 and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV1 and VL, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV1 and position 80 of VL. In some embodiments, the TRAV2 and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV2 and VL, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV2 and position 80 of VL. In some embodiments, the TRBV1 and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRBV1 and VL, preferably, the cysteine ​​mutation is at the following positions: position 84 of TRBV1 and position 80 of VL. In some embodiments, the TRBV2 and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRBV2 and VL, preferably, the cysteine ​​mutation is at the following positions: position 84 of TRBV2 and position 80 of VL. In some embodiments, the TRAV1 and VH comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV1 and VH, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV1 and position 89 of VH. In some embodiments, the TRAV2 and VH comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV2 and VH, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV2 and position 89 of VH.

[0734] In some embodiments, TRAV1 and / or TRAV2 can be native TRAV or a functional variant thereof, as long as the functional variant retains the ability to bind to the target antigen. For example, TRAV1 and / or TRAV2 can be a truncated variant of native TRAV, such as a variant with 8 amino acids truncated at the N-terminus or C-terminus.

[0735] In some embodiments, TRBV1 and / or TRBV2 may be a naturally occurring TRBV or a functional variant thereof, as long as the functional variant retains the ability to bind to the target antigen.

[0736] In some embodiments, the polypeptide molecule further comprises one or more amino acid mutations, insertions or deletions 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 post-translational modification (e.g., glycosylation modification), such as 1, 2, 3, 4, 5 or more amino acid mutations, insertions or deletions. The 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: Ala, Gly. In some embodiments, the amino acid mutations, insertions or deletions are located at the following amino acid positions: the beginning of TRAV, TRBV, VL, VH, the boundary amino acids of TRAV / TRAC, the boundary amino acids of TRBV / TRBC, the boundary amino acids of VL / CL, the boundary amino acids of VH / CH1, the hinge region and its adjacent amino acids, the boundary amino acids of CH2 / CH3, the C-terminus of CH3, and 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 that enhance binding to FcRn include, for example, one or more of the following mutations: L234A, L235A, M252Y, S254T, T256E, M428L, N434S, T250R, M428L, N434A in the CH2 and CH3 domains, preferably L234A L235A, M252Y S254T T256E, M428L N434S and T250R M428L N434A. In some embodiments, the effector function silent 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 silent mutation is generated by replacing at least one residue at positions 233, 234, 235, 236, and 331 with the corresponding residue derived from IgG2 or IgG4. In some embodiments, amino acid mutations that can reduce post-translational modification of the polypeptide molecule include, for example, N113K in TRAC and N210D in TRBC.

[0737] In embodiments of the bispecific polypeptide molecules of the present invention, when the polypeptide molecules comprise more than one linker, the linkers may be the same or different. In some embodiments, the linkers in the polypeptide molecules are each independently selected from linkers consisting of 1-35 amino acids.

[0738] In some embodiments, the linkers in the polypeptide molecule are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GQPKAAP, TVLRT, TVSSAS, GGEGG, GSEGGGS, GGEGGGSEGGGS, RTSGPGDGGKGGPGKGPGGEGTKGTGPGG, GDGGKGGPGKGPGGEGTKGTGPGG, GKGPGGEGTKGTGPGG, TVLSSAS, EDLKN or a variant thereof, EDLNK or a variant thereof, and ANIQK or a variant thereof, or any combination thereof.

[0739] In some embodiments, the variant of EDLKN is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to ANIQK.

[0740] Preferably, the variant of EDLKN is an amino acid sequence formed by replacing one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by replacing one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by replacing one or several amino acids of ANIQK.

[0741] Preferably, the variant of EDLKN is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLKN; and / or the variant of EDLNK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLNK; and / or the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from ANIQK.

[0742] Preferably, the variant of EDLKN is EDL, DL, L, ED, E, D; and / or the variant of ANIQK is ANI, NI, I, AN, N, A, PNI, P, PN.

[0743] 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 an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to EDLNK (preferably at the N-terminus); and / or the variant of ANIQK is an amino acid sequence consisting of 6-35 amino acids formed by adding one or several amino acids to ANIQK (preferably at the N-terminus).

[0744] In some embodiments, the polypeptide molecule comprises one or more domains selected from the group consisting of:

[0745] CH3 domain, TCR constant region or fragment thereof, hinge region, CH2, CH3, CL, CH1, cysteine-containing peptide segment and combinations thereof, when the N-terminus of one or more domains is connected to the C-terminus of its upstream sequence through a linker, the linkers are each independently selected from a linker consisting of no more than 12 amino acids.

[0746] Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, EDLKN or a variant thereof, EDLNK or a variant thereof and ANIQK or a variant thereof.

[0747] In some embodiments, the variant of EDLKN is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids to ANIQK.

[0748] Preferably, the variant of EDLKN is an amino acid sequence formed by replacing one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by replacing one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by replacing one or several amino acids of ANIQK.

[0749] Preferably, the variant of EDLKN is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLKN; and / or the variant of EDLNK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from EDLNK; and / or the variant of ANIQK is an amino acid sequence consisting of 1-3 amino acids formed by deleting one or several amino acids from ANIQK.

[0750] Preferably, the variant of EDLKN is EDL, DL, L, ED, E, D; and / or the variant of ANIQK is ANI, NI, I, AN, N, A, PNI, P, PN.

[0751] 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 an amino acid sequence consisting of 6-12 amino acids formed by adding one or several amino acids to EDLNK (preferably at the N-terminus); and / or the variant of ANIQK is an amino acid sequence consisting of 6-12 amino acids formed by adding one or several amino acids to ANIQK (preferably at the N-terminus).

[0752] In some embodiments, the polypeptide molecule comprises TRAC or a fragment thereof, which is not connected to TRAV via a linker. Preferably, 1-5 amino acids at the N-terminus of TRAC may be substituted.

[0753] In some embodiments, the polypeptide molecule comprises TRBC or a fragment thereof, which is not connected to TRBV via a linker. Preferably, 1-5 amino acids at the N-terminus of TRBC may be substituted.

[0754] Unless otherwise indicated, the term "cis-linked" refers to the linkage between the TCR constant region and the variable region in the natural state.

[0755] In some embodiments, the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or more polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain, such as a signal peptide sequence derived from albumin or immunoglobulin, preferably MGWSCIILFLVATATGVHS.

[0756] In some embodiments, the first antigen and the third antigen are each independently a TAA selected from the following: melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A and an endogenous reverse transcriptase antigen.

[0757] In some embodiments, the first antigen and the third antigen are each independently a TSA selected from the following: KRAS (e.g., G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (e.g., R175H, R173H, R273C, R248W, R248Q, R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (e.g., E542K, E545K, H1047R), CTNNB1 (e.g., S45P, T41A), EGFR (e.g., L858R, T790M), BRAF (e.g., V600E), and GNAS (e.g., R201C, R201H).

[0758] In some embodiments, the first antigen and the third antigen are each independently a viral antigen selected from the group consisting of HPV E6 or E7 antigens, CMV antigens, HBV antigens, EBV antigens, herpes virus antigens, human immunodeficiency virus (HIV) antigens, influenza virus antigens, and coronavirus antigens.

[0759] In some embodiments, the first antigen and the third antigen are each independently an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Ins ulin, 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.

[0760] In some embodiments, the second antigen is selected from CD3 (e.g., CD3γ, CD3δ, and CD3ε chain), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, FcεR I. 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.

[0761] In some embodiments, the first antigen and the third antigen are each independently a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen; and the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and C D3ε chain), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b , CD42a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, F cεRI, TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD 244), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT and B7-H3.

[0762] In some embodiments, the first antigen and the third antigen are each independently a TSA selected from the group consisting of KRAS (e.g., G12D, G12V, G12C, G12R, G12A, G13D, Q61H, G125), TP53 (e.g., R175H, R173H, R273C, R248W, R248Q, R282W, Y220C, V157F, G245S, Y163C, R249S), PIK3CA (e.g., E542K, E545K, H1047R), CTNNB1 (e.g., S45P, T41A), EGFR (e.g., L858R, T790M), BRAF (e.g., V600E), and GNAS (e.g., R201C, R201H); and the second antigen is selected from the group consisting of CD3 (e.g., CD3γ, CD3δ, and CD3ε). chain), CD4, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD25, CD32a, CD32b, CD41, CD41b, CD 42a, CD42b, CD44, CD45RA, CD49, CD61, CD64, CD68, CD94, CD90, CD117, Nkp46, NKG2D, Fcε RI, TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD2 44), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT and B7-H3.

[0763] In some embodiments, the first antigen and the third antigen are each independently a viral antigen selected from the group consisting of HPV E6 or E7 antigens, CMV antigens, HBV antigens, EBV antigens, herpes virus antigens, human immunodeficiency virus (HIV) antigens, influenza virus antigens, and coronavirus antigens; 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(CD 134), ICOS (CD278), 2B4 (CD244), HVEM, LAG3, DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT and B7-H3.

[0764] In some embodiments, the first antigen and the third antigen are each independently an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin, 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.

[0765] In some embodiments, the first antigen and the third antigen are each independently selected from gp100, MAGEA1, KRAS, and HPVE7.

[0766] In some embodiments, the second antigen is CD3, CD28, or 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 CD34-1BB (CD137). In some embodiments, the second antigen is CD3, and the second binding region comprises a VH1 and a VL1 derived from an anti-CD3 antibody selected from the group consisting of OKT3, UCHT-1, BMA031, and 12F6.

[0767] In some embodiments, the polypeptide molecule comprises a polypeptide chain having an amino acid sequence selected from any one of SEQ ID NOs: 9 and 10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 9 and 10.

[0768] In some embodiments, the polypeptide molecule comprises:

[0769] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2;

[0770] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:3, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:3, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:4;

[0771] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:5, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:5, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:6, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:6;

[0772] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:7, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:8;

[0773] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 13, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 14;

[0774] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 15, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 16, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 16;

[0775] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:29, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:29, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:30, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:30;

[0776] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:31, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:31, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:32, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:32;

[0777] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:33, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:33, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:34, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:34;

[0778] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:37, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:37, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:38, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:38;

[0779] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:39, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:39, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:40, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:40;

[0780] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:41, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:41, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:42, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:42; or

[0781] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:47, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:47, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:48, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:48; or

[0782] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 129, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 129, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 130, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 130; or

[0783] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 131, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 131, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 132, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 132; or

[0784] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 141, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 141, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 142, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 142; or

[0785] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 143, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 143, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 144, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 144; or

[0786] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 145, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 145, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 146, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 146; or

[0787] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 147, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 147, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 148, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 148; or

[0788] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 149, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 149, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 150, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 150; or

[0789] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 167, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 167, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 168, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 168; or

[0790] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 169, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 169, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 170, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 170; or

[0791] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 173, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 173, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 174, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 174; or

[0792] A first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:299, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:299, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO:300, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:300.

[0793] On the other hand, the bispecific polypeptide molecules of the present invention utilize a specific antigen-binding region connection method and are combined with TCR constant regions (TRAC and TRBC). This combination with the TCR constant region significantly improves the stability of the molecule. This specific combination of domains achieves efficient immune cell activation and has significantly better effects than using antibody constant regions (CH3, hinge region-CH2-CH3, or CH1 / CL).

[0794] Accordingly, the present invention provides a bispecific polypeptide molecule comprising a first binding region that binds to a first antigen and a second binding region that binds to a second antigen on two polypeptide chains.

[0795] wherein the first binding region comprises an α chain variable region (TRAV) and a β chain variable region (TRBV) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex;

[0796] 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;

[0797] wherein the first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the second antigen is an immune cell surface molecule;

[0798] The two polypeptide chains of the bispecific polypeptide molecule respectively comprise:

[0799] TRAV-VH and VL-TRBV;

[0800] TRAV-VL and VH-TRBV;

[0801] TRBV-VH and VL-TRAV; or

[0802] TRBV-VL and VH-TRAV;

[0803] Adjacent variable regions in the two polypeptide chains are connected by a linker; and

[0804] The bispecific polypeptide molecule further comprises a TCR constant region or fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker.

[0805] In a preferred embodiment, the two polypeptide chains of the bispecific polypeptide molecule comprise: TRAV-VH and VL-TRBV, respectively.

[0806] In a preferred embodiment, the two polypeptide chains of the bispecific polypeptide molecule comprise: TRAV-VH and VL-TRBV, respectively, and the 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 a fragment thereof connected to the C-terminus of each polypeptide chain via an optional linker.

[0807] In some embodiments, the TCR constant region is selected from the group consisting of a TCR alpha chain constant region (TRAC) and a 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.

[0808] In some embodiments, the N-terminal 1-5 amino acids in TRAC may be substituted.

[0809] In some embodiments, 1-5 amino acids at the N-terminus of TRBC may be substituted.

[0810] TRAC and TRBC can be human or murine. TRAC and TRBC can be wild-type or variants thereof. For example, a TRAC variant can include one or more of T48C, N113K, PESS deletion mutation, or FFPSPESS deletion mutation relative to the wild-type sequence. For example, a TRBC variant can include one or more of S57C, C187A, N210D, or FG loop deletion mutation relative to the wild-type sequence. In a preferred embodiment, TRAC and / or TRBC include at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC. More preferably, the cysteine ​​mutation is at position 48 of the wild-type TCR α chain constant region and position 57 of the wild-type TCR β chain constant region.

[0811] In some embodiments, the polypeptide molecule comprises TRAC or a fragment thereof, which is not connected to TRAV via a linker. Preferably, 1-5 amino acids at the N-terminus of TRAC can be substituted.

[0812] In some embodiments, the polypeptide molecule comprises TRBC or a fragment thereof, which is not connected to TRBV via a linker. Preferably, 1-5 amino acids at the N-terminus of TRBC may be substituted.

[0813] Unless otherwise indicated, the term "cis-linked" refers to the linkage between the TCR constant region and the variable region in the natural state.

[0814] 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC (format 22-2).

[0815] 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 comprises from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC.

[0816] 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC.

[0817] 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC.

[0818] 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC.

[0819] 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC.

[0820] 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC.

[0821] 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC.

[0822] 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC.

[0823] In some embodiments, the bispecific polypeptide molecule further comprises at least one of the following functional domains:

[0824] 1) Hinge region derived from an antibody;

[0825] 2) Fc domain or its dimerization portion derived from an antibody;

[0826] 3) Albumin (Alb).

[0827] The introduction of the functional domain can not only prolong the half-life of the polypeptide molecule, but also enhance the killing activity of the polypeptide molecule.

[0828] In some embodiments, the functional domain is connected 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 connected to the C-terminus of either polypeptide chain of the bispecific polypeptide molecule via an optional linker. In some embodiments, the functional domain is connected to the C-terminus of both polypeptide chains of the bispecific polypeptide molecule via an optional linker.

[0829] 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 preferred embodiments, albumin or a fragment thereof is linked to the C-terminus of either polypeptide chain of the bispecific polypeptide molecule via an optional linker. In some embodiments, the albumin can be any mammalian-derived albumin, such as human albumin, bovine albumin, mouse albumin, etc. In some embodiments, the albumin is a 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, a fragment having a length of 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, In a preferred embodiment, the albumin is human serum albumin having an amino acid sequence as shown in SEQ ID NO: 201.

[0830] 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-Alb (Format 64).

[0831] 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 comprises, from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC-linker-Alb.

[0832] 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-optional linker-TRAC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC.

[0833] 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-optional linker-TRBC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC.

[0834] 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC-optional linker-Alb.

[0835] 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-optional linker-TRAC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC.

[0836] 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC-optional linker-Alb.

[0837] 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-optional linker-TRBC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC.

[0838] 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRAC-optional linker-Alb.

[0839] 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-optional linker-TRAC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC.

[0840] 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRBC-optional linker-Alb.

[0841] 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-optional linker-TRBC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC.

[0842] 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRAV-optional linker-TRAC-optional linker-Alb.

[0843] 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-optional linker-TRAC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC.

[0844] 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-optional linker-TRAC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRBC-optional linker-Alb.

[0845] 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-optional linker-TRBC-optional linker-Alb, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC.

[0846] 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-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV-optional linker-TRAC-optional linker-Alb.

[0847] 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 connected to the C-termini of the two polypeptide chains of the bispecific polypeptide molecule via an optional linker.

[0848] The hinge region may comprise part or all of the wild-type hinge sequence of an antibody or a variant thereof having one or more substitutions. In some embodiments, the hinge region of the antibody is the hinge region of a human antibody. The hinge region of the antibody may be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. For example, the hinge region of the antibody may be derived from the hinge domain of human IgG1, 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: 202), EPKSSDKTHTCPPCP (SEQ ID NO: 203), PKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 204), KSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 205), SSDKTHTCPPCPAPPVAGP (SEQ ID NO: 206), SDKTHTCPPCPAPPVAGP (SEQ ID NO: 207), DKTHTCPPCPAPPVAGP (SEQ ID NO: 208), KTHTCPPCPAPPVAGP (SEQ ID NO: 209), THTCPPCPAPPVAGP (SEQ ID NO: 210), HTCPPCPAPPVAGP (SEQ ID NO: 211), TCPPCPAPPVAGP (SEQ ID NO: 212), CPPCPAPPVAGP (SEQ ID NO: 213), PPCPAPPVAGP (SEQ ID NO: 214). NO: 214), CPAPPVAGP (SEQ ID NO: 215), or PAPPVAGP (SEQ ID NO: 216); or variants thereof having one or more substitutions (e.g., 1-6 substitutions, e.g., 1-5, 1-4, 1, 2, 3, 4, 5, or 6 substitutions).

[0849] The Fc domain of an antibody comprises CH2 and CH3. In some embodiments, the Fc domain of an antibody or its dimerization portion is the Fc domain of a human antibody or its dimerization portion. The Fc domain of an antibody can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In some embodiments, the Fc domain of an antibody or its dimerization portion is derived from the Fc domain of human IgG1, IgG2, or IgG4, or a portion thereof, and preferably comprises at least two cysteine ​​residue mutations, such as S354C and Y349C or L242C and K334C. In one embodiment, the Fc domain is an IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In another embodiment, the Fc domain is an IgG4 isotype or derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc domain is human IgG1Fc or human IgG4Fc.

[0850] 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 the hinge region - CH2-CH3. In some embodiments, the CH3 in both polypeptide chains of the bispecific polypeptide molecule comprises at least one mutation that promotes heterodimer formation of the polypeptide molecule. In some embodiments, the CH3 in each of the two polypeptide chains independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405, and 407, and the two CH3s do not comprise the same mutation. Preferably, one of the CH3s comprises a T366W mutation and the other comprises a T366S, L368A, and Y407V mutation. In a preferred embodiment, the amino acid sequence of the hinge region - CH2-CH3 is as shown in SEQ ID NO: 217 or 218.

[0851] 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-optional linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3 (format 63).

[0852] 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 comprises, from N-terminus to C-terminus: VL-linker-TRBV-linker-TRBC-hinge region-CH2-CH3.

[0853] 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-optional linker-TRBC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV-optional linker-TRAC-optional linker-hinge region-CH2-CH3.

[0854] 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-optional linker-TRAC-optional linker-hinge region-CH2-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV-optional linker-TRBC-optional linker-hinge region-CH2-CH3.

[0855] 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-optional linker-TRBC-optional linker-hinge region-CH2-CH3, and the second...

Claims

1. A trispecific polypeptide molecule, wherein the trispecific polypeptide molecule comprises, on one or more polypeptide chains, a first binding region that binds a first antigen, a second binding region that binds a second antigen, and a third binding region that binds a third antigen, wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex; wherein the second binding region comprises a heavy chain variable region VH1 and a light chain variable region VL1 derived from an antibody that binds to the second antigen; and wherein the third binding region comprises a heavy chain variable region VH2 and a light chain variable region VL2 derived from an antibody that binds to the third antigen; in, The TRAV1, TRBV1, VH1, VL1, VH2 and VL2 are distributed on the one or more polypeptide chains such that when the one or more polypeptide chains are folded, TRAV1 and TRBV1 are spatially close to form a first binding region, the VH1 and VL1 are spatially close to form a second binding region, and the VH2 and VL2 are spatially close to form a third binding region; The first antigen is selected from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens and self-antigens, the second antigen is a first immune cell surface molecule, and the third antigen is a second immune cell surface molecule; the first immune cell surface molecule and the second immune cell surface molecule are different.

2. The trispecific polypeptide molecule according to claim 1, wherein the TRAV1, TRBV1, VH1, VL1, VH2 and VL2 are distributed on the one or more polypeptide chains and adjacent variable regions on the same chain are separated by a linker sequence.

3. The trispecific polypeptide molecule according to claim 1 or 2, wherein the polypeptide molecule further comprises at least one pair of TRAV2 and TRBV2, which form a fourth binding region that binds to a fourth antigen; in, The fourth antigen is selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the fourth antigen is the same as or different from the first antigen.

4. The trispecific polypeptide molecule according to any one of claims 1 to 3, wherein the polypeptide molecule further comprises one or more of the following structures: (1) a CH3 domain, wherein the CH3 domain is derived from the CH3 domain of an antibody, and the CH3 of any one polypeptide chain or both polypeptide chains comprises at least one mutation that promotes the formation of heterodimers of the polypeptide molecules; (2) a TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC); (3) hinge region, CH2 and CH3, wherein the hinge region is derived from the hinge domain of an antibody, the CH2 and CH3 are derived from the Fc domain of an antibody, and the CH3 in any one polypeptide chain or both polypeptide chains contains at least one mutation that can promote the formation of heterodimers of the polypeptide molecules; (4) CL and CH1, wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain; (5) Cysteine-containing peptide segments, wherein the cysteine-containing peptide segments are capable of forming a disulfide bond between two polypeptide chains and are 3-10 amino acids in length, preferably 4-8 amino acids in length, such as 4 or 6 amino acids.

5. The trispecific polypeptide molecule according to any one of claims 1 to 4, wherein the polypeptide molecule comprises the first binding region, the second binding region, the third binding region and, when present, the fourth binding region on one polypeptide chain.

6. The trispecific polypeptide molecule according to claim 5, wherein the polypeptide molecule comprises the following structure from N-terminus to C-terminus: TRBV1-Linker-VH1-Linker-VH2-Linker-TRAV2-Linker-TRBV2-Linker-VL2-Linker-VL1-Linker-TRAV1.

7. The trispecific polypeptide molecule according to claim 5, wherein the polypeptide molecule further comprises a CH3 domain, wherein the CH3 domain is derived from the CH3 domain of an antibody and preferably comprises at least one mutation capable of inhibiting the formation of homodimers of the polypeptide molecule; Preferably, each of the CH3 independently comprises one or more amino acids selected from amino acid positions 366, 368, 405 and 407. The mutations at the two CH3 positions do not contain the same mutations; More preferably, one of the CH3 comprises a T366W mutation and the other comprises T366S, L368A and Y407V mutations; Further preferably, the polypeptide molecule comprises the following structure from N-terminus to C-terminus: VL1-Linker-TRBV1-Linker-TRAV1-Linker-VH1-Optional Linker-CH3-Linker-VL2-Linker-TRBV2-Linker-TRAV2-Linker-VH2-Optional Linker-CH3.

8. The trispecific polypeptide molecule according to any one of claims 1 to 4, wherein the polypeptide molecule comprises the first binding region, the second binding region, the third binding region and, when present, the fourth binding region on two polypeptide chains.

9. The trispecific polypeptide molecule according to claim 8, wherein the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VL2; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1; or The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH1-linker-VH2-linker TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-VL2-linker-VL1-linker-TRAV1; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-linker-VH1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV2-linker-VL1-linker-VL2-linker-VH2.

10. The trispecific polypeptide molecule according to claim 8, wherein the polypeptide molecule further comprises a TCR constant region or a fragment thereof, wherein the TCR constant region comprises, for example, a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC).

11. The trispecific polypeptide molecule according to claim 10, wherein the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL2-linker-VH2; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH2-linker-VL2; or The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL2-linker-TRAV1-optional linker-TRAC; or The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-TRAC; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRAC-linker-VL2-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-TRBC-linker-TRBV2-linker-TRAV2; or The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-TRBC; or The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-TRBC.

12. The trispecific polypeptide molecule according to claim 8, wherein the polypeptide molecule further comprises a CH3 domain on both polypeptide chains, wherein the CH3 domain is derived from the CH3 domain of an antibody, and the CH3 in either or both polypeptide chains comprises at least one mutation that promotes heterodimer formation.

13. The trispecific polypeptide molecule according to claim 12, wherein the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH3-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-optional linker-CH3-linker-VH2-linker-VL2, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-optional linker-CH3-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-optional linker-CH3-linker-TRBV2-linker-TRAV2; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VH1-linker-VL1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VL2-linker-VH2-optional linker-CH3; or The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-VH1-Linker-VL1-Optional Linker-CH3-Linker-TRAV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-Linker-VL2-Linker-VH2-Optional Linker-CH3-Linker-TRAV2; or The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-CH3.

14. The trispecific polypeptide molecule according to claim 8, wherein the polypeptide molecule further comprises a hinge region, CH2, and CH3 on two polypeptide chains, wherein the hinge region is derived from an antibody hinge domain, and the CH2 and CH3 are derived from an antibody Fc domain, and the CH3 in either or both polypeptide chains comprises at least one mutation that promotes heterodimer formation.

15. The trispecific polypeptide molecule according to claim 14, wherein the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-hinge region-CH2-CH3; and the second polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-hinge region-CH2-CH3; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-hinge region-CH2-CH3-linker-VH2-linker-VL2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-hinge region-CH2-CH3-linker-TRBV2-linker-TRAV2.

16. The trispecific polypeptide molecule according to claim 8, wherein the polypeptide molecule further comprises a cysteine-containing peptide segment on the two polypeptide chains capable of forming a disulfide bond between the two polypeptide chains, wherein the cysteine-containing peptide segment is 3-10 amino acids in length, preferably 4-8 amino acids in length, for example 4 or 6 amino acids in length, and (1) a CH3 domain, wherein the CH3 domain is derived from the CH3 domain of an antibody, and the CH3 of any one polypeptide chain or both polypeptide chains comprises at least one mutation that promotes the formation of heterodimers of the polypeptide molecule; or (2) a hinge region, CH2, and CH3, wherein the hinge region is derived from the hinge domain of an antibody, the CH2 and CH3 are derived from the Fc domain of an antibody, and the CH3 in any one polypeptide chain or both polypeptide chains contains at least one mutation that can promote the formation of heterodimers of the polypeptide molecules.

17. The trispecific polypeptide molecule according to claim 16, wherein the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3-linker-VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-CH3-linker-TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3-linker-VH2-linker -VH1-optional linker-cysteine-containing peptide, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-CH3-linker-VL1-linker-VL2-optional linker-cysteine-containing peptide; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-hinge region-CH2-CH3-linker-VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-hinge region-CH2-CH3-linker-TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment.

18. The trispecific polypeptide molecule according to claim 8, wherein the polypeptide molecule further comprises a TCR constant region or a fragment thereof and CL and CH1, wherein the TCR constant region, for example, comprises a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC), the CL is derived from an antibody light chain CL domain, and the CH1 is derived from an antibody heavy chain CH1 domain.

19. The trispecific polypeptide molecule according to claim 18, wherein the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-optional linker-CL-linker-TRAV1-optional linker TRAC-linker-VH1-optional linker-CH1; and The second polypeptide chain comprises, from N-terminus to C-terminus: VL2-optional linker-CL-linker-TRBV1-optional linker-TRBC-linker-VH2-optional linker-CH1.

20. The trispecific polypeptide molecule according to any one of claims 1 to 4, wherein the polypeptide molecule comprises the first binding region, the second binding region, the third binding region and, when present, the fourth binding region on four polypeptide chains; Preferably, the polypeptide molecule comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1, the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-CH3, the third polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV2, and the fourth polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-cysteine-containing peptide segment, the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-cysteine-containing peptide segment-optional linker-hinge region-CH2-CH3, the third polypeptide chain comprises, from N-terminus to C-terminus: VL2-linker-TRBV2-optional linker-cysteine-containing peptide segment, and the fourth polypeptide chain comprises, from N-terminus to C-terminus: TRAV2-linker-VH2-optional linker-cysteine-containing peptide segment-optional linker-hinge region-CH2-CH3; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL2-optional linker-CL, the second polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-TRBV1-optional linker-CH1-optional linker-hinge region-CH2-CH3, the third polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-VL1-optional linker TRAC, and the fourth polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV2-optional linker TRBC-optional linker hinge region-CH2-CH3.

21. The trispecific polypeptide molecule according to claim 1 , wherein the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-TRBV1-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL1-linker-VL2; or The first polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VL2-linker-VH2; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH1-linker-VL1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH2-linker-VL2; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-CH3; The first polypeptide chain comprises, from N-terminus to C-terminus: VH2-linker-VH1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL1-linker-VL2-linker-TRAV1-optional linker-TRAC; The first polypeptide chain comprises from N-terminus to C-terminus: VH2-linker-VL1-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises from N-terminus to C-terminus: VH1-linker-VL2-linker-TRAV1-optional linker-TRAC; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH1-optional linker-TRAC-linker-VL2-linker-VH2, and the second polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-optional linker-TRBC-linker-TRBV2-linker-TRAV2; or The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRBV1-linker-TRAV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRBV2-linker-TRAV2-linker-VH2-optional linker-TRBC; or The first polypeptide chain comprises from N-terminus to C-terminus: VL1-linker-TRAV1-linker-TRBV1-linker-VH1-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL2-linker-TRAV2-linker-TRBV2-linker-VH2-optional linker-TRBC.

22. The trispecific polypeptide molecule according to any one of claims 1 to 21, wherein the polypeptide molecule comprises one or more features selected from the group consisting of: (a) the polypeptide molecule comprises a TCR constant region or a fragment thereof, wherein the TCR constant region comprises a TCR α chain constant region (TRAC) and / or a β chain constant region (TRBC), wherein the TRAC and / or TRBC comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, preferably, the cysteine ​​mutation is at the following positions: position 48 of the wild-type TCR α chain constant region and position 57 of the wild-type TCR β chain constant region; (b) the polypeptide molecule comprises a CH3 domain, wherein the CH3s each independently comprise a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407 and the two CH3s do not comprise the same mutation, preferably, one of the CH3s comprises a T366W mutation and the other comprises T366S, L368A and Y407V mutations; (c) the polypeptide molecule comprises CH2 and CH3, wherein the CH2 and CH3 are derived from the Fc domain of human IgG1, IgG2 or IgG4 or a portion thereof, and preferably comprise at least two additional cysteine ​​residue mutations, such as S354C and Y349C or L242C and K334C; (d) the polypeptide molecule comprises a hinge region, wherein the hinge region is derived from the hinge domain of human IgG1, IgG2 or IgG4 or a portion thereof; (e) the polypeptide molecule comprises a cysteine-containing peptide segment, wherein the cysteine-containing peptide segment is derived from an antibody hinge domain, such as the C-terminus of a kappa or lambda chain or the hinge region of IgG1; preferably, the cysteine-containing peptide segment is selected from SSGGC, LGGC, FNRGEC, VAPTEC and VEPKSC; (f) The polypeptide molecule comprises CL and CH1, wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain.

23. The trispecific polypeptide molecule according to any one of claims 1 to 22, wherein said VH1 and VL1 comprise at least one cysteine ​​mutation to form a disulfide bond between VH1 and VL1, preferably, said cysteine ​​mutation is at the following positions: position 44 of VH1 and position 100 of VL1.

24. The trispecific polypeptide molecule according to any one of claims 1 to 23, wherein the polypeptide molecule further comprises one or more amino acid mutations, insertions or deletions that can enhance its affinity or biological activity, enhance its stability, extend its half-life, reduce its immunogenicity or reduce its post-translational modification; for example, the amino acid mutations include mutations that can enhance binding to FcRn and / or effector function silencing mutations.

25. The trispecific polypeptide molecule according to any one of claims 1 to 24, wherein the linkers in the polypeptide molecule are each independently selected from linkers consisting of 1 to 35 amino acids; Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGS, GGGGSGGGGSGGGGSGGGGSGGGGS, GQPKAAP, TVLRT, TVSSAS, GGEGG, GSEGGGS, RTSGPGDGGKGGPGKGPGGEGTKGTGPGG, GDGGKGGPGKGPGGEGTKGTGPGG, GKGPGGEGTKGTGPGG, TVLSSAS, EDLKN or a variant thereof, EDLNK or a variant thereof, and ANIQK or a variant thereof, or any combination thereof; Preferably, the variant of EDLKN is an amino acid sequence formed by substituting, deleting or adding one or more amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substituting, deleting or adding one or more amino acids of EDLNK. and / or a variant of ANIQK is an amino acid sequence formed by substitution, deletion or addition of one or more amino acids to ANIQK; and / or The polypeptide molecule comprises one or more domains selected from the following: CH3 domain, TCR constant region or fragment thereof, hinge region, CH2, CH3, CL, CH1, cysteine-containing peptide segment and combinations thereof, when the N-terminus of one or more domains is connected to the C-terminus of its upstream sequence via a linker, the linkers are each independently selected from linkers 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 a variant thereof, EDLNK or a variant thereof and ANIQK or a variant thereof; Preferably, the variant of EDLKN is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of ANIQK.

26. The trispecific polypeptide molecule according to any one of claims 1 to 25, wherein the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or more polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain, such as a signal peptide sequence derived from albumin or immunoglobulin, preferably MGWSCIILFLVATATGVHS.

27. The trispecific polypeptide molecule according to any one of claims 1 to 26, wherein the first antigen and the fourth antigen are each independently a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen; or wherein the first antigen and the fourth antigen are each independently a TSA selected from the group consisting of KRAS, TP53, PIK3CA, CTNNB1, EGFR, BRAF and GNAS; or wherein the first antigen and the fourth antigen are each independently 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 antigen; or The first antigen and the fourth antigen are each independently an autoantigen selected from the following: 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.

28. The trispecific polypeptide molecule according to any one of claims 1 to 27, wherein the second antigen and the third antigen are each independently selected from 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, N kp46, NKG2D, FcεRI, TCRα / β, TCRγ / δ, HLA-DR, CD28, 4-1BB(CD137), OX40(CD134), ICOS(CD278), 2B4(CD244), HVEM, LAG 3. DAP10, DAP12, CD27, CD40, GITR, LFA-1, MyD88, CD2, CD7, LIGHT, B7-H3, CTLA-4, PD-1, CD80, BTLA, TIM3, TIGIT and LAG-3.

29. The trispecific polypeptide molecule according to any one of claims 1 to 28, wherein the second antigen and the third antigen are different and are each independently CD3, CD28 and 4-1BB (CD137), and / or the first antigen and the fourth antigen are each independently selected from gp100, MAGEA1, KRAS and HPVE7.

30. The trispecific polypeptide molecule according to claim 8, wherein said polypeptide molecule comprises: a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 157, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 158; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 135, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 136; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 137, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 138; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 139, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 140; a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 11, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 12; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 151, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 152; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 17, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 18; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 19, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 20; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 23, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 24; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 27, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 28; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:35, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:36; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:43, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:44; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:45, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:46; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:49, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:50; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:51, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:52; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:53, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:54; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:55, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:56; A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 57, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 58; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 59, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 60; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 127, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO:

128.

31. The trispecific polypeptide molecule according to claim 20, wherein said polypeptide molecule comprises: A first polypeptide chain having the amino acid sequence shown in SEQ ID NO:61, a second polypeptide chain having the amino acid sequence shown in SEQ ID NO:62, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO:63, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO:

64.

32. 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, wherein the first binding region comprises an α chain variable region (TRAV1) and a β chain variable region (TRBV1) derived from a T cell receptor (TCR) that binds to the first antigen-MHC complex; 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; in, The TRAV1, TRBV1, VH and VL are distributed on the one or more polypeptide chains such that when the one or more polypeptide chains are folded, the TRAV1 and TRBV1 are spatially close to form the first binding region, and the VH and VL are spatially close to form the second binding region; The first antigen is selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and a self-antigen, and the second antigen is an immune cell surface molecule.

33. The bispecific polypeptide molecule according to claim 32, wherein the TRAV1, TRBV1, VH and VL are distributed on the one or more polypeptide chains and adjacent variable regions on the same chain are separated by a linker sequence.

34. The bispecific polypeptide molecule according to claim 32 or 33, wherein the polypeptide molecule further comprises at least one pair of TRAV2 and TRBV2, which form a third binding region that binds to a third antigen; in, The third antigen is selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a viral antigen, and an autoantigen, and the third antigen is the same as or different from the first antigen.

35. The bispecific polypeptide molecule according to any one of claims 32 to 34, wherein said polypeptide molecule comprises an additional copy of said VH and VL, which form a fourth binding region that binds to said second antigen.

36. The bispecific polypeptide molecule according to any one of claims 32 to 35, wherein the polypeptide molecule comprises the first binding region, the second binding region, when present, the third binding region, and when present, the fourth binding region on one polypeptide chain.

37. The bispecific polypeptide molecule according to claim 36, wherein the polypeptide molecule does not comprise the constant region of a TCR and does not comprise the hinge domain and Fc domain of an antibody.

38. The bispecific polypeptide molecule according to claim 37, wherein the polypeptide molecule comprises the following structure from N-terminus to C-terminus: TRAV1-Linker-TRBV1-Linker-VH-Linker-VL; or VL-Linker-TRAV1-Linker-TRBV1-Linker-VH; or TRAV1-Linker-VH-Linker-TRAV2-Linker-TRBV2-Linker-VL-Linker-TRBV1.

39. The bispecific polypeptide molecule according to any one of claims 32 to 35, wherein the polypeptide molecule comprises the first binding region, the second binding region, when present, the third binding region, and when present, the fourth binding region on two polypeptide chains.

40. The bispecific polypeptide molecule according to claim 39, wherein the polypeptide molecule does not comprise the constant region of a TCR and does not comprise the hinge domain and Fc domain of an antibody.

41. The bispecific polypeptide molecule according to claim 40, wherein said polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1; or The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VH, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRAV1; or The first polypeptide chain comprises from N-terminus to C-terminus: VH-linker-TRBV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VL; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRBV1-optional linker-cysteine-containing peptide segment, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-linker-VL-optional linker-cysteine-containing peptide segment; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV2-linker-VH, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-TRAV2-linker-VL; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-linker-TRAV2, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV1-linker-VL-linker-TRBV2; The cysteine-containing peptide segment is capable of forming a disulfide bond between the first polypeptide chain and the second polypeptide chain, and has a length of 3-10 amino acids, preferably a length of 4-8 amino acids, such as 4 or 6 amino acids.

42. The bispecific polypeptide molecule according to claim 41, wherein the cysteine-containing peptide is derived from an antibody hinge domain, such as the C-terminus of a kappa or lambda chain or the IgG1 hinge region; preferably, the cysteine-containing peptide is selected from the group consisting of SSGGC, LGGC, FNRGEC, VAPTEC and VEPKSC.

43. The bispecific polypeptide molecule according to claim 39, wherein the polypeptide molecule does not comprise an antibody hinge domain and a CH2 domain, and comprises an antibody-derived CH3 domain in the two polypeptide chains; The CH3 domains in the two polypeptide chains contain at least one mutation that can promote the polypeptide molecules to form heterodimers.

44. The bispecific polypeptide molecule according to claim 43, wherein said polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-CL-linker-VH-optional linker-CH3-linker-TRBV1-optional linker-CH1, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-CH1-linker-VL-optional linker-CH3-linker-TRAV2-optional linker-CL; or The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH-optional linker-CH3-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-TRBC-linker-VL-optional linker-CH3-linker-TRAV2-optional linker-TRAC; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-linker-VL-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-linker-VH-optional linker-CH3; or The first polypeptide chain comprises from N-terminus to C-terminus: TRBV1-Linker-VL-Optional Linker-CH3-Linker-TRAV1, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-Linker-VH-Optional Linker-CH3-Linker-TRAV2; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: VL-optional linker-CH3-linker-TRAV2-linker-TRBV2, and the second polypeptide chain comprises, from N-terminus to C-terminus: VH-optional linker-CH3-linker-TRAV1-linker-TRBV1; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: TRAV2-linker-TRBV2-optional linker-CH3-linker-VH-linker-VL; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VH-linker-VL-optional linker-CH3-linker-TRAV2-linker-TRBV2; wherein the CL is derived from the antibody light chain CL domain, and the CH1 is derived from the antibody heavy chain CH1 domain; The TRAC is derived from the constant region of the TCRα chain, and the TRBC is derived from the constant region of the TCRβ chain.

45. The bispecific polypeptide molecule according to claim 44, wherein: Each of the CH3 independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407 and the two CH3s do not comprise the same mutation, preferably, one of the CH3s comprises a T366W mutation and the other comprises T366S, L368A and Y407V mutations; and / or The TRAC and / or TRBC comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC; preferably, the cysteine ​​mutation is 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.

46. ​​The bispecific polypeptide molecule according to claim 39, wherein the polypeptide molecule does not comprise the hinge domain and Fc domain of an antibody, and comprises the α chain constant region (TRAC) and the β chain constant region (TRBC) of a TCR.

47. The bispecific polypeptide molecule according to claim 46, wherein said polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-TRBC; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-TRBC; or The first polypeptide chain comprises, from N-terminus to C-terminus: VH-linker-TRAV1-optional linker-TRAC-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: VL-linker-TRBV2-optional linker-TRBC-linker-TRAV2-optional linker-TRAC; or The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH-optional linker-TRAC-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-TRBC-linker-VL-optional linker-TRBC-linker-TRAV2-optional linker-TRAC.

48. The bispecific polypeptide molecule according to claim 39, wherein the polypeptide molecule does not comprise the hinge domain and Fc domain of an antibody, and comprises the α chain constant region (TRAC) and the β chain constant region (TRBC) of a TCR, and the TRAC and TRBC are respectively connected to the C-termini of the two polypeptide chains of the polypeptide molecule via an optional linker.

49. The bispecific polypeptide molecule according to claim 48, wherein said polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-TRBV1-optional linker-TRBC; or The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-VH-optional linker-TRAC, and the second polypeptide chain comprises from N-terminus to C-terminus: TRBV2-linker-TRAV2-linker-VL-linker-TRBV1-optional linker-TRBC; or The first polypeptide chain comprises, from N-terminus to C-terminus: TRAV1-optional linker-TRAC-linker-VH-optional linker-TRAC-linker-TRBV1-optional linker-TRBC, and the second polypeptide chain comprises, from N-terminus to C-terminus: TRBV2-optional linker-TRBC-linker-VL-optional linker-TRBC-linker-TRAV2-optional linker-TRAC.

50. The bispecific polypeptide molecule according to any one of claims 46 to 49, wherein: The TRAC and / or TRBC comprises at least one cysteine ​​mutation relative to the wild-type sequence to form a disulfide bond between TRAC and TRBC, preferably, the cysteine ​​mutation is 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.

51. The bispecific polypeptide molecule according to claim 39, wherein the polypeptide molecule does not comprise a TCR constant region, and the two polypeptide chains comprise a hinge domain and CH2 and CH3 domains derived from an antibody; The CH3 domains in the two polypeptide chains contain at least one mutation that can promote the polypeptide molecules to form heterodimers.

52. The bispecific polypeptide molecule according to claim 51, wherein said polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises from N-terminus to C-terminus: TRAV1-linker-TRBV1-optional linker-hinge domain-CH2-CH3, and the second polypeptide chain comprises from N-terminus to C-terminus: VL-linker-VH-optional linker-hinge domain-CH2-CH3; or The first polypeptide chain comprises, from N-terminus to C-terminus: hinge domain-CH2-CH3-linker-TRBV1-linker-TRAV1, and the second polypeptide chain comprises, from N-terminus to C-terminus: hinge domain-CH2-CH3-linker-VH-linker-VL; or The first polypeptide chain comprises from N-terminus to C-terminus: hinge domain-CH2-CH3-linker-TRAV1-linker-VH, and the first The two polypeptide chains comprise from N-terminus to C-terminus: hinge domain-CH2-CH3-linker-VL-linker-TRBV1.

53. The bispecific polypeptide molecule according to claim 51 or 52, wherein: Each of the CH3 independently comprises a mutation at one or more positions selected from amino acid positions 366, 368, 405 and 407 and the two CH3s do not comprise the same mutation, preferably, one of the CH3s comprises a T366W mutation and the other comprises T366S, L368A and Y407V mutations; and / or Said CH2 and CH3 are derived from the Fc domain of human IgG1, IgG2 or IgG4 or part thereof, and preferably contain at least two additional cysteine ​​residue mutations, such as S354C and Y349C or L242C and K334C; and / or The hinge region is derived from the hinge domain of human IgG1, IgG2 or IgG4, or a portion thereof.

54. The bispecific polypeptide molecule according to any one of claims 32 to 53, wherein The VH and VL comprise at least one cysteine ​​mutation to form a disulfide bond between VH and VL, preferably, the cysteine ​​mutation is at the following positions: position 44 of VH and position 100 of VL; and / or The TRAV1 and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV1 and VL, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV1 and position 80 of VL; and / or The TRAV2 and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV2 and VL, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV2 and position 80 of VL; and / or The TRBV1 and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRBV1 and VL, preferably, the cysteine ​​mutation is at the following positions: position 84 of TRBV1 and position 80 of VL; and / or The TRBV2 and VL comprise at least one cysteine ​​mutation to form a disulfide bond between TRBV2 and VL, preferably, the cysteine ​​mutation is at the following positions: position 84 of TRBV2 and position 80 of VL; and / or The TRAV1 and VH comprise at least one cysteine ​​mutation to form a disulfide bond between TRAV1 and VH, preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV1 and position 89 of VH; and / or The TRAV2 and VH contain at least one cysteine ​​mutation to form a disulfide bond between TRAV2 and VH. Preferably, the cysteine ​​mutation is at the following positions: position 104 of TRAV2 and position 89 of VH.

55. The bispecific polypeptide molecule according to any one of claims 32 to 54, wherein the polypeptide molecule further comprises one or more amino acid mutations, insertions or deletions that can enhance its affinity or biological activity, enhance its stability, extend its half-life, reduce its immunogenicity or reduce its post-translational modification; for example, the amino acid mutations include mutations that can enhance binding to FcRn and / or effector function silencing mutations.

56. The bispecific polypeptide molecule according to any one of claims 32 to 55, wherein the linkers in the polypeptide molecule are each independently selected from linkers consisting of 1 to 35 amino acids; Preferably, the linkers are each independently selected from S, GGGS, GGGGS, GGGSGGGG, GGSGGS, GGSGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGS, GGGGSGGGGSGGGGSGGGGSGGGGS, GQPKAAP, TVLRT, TVSSAS, GGEGG, GSEGGGS, RTSGPGDGGKGGPGKGPGGEGTKGTGPGG, GDGGKGGPGKGPGGEGTKGTGPGG, GKGPGGEGTKGTGPGG, TVLSSAS, EDLKN or a variant thereof, EDLNK or a variant thereof, and ANIQK or a variant thereof, or any combination thereof; Preferably, the variant of EDLKN is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of ANIQK; and / or The polypeptide molecule comprises one or more domains selected from the following: CH3 domain, TCR constant region or fragment thereof, hinge region, CH2, CH3, CL, CH1, cysteine-containing peptide segment and their combination, when the N-terminus of one or more domains is connected to the C-terminus of its upstream sequence through a linker, the linkers are independently 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 a variant thereof, EDLNK or a variant thereof and ANIQK or a variant thereof; Preferably, the variant of EDLKN is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLKN; and / or the variant of EDLNK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of EDLNK; and / or the variant of ANIQK is an amino acid sequence formed by substitution, deletion or addition of one or several amino acids of ANIQK.

57. The bispecific polypeptide molecule according to any one of claims 32 to 56, wherein the polypeptide molecule further comprises a signal peptide sequence at the N-terminus of one or more polypeptide chains, preferably a signal peptide sequence at the N-terminus of each chain, such as a signal peptide sequence derived from albumin or immunoglobulin, preferably MGWSCIILFLVATATGVHS.

58. The bispecific polypeptide molecule according to any one of claims 32 to 57, wherein the first antigen and the third antigen are each independently a TAA selected from the group consisting of melanoma-associated antigens (e.g., gp100, MAGEA1, MAGEA3, MAGEA6, MAGEA4, MAGEA2, MAGEA12, MAGEA2B, MAGEA9B, MAGEA10, MAGEA11, MAGEB2, MAGEC1, MAGEC2), IGF2BP1, GNGT1, PI4K2B, CCR8, NPSR1, COX7B2, ONECUT3, SMC1B, FOXI3, GAGE2A, FBXO43, BRDT, PAGE2, GAGE13, POU5F1B, CTAG1A, and an endogenous reverse transcriptase antigen; or wherein the first antigen and the third antigen are each independently a TSA selected from the group consisting of KRAS, TP53, PIK3CA, CTNNB1, EGFR, BRAF and GNAS; or wherein the first antigen and the third antigen are each independently 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 antigen; or The first antigen and the third antigen are each independently an autoantigen selected from the group consisting of AFP, CEA, CD19, CD20, BCMA, CD22, CD30, SLAM, CLDN18.2, GD2, mesothelin, CD38, Her2, GPC3, MUC1, Ro52, Ro60, La, Jo-1, SRP, IFIH1, CENPA, CENPB, SNRPA1, SNRNP70, SNR-PD3, RNAP3, TOPO1, Insulin , 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.

59. The bispecific polypeptide molecule according to any one of claims 32 to 58, wherein 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.

60. The bispecific polypeptide molecule according to any one of claims 32 to 59, wherein the second antigen is CD3, CD28 or 4-1BB (CD137), and / or the first antigen and the third antigen are each independently selected from gp100, MAGEA1, KRAS and HPVE7.

61. The bispecific polypeptide molecule according to claim 36, wherein the polypeptide molecule comprises a polypeptide chain having an amino acid sequence selected from any one of SEQ ID NOs: 9 and 10.

62. The bispecific polypeptide molecule according to claim 39, wherein said polypeptide molecule comprises: a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 141, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 142; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 143, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 144; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 145, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 146; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 147, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 148; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 149, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 150; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 299, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 300; a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 1, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 2; a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 3, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 4; a first polypeptide chain having the amino acid sequence shown in SEQ ID NO:5, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO:6; a first polypeptide chain having the amino acid sequence shown in SEQ ID NO:7, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO:8; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 13, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 14; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 15, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 16; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:29, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:30; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:31, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:32; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 33, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 34; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 37, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 38; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:39, and a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:40; A first polypeptide chain having the amino acid sequence shown in SEQ ID NO:41, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO:42; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 47, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 48; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 129, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 130; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 131, and a second polypeptide chain having the amino acid sequence shown in SEQ ID NO:

132.

63. A nucleic acid comprising a nucleotide sequence encoding each chain of the trispecific polypeptide molecule of any one of claims 1-31 or the bispecific polypeptide molecule of any one of claims 32-62.

64. A vector comprising the nucleic acid of claim 63.

65. A vector system comprising, on one or more vectors, a nucleotide sequence encoding each chain of the trispecific polypeptide molecule of any one of claims 1-31 or the bispecific polypeptide molecule of any one of claims 32-62.

66. A host cell comprising the nucleic acid of claim 63, the vector of claim 64, or the vector system of claim 65.

67. A conjugate comprising the trispecific polypeptide molecule of any one of claims 1-31 or the bispecific polypeptide molecule of any one of claims 32-62, and a chemical moiety conjugated to the trispecific polypeptide molecule or the bispecific polypeptide molecule.

68. The conjugate according to claim 67, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, an immunostimulatory molecule, and a detectable label; Preferably, the therapeutic agent is selected from the group consisting of an immunomodulator, a radioactive compound, an enzyme, a chemotherapeutic agent, and a toxin. Preferably, the immunostimulatory molecules are selected from cytokines, chemokines, platelet factors and complement initiators; Preferably, the detectable label is selected from the group consisting of biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, a nucleic acid probe, a contrast agent, and a fluorescent, phosphorescent or chemiluminescent molecule.

69. A pharmaceutical composition comprising the trispecific polypeptide molecule of any one of claims 1-31, the bispecific polypeptide molecule of any one of claims 32-62, the nucleic acid of claim 63, the vector of claim 64, the vector system of claim 65, the host cell of claim 66, the conjugate of claim 67 or 68, and a pharmaceutically acceptable excipient.

70. The pharmaceutical composition according to claim 69, wherein the pharmaceutical composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent and a small molecule drug.

71. A method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the trispecific polypeptide molecule of any one of claims 1-31, the bispecific polypeptide molecule of any one of claims 32-62, the nucleic acid of claim 63, the vector of claim 64, the vector system of claim 65, the host cell of claim 66, the conjugate of claim 67 or 68, or the pharmaceutical composition of claim 69 or 70, wherein the disease is selected from cancer, an infectious disease, an autoimmune disease, and an inflammatory disease.

72. The method according to claim 71, further comprising administering to the subject a second therapeutic agent, preferably, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

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